British Pharmacopoeia 2020

Volume I

The British Pharmacopoeia Commission has caused this British

Pharmacopoeia 2020 to be prepared under regulation 317(1) of the Human
Medicines Regulations 2012 and, in accordance with regulation 317 (4), the
Ministers have arranged for it to be published. It has been notified in draft
to the European Commission in accordance with Directive 98/34/EEC.
The monographs of the Ninth Edition of the European Pharmacopoeia
(2016), as amended by Supplements 9.1 to 9.8, published by the Council
of Europe are reproduced either in this edition of the British
Pharmacopoeia or in the associated edition of the British Pharmacopoeia
. (Veterinary).
See General Notices

Effective date: 1 January 2020

see Notices

London: The Stationery Office

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 In respect of Great Britain:

THE DEPARTMENT OF HEALTH AND SOCIAL CARE

In respect of Northern Ireland:

THE DEPARTMENT OF HEALTH (NI)

© Crown Copyright 2019

Published by The Stationery Office on behalf of the Medicines and
Healthcare products Regulatory Agency (MHRA) except that:

European Pharmacopoeia monographs are reproduced with the permission

of the Council of Europe and are not Crown Copyright, These are
identified in the publication by a chaplet of stars.

This publication is a 'value added' product. If you wish to re-use the

Crown Copyright material from this publication, applications must be made
. in writing, clearly stating the material requested for re-use, and the purpose
for which it is required. Applications should be sent to: Mr J Pound,
MHRA, 10th Floor, 10 South Colonnade, Canary Wharf, London
E144PU.

First Published 2019

ISBN 978 011 3230 761

British Pharmacopoeia Commission Office:

Medicines and Healthcare products Regulatory Agency
10 South Colonnade,
Canary Wharf,
London E14 4PU
Telephone: +44 (0)20 3080 6561
E-mail: [email protected]
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Laboratory:
British Pharmacopoeia Commission Laboratory
Queen's Road
Teddington
Middlesex TWII OLY
Telephone: +44 (0)20 8943 8960
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Foreword

Patients rightly expect that their medicines work and are acceptably safe
and this is underpinned by the assurance of medicines quality. The British
Pharmacopoeia (BP), as part of the Medicines and Healthcare Products
Regulatory Agency (MHRA), plays an important role in the assurance of
medicines quality and supporting innovation through the development of its
quality standards. The BP shares the MHRA's· commitment to working
with our stakeholders, from our partners in industry to international
regulatory and pharmacopoeial peers.
The BP excels at collaboration and this can be seen across its work with
international partners, support for European standards via its role as a
National Pharmacopoeial Authority to the European Pharmacopoeia
Commission and its constructive approach to consultation, with recent
examples being the BP's consultation on Dissolution testing and its input
into the MHRA's Strategy for Pharmacopoeial Public Quality Standards for
Biological Medicines.
Just as the BP's partners and stakeholders are diverse and varied, so too is
the breadth of expertise needed to produce standards that ensure patients'
expectations of quality medicines are met.
Some of our most important partners are the independent experts that form
the BP Commission and its working groups, panels and parties. These
experts volunteer from a wide range of backgrounds such as Industry,
Academia and the UK's National Health .Service (NHS) and bring a diverse
array of skills, including pharmaceutics, synthetic chemistry, biology and
statistics, to our work.
It is these experts who work to help us overcome challenges and develop
the standards and policies that continue to ensure that our work meets the
needs of our users, and ultimately, patients. They all share our commitment
to the assurance of quality and the role of standards in supporting this.
It is only by working together collectively with our experts, peers and
stakeholders that we can realise our shared mission to protect public health.

Professor Sir Michael· Rawlins

Chairman
Medicines and Healthcare products Regulatory Agency

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Contents

Contents of Volume I

FOREWORD
NOTICES
PREFACE
BRITISH PHARMACOPOEIA COMMISSION
EXPERT ADVISORY GROUPS, PANELS OF EXPERTS AND
WORKING PARTIES
CODE OF PRACTICE
MEMBERSHIP
BP Commission, Expert Advisory Groups, Panels of Experts, Working
Parties
STAFF
British Pharmacopoeia, BP Laboratory, Publisher
INTRODUCTION
Additions, Omissions, Technical Changes, Changes in Title
GENERAL NOTICES
MONOGRAPHS
Medicinal and Pharmaceutical Substances (A - I)
Contents of Volume II

NOTICES
GENERAL NOTICES
MONOGRAPHS
Medicinal and Pharmaceutical Substances a- Z)
Contents of Volume III

NOTICES
GENERAL NOTICES
MONOGRAPHS
Formulated Preparations: General Monographs
Formulated Preparations: Specific Monographs

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 Contents of Volume IV

NOTICES
GENERAL NOTICES
MONOGRAPHS
Herbal Drugs, Herbal Drug Preparations and Herbal Medicinal Products

Materials for use in the Manufacture of Homoeopathic Preparations

Blood-related Products

Immunological Products
Radiopharmaceutical Preparations

Surgical Materials

Contents of Volume V

NOTICES
GENERAL NOTICES
INFRARED REFERENCE SPECTRA
APPENDICES
SUPPLEMENTARY CHAPTERS

INDEX

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 Notices

Monographs of the European Pharmacopoeia are distinguished by a chaplet

of stars against the title. The term European Pharmacopoeia, used without
qualification, means the ninth edition of the European Pharmacopoeia
comprising, unless otherwise stated, the main volume, published in 2016, as
amended by any subsequent supplements and revisions. "

Patents In this Pharmacopoeia certain drugs' and preparations have been included
notwithstanding the existence of actual or potential patent rights. In so far
as such substances are protected by Letters Patent their inclusion in this
Pharmacopoeia neither conveys, nor implies, licence to manufacture.

Effective dates New and revised monographs of national origin enter into force on
1 January 2020. The monographs are brought into effect under regulation
320(2) of the Human Medicines Regulations 2012.

Monographs of the European Pharmacopoeia have previously been

published by the European Directorate for the Quality of Medicines &
HealthCare, in accordance with the Convention on the Elaboration of a
European Pharmacopoeia, and have been brought into effect under
European Directives 2001/82IEC, 200 1/83/EC .and 2003/63IEC, as
amended, on medicines for human and veterinary use.

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 Preface

The British Pharmacopoeia Commission has caused this British

Pharmacopoeia 2020 to be prepared under regulation 317(1) of the Human
Medicines Regulations 2012 and, in accordance with regulation 317(4), the
Ministers have arranged for it to be published.
The British Pharmacopoeia 2020 contributes significantly to the quality
control of medicinal products for human use. It contains publicly available,
legally enforceable standards that provide an authoritative statement of the
quality that a product, material or article is expected to meet at any time
during its period of use. The Pharmacopoeia! standards are designed to
complement and assist the licensing and inspection processes and are part
of the overall system for safeguarding the health of purchasers and users of
medicinal products in the UK.
The British Pharmacopoeia Commission wishes to record its appreciation of
the services of all those who have contributed to this important work.

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British Pharmacopoeia
Commission

The British Pharmacopoeia Commission is appointed, on behalf of the

Secretary of State for Health and Social Care, by the Department of Health
and Social Care's Appointments Team who are responsible for
appointments to all of the Advisory Bodies appointed under the Human
Medicines Regulations 2012.

Under the terms of the Human Medicines Regulations 2012, the duties of
. the British Pharmacopoeia Commission are as follows:
(a) the preparation and publication of any new edition of the British
Pharmacopoeia [regulations 317(1) and 317(4)];
(b) the preparation and publication of any compendium containing
information relating to substances and articles which are or may be
used in the practice of veterinary medicine or veterinary surgery
[regulations 317(3)(b) and 317(4)];
(c) the preparation and publication of a list of names to be used as the
headings to monographs in the British Pharmacopoeia [regulations 318
(1) and 318(2)];
(d) the preparation of any amendments to the above publications
[regulation 317(5)(a)].

Members of the British Pharmacopoeia Commission are appointed for a

renewable term of 4 years and, under the requirements laid down by the
Office of the Commissioner for Public Appointments; can serve for a
maximum of 10 years.

In order to ensure that the British Pharmacopoeia Commission fulfils its

duties under the Human Medicines Regulations 2012, the members also
have the following duties:
(1) to frame clear and unequivocal technical advice in order to discharge
the Commission's responsibilities both for the British Pharmacopoeia,
the British Pharmacopoeia (Veterinary) and British Approved Names
and as the national pharmacopoeial authority with respect to the
European Pharmacopoeia;
(2) to develop clear policies for the preparation and publication of the
British Pharmacopoeia and its related publications;
(3) to serve on one or more Expert Advisory Groups or Panels of Experts
of the BP Commission, usually in the position of Chair or Vice-Chair;
(4) to approve new and revised text for inclusion in new editions of the
British Pharmacopoeia and British Pharmacopoeia (Veterinary);
(5) to approve new and revised names for inclusion in new editions of
British Approved Names and its annual supplements.

In addition to the duties listed above, the Chair of the British

Pharmacopoeia Commission has the following additional duties:
(1) To chair all scheduled and unscheduled meetings;

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 (2) To carry out members appraisals in accordance with policies and
timelines laid down by the Department of Health and Social Care;
(3) To participate in the process to appoint/re-appoint members of the
British Pharmacopoeia Commission.

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Expert Advisory Groups, Panels
of Experts and Working Parties

Members of Expert Advisory Groups, Panels of Experts and Working

Parties are appointed by the British Pharmacopoeia Commission.
The duties of the members are as follows:
(a) to collaborate in the preparation and revision of Monographs,
Appendices and Supplementary Chapters for inclusion in the British
Pharmacopoeia and British Pharmacopoeia (Veterinary);
(b) to collaborate in the preparation and revision of Monographs, Methods
and General Chapters of the European Pharmacopoeia;
(c) to review reports from the British Pharmacopoeia Laboratory in terms
of technical content and, where possible, provide independent
experimental data to assist in decision making;
(d) to collaborate in the preparation and revision of the list of names to be
used as titles for monographs of the British Pharmacopoeia and British
Pharmacopoeia (Veterinary).
Members of Expert Advisory Groups, Panels of Experts and Working
Parties are usually appointed for a renewable term of 4 years.

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 Code of Practice

Members of the British Pharmacopoeia Commission and its supporting

Expert Advisory Groups, Panels of Experts and Working Parties are
required to comply with a Code of Practice on Declaration of Interests in
the Pharmaceutical Industry.
British Pharmacopoeia Commission

The Chair and members of the British Pharmacopoeia Commission are

required to make a full declaration of interests on appointment and
annually thereafter. They must also inform the BP Secretariat promptly of
any changes to these interests during the year. These interests are published
in the Medicines Advisory Bodies Annual Reports.
Relevant interests must be declared at meetings and are recorded in the
Minutes.
Expert Advisory Groups, Panels of Experts and Working Parties

Chairs and members are required to make a full declaration of interests on

appointment and to update the Secretariat if these interests change during
their term of office. A record is kept of those experts who have declared
specific interests, but these are not published.
Relevant interests must be declared at meetings and are recorded in the
Minutes.

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 Metnbership of the British
Pharmacopoeia Commission

The list below includes those members who served during the period 2018
to 2019.

Chair .Professor Kevin M G Taylor BPharm PhD FRPharmS

Professor of Clinical Pharmaceutics, UCL School of Pharmacy

Vice-Chair Professor Alastair G Davidson BSc PhD FRPharmS

Visiting Professor of Pharmaceutical Sciences, University of Strathclyde

Professor Matthew Almond BSc DPhil DSc CChem FRSC PFHEA NTF
Professor of Chemistry Education, University of Reading

Dr Jon Beaman BSc PhD MBA CChem MRSC

Head of Development Analytical Group, Pfizer UK

Dr Anna-Maria Brady BSc PhD

Former Head of Biologicals and Administration, Veterinary Medicines Directorate

Dr Graham D Cook BPharm PhD MRPharmS

Senior Director, Process Knowledge/Quality by Design, Pfizer

Dr Andrew Coulson! BVetMed MSc MRCVS MA

Member of the Royal College of Veterinary Surgeons; Non-Executive Director,
Veterinary Medicines Directorate; former Superintending Inspector, Science &
Research Group, The Home Office

Dr Alison Gleadle BSc PhD (Lay representative)

Former Group Product Risk Director, Tesco Stores Ltd.

Dr Gerard Lee BPharm PhD FRPharmS MRSC CChem

Fonner Group Manager, British Pharmacopoeia and Laboratory Services,
MHRA; former Secretary & Scientific Director of the British Pharmacopoeia
Commission

Mr Robert Lowe BPharm FRPharmS

Director of Pharmacy Quality Assurance Specialist Services, NHS East of
England & Northamptonshire

Dr Brian R Matthews/ BPharm PhD FRPharmS FTOPRA

Consultant on pharmaceutical and medical device regulatory affairs; former Senior
Director, EC Registration, Alcon Laboratories

Professor John Miller MSc PhD MRSC CChem

Visiting Professor, Strathclyde Institute of Pharmacy and Biomedical Sciences;
former Head of the EDQM Laboratory

1 Deceased.
2 Retired, 31 December 2018.

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 . Ms Sharon Palser MSc (Lay Representative)
Former Director of Development, NHS Plymouth

Professor Monique Simmonds OBE JP BSc PhD FLS FBS FRES FWIF
Deputy Director of Science, Royal Botanic Gardens, Keui

Dr Ronald Torano BSc PhD MRSC CChem

Pharmacopoeial Intelligence and Advisory Specialist; GlaxoSmithKline

Dr Paul Varley BSc PhD

Vice President of Biopharmaceutical Development, Medimmune Limited

Secretary and Mr James Pound BSc

Scientific Director Group Manager, British Pharmacopoeia and Laboratory Services, MHRA

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 Metnbership of Expert Advisory
Groups, Panels of Experts and
Working Parties

The Commission appointed the following Expert Advisory Groups, Panels

of Experts and Working Parties to advise it in carrying out its duties.
Membership has changed from time to time; the lists below include all who
have served during the period 2018 to 2019.

EXPERT ADVISORY GROUPS

ABS: Antibiotics R L Harder (Chair), G Cook (Vice-Chair), G Blake, E Flahive, V jaitely,
W Mann, J Miller, M Pires, J Sum al, I R Williams

BIO: Biological and P Varley (Chair), A-MBrady (Vice-Chair), L Bisset", C. Braxton, C Bums,
Biotechnological K Chidwick', A Cook", J Cook', B Cowper, S Gill, E Griffiths, C jones", V
Products Loh, A Kippen, K Nordgren,B Patel, A M Pickett", T Pronce, L Randon,
I Rees l, S Schepe1mann l, P Sheppard, P Stickings l, A H Thomas'',
R Thorpe, L Tsang, M Wadhwal, W Zunic

HCM: Herbal and M Simmonds (Chair), R Middleton (Vice-Chair), L A Anderson,

Complementary P Anderson, A Booker, C Etheridge, C Leon, B Moore, M Pires, E Reich,
Medicines M Rowan, A Slater, K Strohfeldt-Venables, J Sumal", C Welham,
E Williamson, K Zhao
(Corresponding members SS Handa, A Krauss, Z-T Wang)

MCl: Medicinal A G Davidson (Chair), D Cairns (Vice-Chair), S Bale, H Batchelor,

Chemicals J C Berridge, E Bush, A J Caws,D Deutsch, P Fleming, E Gray,
W J Lough, D Malpas, S Nolan

Me2: Medicinal G Cook (Chair), C T Goddard (Vice-Chair), J Birchall, K Bracht, J Cowie,

Chemicals D Edwards, K Foster, E Hook, J Lim, J Miller, P Murray'',
A Ruggiero, M Turgoose, N Wynne
(Corresponding members M Brit~,)V Sherwin)

MC3: Medicinal M Almond (Chair), J Beach (Vice-Chair, from 1 January 2019), J Beaman, K
Chemicals Foster, C T Goddard, P Hampshire, W K L Pugh, B Rackstraw, R Torano,
M Tubby, I R Williams

NOM: J K Aronson (Chair), L Tsang (Vice-Chair, until 31 December 2018),

Nomenclature M Ahmed, A McFarlane, D Mehta, G P Moss, R Thorpe
(Corresponding members R G Balocco Mattavelli, J S Robertson)

1 Specialist member.
2 Deceased.

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 PCY: Pharmacy R L Horder (Chair), B R Matthews (Vice-Chair~ until 31 December 2018),
R Lowe (Vice-Chair, from 1 January 2019), M Ahmed", E Baker, J Beach,
D Elder, J Lim", J MacDonald, A McFarlane, J F McGuire, T Purewal,
K M G Taylor, S Wicks
(Corresponding member J Churchill)

ULM: Unlicensed M G Lee (Chair), V Fenton-May (Vice-Chair), A Bosley, S Branch,

Medicines D Caulfield, M Godber,W Goddard, S Hartley, SHo, J Rickard, D Kirby,
M Santillo, J Ramada-Magalhaes, J Smith, A Sully, P Weir, M Westwood

PANELS OF EXPERTS
BLP: Blood K Chidwick, A R Hubbard, J More, P Varley
Products

CX: Excipients B R Matthews (Chair~ until 31 December 2018), C Mroz (Vice-Chair), H

Batchelor, R Cawthorne, D Deutsch

DNA: Identification A Slater (Chair), M Carine, I Feavers, J Hawkins, E Mee, E \1V'illiamson

Techniques
(Disbanded~ 30 June 2019)

IGC: Inorganic and C T Goddard (Chair), M Almond, S Atherton, S Boland, D Caulfield,

General Chemicals P Henrys, G Lay

MIC: Microbiology V Fenton-May (Chair), B Alexander, S Denyer, P Hargreaves, C Iverson, V

jaitely, B R Matthews, J Silva

RAD: Radioactive I Boros, J Brain, D Graham, G Inwards, R D Pickett, R Smith

Materials

VET: Veterinary E Williamson (Chair), A Coulson/ (Vice-Chair), A Cairns, S Cockbill,

" Medicines D Evans, E Flahive, B Ward

VIP: Veterinary A M Brady (Chair), R Banks, R Cooney, M Johnson, K Redhead, J Salt, C

Immunological Stirling, R Woodland
Products

WORKING PARTIES
AQbD: Analytical G Cook (Chair), S Brown, M Chatfield, S Ellison, C Gray, M Hanna-
Quality by Design Brown, S Jones, P Nethercote, E Razzano
(Corresponding members K Barnett, B Harrington, W Sherwin)

BIO-DPS: P Varley (Chair), A-M Brady (Vice-Chair),B Cowper, C Burns, N Czeloth,

Documentary and L Duhau, V Ganeva, C E Giartosio, A Ramzan, B Rellahan, M Wild
Physical Standards*
* BIO-DPS: Alternative Approaches for Documentary and Physical Standards
for Biotechnological Products

1 Specialist member.
2 Deceased.

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MCS: Microscopy E Williamson (Chair), R Arroo, R Fleck, IZ Helliwell, K MacLellan Gibson
(Disbanded, 31 December 2018)

AD-HOC GROUP
New Analytical M Almond, J Beaman, G Cook, J Miller, R Torano, M Simmonds
Technologies

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 Current British Pharmacopoeia
Staff

Secretariat J Pound (Secretary and Scientific Director)

A Gibb (Editor-in-Chief)
S Young (Head of Analytical Science)
H Ashraf, H Corns, P Crowley, L Elanganathan, A Evans, A Gardiner,
S Gomersal, G Kemp, C Lenihan, G Li-Ship, S Maddocks, H Makwana,
F J Swanson, M-L Wall, M Whaley

NIBSC-based Staff L Gibson, C Gkouva, C Howard, C Lockie-Williams

Administrative F Chughtai, B F Delahunty, J Paine, U Rothna, N Siddika

ISO 9001
F527268

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Current British Pharmacopoeia
Laboratory Staff

R Adams (Operations Manager)

D Ballottin, C Balsa, 0 Bernabe.A Biesenbruch, M Boardman, H Bowden,
K Busuttil, A Cepeda, S Choudhury, A Ciesluk, J Couzins, Y EI Dabh, S
Doyle, S Ganguli, M Goode, R Griffiths, D Holcombe, N Ionescu,
PMakhomu, K Meyer de Figueiredo, W Mohammed,G Naar, M Nanasi,
A Paul, M Petrova, L Piare, S Planou, R Ravishankar, S Reeves,
I Reydellet, D Rutty, M Sciberras, G Searle, C Smart, B Smith,
C Thompson, M Threadgold, V Vekereya

ISO 9001
F527613

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Current Staff of the Publisher of
the British Pharmacopoeia

A Prince (Business Director)

P Allard (Service Delivery Manager)

A Allen (Director, Parliament and Publishing)

N Billington, C Cole, A Dampier, C Gaines, N Griffiths, A Hughes, I
Ichongiri, N joisa, J Kharuna, SPage, M Parka, N Pope, P Relfe, J Stoker,
V Verma, T Wheeler

ISO 9001
F522428

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2020 Introduction I-xxiii

Introduction

British Pharmacopoeia 2020

The British Pharmacopoeia 2020 supersedes the British Pharmacopoeia
2019. It has been prepared by the British Pharmacopoeia Commission, with
the collaboration and support of its Expert Advisory Groups, Panels of
Experts and Working Parties and contains approximately 4000 monographs
for substances, preparations and articles used in the practice of medicine.
Some of these monographs are of national origin and have been elaborated
or revised under the auspices of the British Pharmacopoeia Commission
whilst others (indicated to users by a chaplet of stars) have been elaborated,
or revised, under the auspices of the. European Pharmacopoeia
Commission, supported by its Groups of Experts and Working Parties, and
- are reproduced from the European Pharmacopoeia. This edition, together
with its companion volume, the British Pharmacopoeia (Veterinary) 2020,
incorporates all the monographs of the 9th Edition of the European
Pharmacopoeia, as amended by Supplements 9.1 to 9.8. Users of the
British Pharmacopoeia thereby benefit by finding within this
comprehensively indexed compendium all current United Kingdom
pharmacopoeial standards for medicines for human. use.
The BP 2020 comprises six volumes as follows.

Volumes I and II Medicinal Substances

Volume III Formulated Preparations: General Monographs
Formulated Preparations: Specific Monographs
Volume N Herbal Drugs, Herbal Drug Preparations and
Herbal Medicinal Products
Materials for use in the Manufacture of
Homoeopathic Preparations
Blood-related Products
Immunological Products
Radiopharmaceutical Preparations
Surgical Materials
Volume V Infrared Reference Spectra
Appendices
Supplementary Chapters
Index
Volume VI British Pharmacopoeia (Veterinary) 2020

Effective Date The effective date for British Pharmacopoeia monographs in this edition is
1 January 2020.
National monographs omitted from this or earlier editions of the British
Pharmacopoeia remain effective in accordance with Regulation 252(2)(c) of
the Human Medicines Regulations 2012.

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I-xxiv Introduction 2020

Implementation dates regarding European Pharmacopoeia publications are

provided in Supplementary Chapter IV B: Dates of Implementation.
European Pharmacopoeia monographs are identified by a chaplet of stars
alongside the title.

Additions A list of monographs included for the first time in the British
Pharmacopoeia 2020 is given at the end of this introduction. It includes 35
new monographs of national origin and 40 new monographs reproduced
from the 9th Edition of the European Pharmacopoeia, as amended by
Supplements 9.1 to 9.8.

Pharmacopoeial The Medicines and Healthcare products Regulatory Agency (MHRA) has
Public Quality continued to implement its strategy for pharmacopoeial public quality
Standards for standards for biological medicines as published in 2017 1 . The strategy
Biological acknowledges the importance of biological medicines, the value of
Medicines pharmacopoeial public quality standards and the unique position of the
MHRA to lead in this field through its alignment of regulatory,
documentary (BP) and physical (NIBSe) standard setting functions.

Part of the published strategy was to investigate alternative approaches to

standards for biological medicines. This has led to the establishment of the
Alternative Approaches for Documentary and Physical Standards for
Biotechnological Products Working Party (WP BIO-DPS) in 2018. WP
BIO-DPS aims to better understand the challenges of implementing
pharmacopoeial standards and how alternative approaches can respond to
these issues whilst continuing to support innovation throughout the product
lifecycle. The working party are exploring ideas for new standards concepts,
including performance and class based standards, and how to assess the
value and potential implementation of such concepts.

The strategy also highlighted the need to investigate and take forward
documentary and physical standard setting opportunities for Advanced
Therapy Medicinal Products (ATMPs).The MHRA has engaged with
groups across the ATMP community to improve its understanding of the
challenges and needs faced by the ATMP community and the role of
standards in supporting innovation and the assurance of product quality.

Dissolution The dissolution consultation response was published in January 2019 and
Consultation incorporates the feedback from stakeholders and proposals put forward by
the Pharmacy Expert Advisory Group and British Pharmacopoeia
Commission. The response document outlines a revised policy on
dissolution testing. The BP has addressed each of the points raised with a
clear action plan that defines how this policy will be implemented.

https://www.pharmacopoeia.com/content/file/Dissolution-Testing-
Consultation-Response-Document.pdf
The Bl" 2020 includes an initial revision to Supplementary Chapter I E
(Dissolution Testing of Solid Oral Dosage Forms) and a change to the
order of the appendices. These initial changes to the supporting information
-t for dissolution testing in the pharmacopoeia are the first stage of the
implementation of this .revised policy. '

1 The strategy and workprogramme can befound on the following webpage: https://www.gov.uk/
govemmentlconsultations/strategy-for-pharrnacopoeial-public-quality-standards-for-biological-medicines.

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2020 Introduction I-xxv

Traditional Herbal Two new BP monographs for herbal medicines are included in this edition.
Medicines; They reflect a continued commitment to providing standards for herbal
Homoeopathic drugs commonly used in the UI( and for those known to be used for the
Preparations preparation of traditional medicines.

Appendix XI V: Deoxyribonucleic Acid (DNA) Based Identification

Techniques for Herbal Drugs has been updated to include a worked
example of the DNA-based identification of Tribulus terrestris Fruit.
Supplementary Chapter VII D:DNA Barcoding as a tool for Botanical
Identification of Herbal Drugs has also been updated to include the
reference sequences for Tribulus terrestris Fruit and Galium aparine L.
(Clivers). Where individual monographs have a reference sequence, a non-
mandatory annex has been included in the monograph to inform users of
the published reference sequence in Supplementary Chapter VII D.

Unlicensed With this new edition, a further 5 monographs for unlicensed formulations
Medicines have been added. All monographs for such formulations are characterised
by a statement that the monograph has been prepared to cover unlicensed
formulations. The general and individual monographs are intended to apply
to all types of Unlicensed Medicines, that is, those formulations prepared
under a Manufacturer's 'Specials' Licence and those prepared
extemporaneously under the supervision of a pharmacist.

The General Monograph on Unlicensed Medicines has been amended to

reflect that alternative approaches to the BP test for Sterility might be
required for sterile preparations that are prepared extemporaneously. or in
small batches.

Analytical Quality The British Pharmacopoeia, working with the MHRA and stakeholders, is
by Design (AQbD) investigating the application of the Quality by Design concept to analytical
methods and the pharmacopoeia. Several AQbD concepts have been
practically assessed in conjunction with the British Pharmacopoeia
Laboratory, including: risk analysis, Design of Experiments (DoE) and
Analytical Target Profiles (ATPs). The Australian Therapeutic Goods
Administration have also been a key collaborator in the project.

The MHRA recognises the importance of alignment between regulators,

pharmacopoeias and stakeholders in the. development . of new and innovative
policy, such as the application of AQbD principles. Therefore, the
outcomes of this work are to be published ahead of the publication of the
BP 2020. The MHRA will also publish an accompanying consultation to
seek stakeholders' views on how AQbD concepts could be applied in a
pharmacopoeial context.

Revisions A significant number (331, comprising 103 technical revisions and 228
editorial revisions). of national monographs have been amended by means of
this edition. Of these monographs, those with major technical revisions are
listed at the end of this Introduction. For the benefit of the reader this list
indicates the section, or sections, of each monograph which has/have been
revised.

The list of revisions appended to this Introduction is as comprehensive as

practicable. However, to ensure that the reader uses the current standard, it
is essential to refer to the full text of each individual monograph.

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I-xxvi Introduction 2020

For those texts reproduced from the European Pharmacopoeia, the

European Directorate for the Quality of Medicines & HealthCare (EDQM)
database (see below, under Web sites) provides information on revisions of
the monographs or other texts on a historical basis, beginning from the 5th
Edition of the European Pharmacopoeia.

British The British Pharmacopoeia continues to expand the catalogue of

Pharmacopoeia BPCRS which are essential parts of the published monographs. The
Chemical Reference catalogue currently contains well over 800 items. The British
Substances Pharmacopoeia Commission Laboratory continuously strives to improve the
(BPCRS) percentage of BPCRS in stock and is working towards making the
BPCRS to support new monographs for the BP 2020 and future editions
available for our users at the same time as the publication becomes available
and ahead of the implementation date.

Tide Changes 108 monograph titles have been amended in this edition. In accordance
with the British Pharmacopoeia Commission decision to amend monograph
titles that include a split in the standard term, changes have been made to
affected monograph titles in this edition and are included within the
appended list.

Omissions 73 monographs have been omitted from the British Pharmacopoeia 2020.
The list of omissions is appended at the end of this Introduction.

Infrared Reference As with the previous edition, the reference spectra are placed in alphabetical
Spectra order within this edition. Six new spectra have been added to the collection.

Appendices Three new Appendices to harmonise with the European Pharmacopoeia

were first published in the BritishPharmacopoeia 2019 electronic updates.
These have been consolidated in the new edition as follows:
Appendix II M. Direct Amperometric and Pulsed Electrochemical Detection
(Ph. Eur. Method 2.2.63);
Appendix XI O. Foam Index (Ph. Eur. Method 2.8.24);
Appendix XVI H. Microbiological Examination of Live Biotherapeutic
Products (Ph. Eur. Methods 2.6.36 and 2.6.38).

European Co-operation Agreement

Pharmacopoeia As a consequence of the Co-operation Agreement with the EDQM of the
Council of Europe, the British Pharmacopoeia Commission is pleased to
note the integration of European Pharmacopoeia texts for the British
Pharmacopoeia 2019 in-year online updates and for this edition of the
British Pharmacopoeia.
In accordance with previous practice, all monographs and requirements of
the European Pharmacopoeia are reproduced in this edition of the British
Pharmacopoeia or, where appropriate, within its.companion edition, the
British Pharmacopoeia (Veterinary) 2020.
Where a monograph has been reproduced from the European
Pharmacopoeia, this is signified by the presence of a chaplet of stars
alongside its title. Additionally, reference to the European Pharmacopoeia
monograph number is included immediately below the title in italics in the
form 'Ph. Bur. monograph xxxx'. Where the title in the British

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 Introduction I-xxvii

Pharmacopoeia is different from that in the European Pharmacopoeia, an

approved synonym has been created (see Appendix XXI B) and the
European Pharmacopoeia title is included before the monograph number.
The entire European Pharmacopoeia text is delineated by two horizontal
lines bearing the symbol 'Ph. Bur.'.
The European Pharmacopoeia texts have been reproduced in their entirety
but, where deemed appropriate, additional statements of relevance to UK
usage have been added (e.g. action and use statement, a list of British
Pharmacopoeia preparations). It should be noted, however, that in the
event of doubt of interpretation in any text of the European
Pharmacopoeia, the text published in English under the direction of the
Council of Europe should be consulted.
Correspondence between the general methods of the European
Pharmacopoeia and the appendices of the British Pharmacopoeia is
indicated in each appendix and by inclusion of a list at the beginning of the
appendices section.

Pharmacopoeial It should be noted that any article intended for medicinal use which is
Requirements described by a name at the head of a monograph in the current edition of
the Pharmacopoeia must comply with that monograph 'whether or not it is
referred to as BP ..

It is also important to note that no requirement of the Pharmacopoeia can

be taken in isolation A valid interpretation of any particular requirement
e .

depends upon it being read in the context of (i) the monograph as a whole,
(ii) the specified method of analysis, (iii) the relevant General Notices and,
where appropriate, (iv) the relevant General Monograph(s). Familiarity with
the General Notices of the Pharmacopoeia will facilitate the correct
application of the requirements. Additional guidance and information on
the basis of pharmacopoeial requirements is provided in Supplementary
Chapter I. This non-mandatory text describes the general underlying
philosophy and current approaches to particular aspects of pharmacopoeial
control.

Code of Practice Members of the British Pharmacopoeia Commission and its supporting
Expert Advisory Groups, Panels of Experts and Working Parties are
required to comply with a Code of Practice on Declaration of Interests in
the pharmaceutical industry. Details of the Code are published on the
website (pharmacopoeia.com}.

Websites British Pharmacopoeia Website

The British Pharmacopoeia website, pharmacopoeia.com, contains
information relating to the British Pharmacopoeia. It allows subscribers to
access the British Pharmacopoeia 2020 and British Pharmacopoeia
(Veterinary) 2020 online and British Approved Names publications. All
users are also able to view and purchase BPCRS products through the
website.
Chromatograms for information to support new monographs published in
the British Pharmacopoeia 2020 have been added to the example test
results gallery to aid users of British Pharmacopoeia monographs. This
service will increase year-on-year to allow users to examine chromatograms

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I-xxviii Introduction 2020

obtained during the practical evaluation of new monographs by the British

Pharmacopoeia Commission Laboratory.

Aregular review schedule for draft texts is included on the website, with
draft new and revised monographs being posted at the start of each quarter
and available .for comment for a period of three months thereafter. This free
service allows greater visibility of the BP's work programme and enables
stakeholder contributions to monograph development.

Subscribers to the BP online will find that draft texts and example test
results are also linked with relevant texts and directly accessible from the
BP online content. Additionally, BPCRS products are also linked with
relevant BP monographs and subscribers to the BP online will be able to
purchase these directly from the BP online. BPCRS customers are able to
make purchases through invoice or credit card orders.
An email subscription feature allows users to keep abreast with BP news.
Additionally, users can subscribe to receive BPCRS updates, which are now
posted monthly.
Access to previous editions of the BP is available as a BP archive product
for purchase by new and existing BP online subscribers. The content of the
archive starts from the BP 2014 onwards and grows year-on-year as
superseded editions are added to the archive.
The British Pharmacopoeia is committed to continuously improving the
user experience of our products and services. Since 2019 the publication
process has been upgraded. User benefits include searchable tables and
equations, better linking functionality and higher formatting consistency.
Additionally, the reagent requirements have been harmonised with the
European Pharmacopoeia where possible, making it easier for users to
ensure compliance.

A policy of continuous improvement allows the BP website to keep up to

date and respond to users. Customers are therefore invited to provide the
Secretariat with feedback on their experience. Independent user researchers
have developed a programme of research to further understand our users
needs in order to enhance BP products. This has informed the development
of a short guide on how to use the BP with further developments to be
released over the year.
European Pharmacopoeia Websites

For those texts reproduced from the European Pharmacopoeia, the EDQM
website provides access to a database (the Knowledge database: https://
extranet.edqm.eu/publications/recherches_sw.shtml) containing information
of various sorts related to monographs and intended to facilitate their
proper use. Information is provided on chromatographic columns used in
monograph development, suppliers ofreagents and equipment that may be
difficult to find for some users, the status of monographs (in development,
adopted, published, under revision), revisions of the monographs on a
historical basis, beginning from the 5th Edition of the European
. Pharmacopoeia as well as other useful information.
The European Pharmacopoeia Forum, Pharmeuropa, is published quarterly
as an aid for the elaboration of monographs and as a vehicle for information

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 Introduction I-xxix

on pharmacopoeial and related matters. Pharmeuropa is available as a free

online publication: https://pharmeuropa.edqm.eu/home

International Therapeutic Goods Administration, Australia The British

Collaboration Pharmacopoeia Commission is pleased to continue its long-standing co-
operation with the Australian Department of Health Therapeutic Goods
Administration (TGA). The TGA continues to provide advice to British
Pharmacopoeia Commission Expert Advisory Groups, to participate in
inter-laboratory evaluation of British Pharmacopoeia monographs and to
review data jointly. This collaboration has enabled the production of robust,
high quality monographs for users.

Chinese Pharmacopoeia The British Pharmacopoeia Commission is

pleased to continue its collaboration with the Chinese Pharmacopoeia on
the development of monographs and staff exchanges to support mutually
agreed projects.

The Croatian Agency for Medicinal Products and Medical Devices

("HALMED") The Cooperation Agreement between the Medicines and
Healthcare products Regulatory Agency and HALMED provides a licence
for the use of information in the British Pharmacopoeia on unlicensed
medicines.

The Japanese Pharmacopoeia The British Pharmacopoeia has

collaborated with the Japanese Pharmacopeia for the development of
informally harmonised standards and knowledge sharing in a number of
areas of mutual interest.

State Pharmacopoeia of the Republic of Kazakhstan Following the

signing of a Collaboration Agreement in April 2016, the Medicines and
Healthcare products Regulatory Agency has granted the Committee on
Surveillance of Medical and Pharmaceutical Activities of the Ministry of
Health of the Republic. of Kazakhstan a licenceto continue to use' relevant
contents of the British Pharmacopoeia in the State Pharmacopoeia of the
Republic of Kazakhstan,

State Pharmacopoeia of Ukraine Following the signing of a

Collaboration Agreement in 2016, the Medicines and Healthcare products
Regulatory Agency has continued to grant the Ukrainian Scientific
Pharmacopoeial Center for Quality of Medicines a licence to use relevant
contents of the British Pharmacopoeia in the State Pharmacopoeia of
Ukraine.

United States Pharmacopeia Close collaboration with the United States

Pharmacopeia continues, building on the success of the programme of work
to jointly develop and revise drug product monographs, with joint
participation in conferences and symposia and knowledge sharing in areas
of mutual interest.

World Health Organization The collaboration agreement between the

British Pharmacopoeia and the International Pharmacopoeia continues to
support the work of the WHO, including collaboration and information
exchange, contribution to the International Meeting of W orId
Pharmacopoeias, and the international non-proprietary names programme.

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I-xxx Introduction 2020

Forward Look In-year Updates The British Pharmacopoeia 2020 online updates will be
published on the website, pharmacopoeia.com, to enable users to keep up
to date with monographs published in the European Pharmacopoeia. These
updates will be integrated annually with the publication of the main edition
of the British Pharmacopoeia.

Acknowledgements The British Pharmacopoeia Commission is greatly indebted to the members

. of its Expert Advisory Groups, Panels of Experts and Working Parties for
their dedicated enthusiasm and assistance in the preparation of this edition.
In particular Dr Brian Matthews who, after 9 years of providing support
and advice, has ended his term on the British Pharmacopoeia Commission.
We would also like to acknowledge the contributions of Dr Andrew
Coulson, Mr Peter Murray and Dr Adrian Thomas who sadly passed away
during the last year.

Close co-operation has continued with many organisations at home and

overseas. These include the Medicines and Healthcare products Regulatory
Agency, the Veterinary Medicines Directorate, the Royal Pharmaceutical
Society, the Association of the British Pharmaceutical Industry, the British
Association of Homoeopathic Manufacturers; the United Kingdom Herbal
Forum, The China Food and Drug Administration, the Chinese
Pharmacopoeia Commission, the European Pharmacopoeia Commission
and the European Directorate for the Quality of Medicines & HealthCare,
the Therapeutic Goods Administration (Australia), the Health Products and
Food Branch of Health Canada, the United States Pharmacopeia, the
Quality Assurance and Safety: Medicines Department of the World Health
Organization (WHO), the Health Sciences Authority of Singapore and the
Royal Botanic Gardens, Kew.

The British Pharmacopoeia Commission wishes to thank the European

Directorate for the Quality of Medicines & HealthCare for their support
and assistance in the reproduction of the European Pharmacopoeia texts
and monographs. The British Pharmacopoeia Commission acknowledges
the importance of the work of the European Pharmacopoeia Commission
and its Groups of Experts and Working Parties. The British Pharmacopoeia
Commission is also grateful for the generous contribution by the UK
experts to the work of the Groups of Experts and Working Parties of the
European Pharmacopoeia Commission.

The British Pharmacopoeia Commission acknowledges the contribution of

Professor Frederick A Senese, Department of Chemistry, Frostburg State
University, USA, for his kind permission to reproduce the indicator colour
chart.
The British Pharmacopoeia Commission also acknowledges and appreciates
the advice ofthe publishing team at The Stationery Office, in particular, Mr
Paul Allard, Mr Andrew Allen, Ms Nichola Billington, Mr Chris Cole, Mr
Nagaraja joisa, Mr Steve Page, Mr Paul Relfe and Mr Ian Webb, in the
production of this. edition.

The British Pharmacopoeia Commission acknowledges the contribution of

two members of the Civil Service Fast Stream programme, Mr Toby
Gladwin and Ms Naomi Clothier who spent six and twelve months,
respectively, working with the British Pharmacopoeia Secretariat.

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 Introduction J-xxxi

Additions The following monographs of the British Pharmacopoeia 2020 were not
included in the British Pharmacopoeia 2019.
Medicinal and Pharmaceutical Substances
Atazanavir Sulfate l
Boldine.'
Dexamfetamine Sulfate 1
Everolimus 1
Fingolimod Hydrochloride!
Concentrated Solutions for Haemofiltration and Haemodiafiltration 1
Levofloxacin Hemihydrate'
Infliximab Concentrated Solution 1
Magnesium Aluminometasilicate 1
Mebeverine Hydrochloride 1
Nilotinib Hydrochloride Monohydrate!
Phenoxymethylpenicillin (Benzathine) Tetrahydrate 1
Phytomenadione 1
. Podophyllotoxin 1
Regorafenib Monohydrate 1
Rotigorine'
Sulfobutylbetadex Sodium 1
Terpin Monohydrate!
Zoledronic Acid Monohydrate 1
Formulated Preparations: General Monographs
Live Biotherapeutic Products for Human Usel
Formulated Preparations: Specific Monographs
Amitriptyline Oral Solution
Azithromycin Eye.Drops
Cabergoline Tablets
Calcium Carbonate Oral Suspension
Capecitabine Tablets
Celecoxib Capsules
Cilastatin and Imipenem for Infusion
Colistimethate Inhalation Powder, Hard Capsule
Deferiprone Oral Solution 1
Deferiprone Tablets 1
Diltiazem Oral Suspension
Ferric Chloride Injection
Filgrastim Injection'
Galantamine Prolonged-release Capsules
Galantamine Oral Solution
Galantamine Tablets
Ibuprofen Orodispersible Tablets
Lacosamide Infusion 1
Lacosamide Oral Solution 1
Lacosamide Tablets l
Leflunomide Tablets
Letrozole Tablets
Metoprolol Oral Suspension
Minocycline Capsules

1 denotes a monograph of the European Pharmacopoeia

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I-xxxii Introduction 2020

Moxonidine Tablets
Risedronate Sodium Tablets
Ritonavir Oral Solution
Ritonavir Tablets
Rotigotine Transdermal Patches
Salmeterol Inhalation Powder, pre-metered
Salmeterol Pressurised Inhalation, Suspension
Temozolomide Capsules
Temozolomide for Injection
Tobramycin Eye Drops
Tobramycin and Dexamethasone Eye Drops
. Tobramycin Inhalation Powder, Hard Capsule

Herbal Drugs, Herbal Drug Preparations and Herbal Medicinal

Products
Achyranthes Bidendata Root'
Clivers
Corydalis Rhizome I
Gastrodia Elata Rhizome l
Ligusticum Root and Rhizome!
Dwarf Lilyturf Tuber l
Indian Sandalwood Oil
Sophora Flavescens Root l
Typhae Pollen I

Materials for use in the Manufacture of Homoeopathic Preparations

Digitalis for Homoeopathic Preparations I

Immunological Products
Meningococcal Group A, C, W135 and Y Conjugate Vaccine I

Radiopharmaceutical Preparations
Flurodopa eSF) (Prepared by Nucleophilic Substitution) Injection'
Yttrium (90 y ) Chloride Solution for Radiolabelling'

ol1:iiSsi 0:l18 The following monographs of the British Pharmacopoeia 2019 are not
included in the British Pharmacopoeia 2020.
l\iedicinal and Pharmaceutical Substances·'
'¥oxiprin
~.~propazone
B~g()rilate
C~tbaryl
eh1u···· .
Ji~ethlne Hydrochloride
Ch~g7Rr?pamide2
De~~S?quineSulfate
De~g~99r;one Acetate3
D~}{tr()RrOPoxyphene Napsilate
Dihy:~()~.~~otamineTartrate 4
Diloxani~#"'~lU"0ate
Dipip~l1()Ile'l-Iydrochloride

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 Introduction I-xxxiii

Emetine Hydrochloride Pentahydrate 1

Gliquidone
Isometheptene Mucate
Methoxamine Hydrochloride
Mexenone
Nicotinyl Alcohol Tartrate
Oxprenolol Hydrochloride/
Pentagastrin
Phenindamine Tartrate
Phytomenadione 1,3
Poldine Metilsulfate
Polythiazide
Quinidine Bisulfate
Ritodrine Hydrochloride
Tolazamide
Highly Purified Water 2
Formulated Preparations: Specific Monographs
Aloxiprin Tablets
Azapropazone Capsules
Benorilate Oral Suspension
Brompheniramine Tablets
Carbaryl Lotion
Chlormethine Injection
Chloroquine Sulfate Injection
Chlorpropamide Tablets
Cyclopenthiazide Tablets
Dextromoramide Tablets
Dextropropoxyphene Capsules
Digitoxin Tablets
Diloxanide Tablets
Dipipanone and Cyclizine Tablets
Estradiol Injection
Estropipate Tablets
Etodolac Tablets
Fenbufen Capsules
Flucytosine Tablets
Gliquidone Tablets
Guanethidine Tablets
Hydroflumethiazide Tablets
Idoxuridine Eye Drops
Interferon Alfa-2b Injection
Levodopa Capsules
Levodopa Tablets
Mepyramine Tablets
Methyldopate Injection
Nandrolone Decanoate Injection
Nicotinyl Alcohol Tablets

1 Monograph suppressed by the European Pharmacopoeia Commission on 1stJanuary 2019.

2 Monograph suppressed by the European Pharmacopoeia Commission on 1stApril 2019
3 The monograph for Phytomenadione was replaced by the monograph for Racemic Phytomenadione in
Supplement 9.6 of the 9th Edition of the European Pharmacopoeia, but the title "Phytomenadione" has been
retained in the British Pharmacopoeia.

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 Introduction I-xxxiii

Emetine Hydrochloride Pentahydrate '

Gliquidone
Isometheptene Mucate
Methoxamine Hydrochloride
Mexenone
Nicotinyl Alcohol Tartrate
Oxprenolol Hydrochloride/
Pentagastrin
Phenindamine Tartrate
Phytomenadione 1,3
Poldine Metilsulfate
Poly-thiazide
Quinidine Bisulfate
Ritodrine Hydrochloride
Tolazamide
Highly Purified Water2

Formulated Preparations: Specific Monographs

Aloxiprin Tablets
Azapropazone Capsules
Benorilate Oral Suspension
Brompheniramine Tablets
Carbaryl Lotion
Chlormethine Injection
Chloroquine Sulfate Injection
Chlorpropamide Tablets
Cyclopenthiazide Tablets
Dextromoramide Tablets
Dextropropoxyphene Capsules
Digitoxin Tablets
Diloxanide Tablets
Dipipanone and Cyc1izine Tablets
Estradiol Injection
Estropipate Tablets
Etodolac Tablets
Fenbufen Capsules
Flucytosine Tablets
Gliquidone Tablets
Guanethidine Tablets
Hydroflumethiazide Tablets
Idoxuridine Eye Drops
Interferon Alfa-2b Injection
Levodopa Capsules
Levodopa Tablets
Mepyramine Tablets
Methyldopate Injection
Nandrolone Decanoate Injection
Nicotinyl Alcohol Tablets

1 Monograph suppressed by the European Pharmacopoeia Commission on 1stJanuary 2019.

2 Monograph suppressed by the EuropeanPharmacopoeia Commission on 1stApril 2019
3 The monograph for Phytomenadione was replaced by the monograph for Racemic Phytomenadionein
Supplement 9.6 of the 9th Edition of the European Pharmacopoeia, but the title "Phytomenadione" has been
retained in the BritishPharmacopoeia.

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 Introduction I-xxxv

Amitriptyline Tablets Identification; Dissolution; Related

substances;Production
Benzatropine Injection Production
Benzatropine Tablets Production
Benzylpenicillin Injection Requirements for ready-to-use solution -
deleted; Benzylpenicillin for Injection -
Identification; Relatedsubstances; Assay
Bisacodyl Gastro-resistant Tablets Title change; Dissolution
Bisoprolol Tablets Content of bisoprolol fumarate;
Identification testA; Dissolutioru Related
substances; Assay
Bromocriptine Capsules Production
Bromocriptine Tablets Production
Caffeine Citrate Injection Related substances; Assay
Caffeine Citrate Oral Solution Related substances; Assay
Calcium Chloride Injection Assay
Candesartan Tablets Related substances
Carbimazole Tablets Related substances
Ceftriaxone Injection Assay
Clindamycin Capsules Identification testAj Dissolution
Clobazam Oral Suspension Dissolution; Relatedsubstances
Co-amoxiclav Injection Related substances
Co-beneldopa Capsules Related substances testA
Co-beneldopa Dispersible Tablets Title change; Relatedsubstances testA
Colchicine Tablets Related substances
Dalteparin Sodium Injection Identification testAj Related substances
Desferrioxamine Injection Production
Dobutamine Infusion Related substances
Enoxaparin Sodium Injection Identification testAj Related substances
Ferrous Fumarate Capsules Dissolution
Fluvoxamine Tablets Related substances
Griseofulvin Tablets Identification; Related substances; Assay
Heparin Injection Related substances
Hydrochlorothiazide Tablets Related substances
Hydroxycarbamide Capsules Hydroxylamine
Hyoscine Butylbromide Tablets Dissolution; Relatedsubstances
Ibuprofen Prolonged-release Capsules Title change; Relatedsubstances
Ibuprofen Gel Identification; Acidity or alkalinitYj
Related substances; Assay
Ibuprofen Tablets Dissolution; Relatedsubstances
Ibuprofen Prolonged-release Tablets Title change; Relatedsubstances
Interferon Beta-la Injection Content of interferon beta-Ia;
Identification test C; Oxidisedforms;
Dimers and related substances of higher
molecular weight (Method A)
Ipratropium Pressurised Inhalation, Title change; Content of ipratropium
Solution bromide; Identification tests A and.B;
Uniformity of delivered dose; Impurity
A; Related substances; Assay
Ketamine Nasal Spray Identification test C; Related substances;
Assay
Ketoprofen Capsules Related substances

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I-xxxvi Introduction 2020

Ketoprofen Gel Related substances; Impurities

Loprazolam Tablets Production
Lorazepam Injection Related substances
Lorazepam Tablets Related substances
Minocycline Prolonged-release Title change; Content of minocycline;
Capsules Identification; Related substances; Assay
Minocycline Tablets Content of minocycline; Identification;
Dissolution)' Related substances; Assay
Olmesartan Tablets Assay
Pantoprazole Gastro-resistant Tablets Dissolution; Assay
Phenobarbital Injection Related substances
Phenoxymethylpenicillin Oral Related substances; Assay
Solution
Phenoxymethylpenicillin Tablets Related substances; Assay
Phentolamine Injection Production
Phenytoin Capsules Production)' Content of phenytoin
sodium)' Identification)' Related
substances; Assay)' Impurities
Phenytoin Injection Identification; Benzil and benzophenone
(deleted); Ethanol (deleted); Propylene
Glycol (deleted); Ethanol and propylene
glycol (added); Related substances)'
Assay; Impurities
Phenytoin Oral Suspension Content of phenytoin; Identification;
Benziland benzophenone (deleted);
Related substances; Assay)' Impurities
Phenytoin Tablets Production; Identification; Related
substances; Assay)' Impurities
Prochlorperazine Injection Production
Prochlorperazine Oral Solution Production
Progesterone Injection Related substances; Assay
Pyrimethamine Tablets Content of pyrimethamine; Dissolution)'
Related substances)' Impurities
Ranitidine Oral Solution Related substances
Salbutamol Pressurised Inhalation, Title change; Identification; Uniformity
Suspension of delivered dose)' Related substances)'
, Assay; Impurities
Sildenafil Orodispersible Films Related substances
Sildenafil Injection Related substances
Sildenafil Powder for Oral Suspension Related substances
Sildenafil Tablets Related substances; Assay
Sildenafil Chewable Tablets Related substances
Sildenafil Orodispersible Tablets Related substances
Silver Nitrate Sterile Solution Title change; Definition; Italic opening
statement; Sterility; Labelling (deleted)
Simvastatin Oral Suspension Related substances
Simvastatin Tablets Related substances
Compound Sodium Lactate Infusion Identification test C
Sotalol Injection Identification testB
Sumatriptan Injection Related substances
Sumatriptan Nasal Spray Related substances
Tamoxifen Tablets Identification; Related substances; Assay
Tenoxicam Injection . Impurities

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 Introduction T-xxxvii

Tenoxicam Tablets Impurities

Tinzaparin Sodium Injection Identification testAj Related substances
Triamcinolone Acetonide Injection Identification testBj Related substances;
Assay
Triamcinolone Oromucosal Paste Related substances; Assay
Zuclopenthixol Tablets Related substances; Assay

Herbal Drugs, Herbal Drug Preparations and Herbal Medicinal

Products

Anethum Graveolens Sowa Fruit Apiole; DNA Reference sequence

Holy Basil Leaf DNA Reference sequence
Glehnia Littoralis Root DNA Reference sequence
Nutmeg Foreign matter; DNA Reference sequence
Phellodendron Amurense Bark DNA Reference sequence
Phellodendron Chinense Bark DNA Reference sequence
Tribulus Terrestris Fruit DNA Reference sequence
In addition to the changes listed above; the following changes have also
been made to BP monographs in this edition.
The opening statements in monographs for unlicensed medicines have been
amended to reflect that such monographs have been developed to cover
unlicensed formulations. This will avoid any confusion should such
formulations become licensed in the future.
The titles of a large number of monographs have been updated to reflect
the decision of the British Pharmacopoeia Commission to avoid including
split standard terms in the title.

Changes in Title The following list gives the alterations in the titles of monographs of the
British Pharmacopoeia 2019 that have been retained in the British
Pharmacopoeia 2020.

BRITISH PHARMACOPOEIA BRITISH PHARMACOPOEIA

2019 2020

Medicinal and Pharmaceutical Substances

Spray-dried Acacia Acacia, Dried Dispersion

Alfentanil Hydrochloride AlfentanilHydrochloride Hydrate
Benzathine Benzylpenicillin Benzathine Benzylpenicillin
Tetrahydrate
Calcifediol Calcifediol Monohydrate
Calcium Folinate Calcium Folinate Hydrate
Calcium Levofolinate Pentahydrate Calcium Levofolinate Hydrate
Dipotassium Clorazepate Dipotassium Clorazepate
Monohydrate
Lidocaine Hydrochloride Lidocaine Hydrochloride
Monohydrate
Methylthioninium Chloride Methylthioninium Chloride Hydrate
Procaine Benzylpenicillin Benzylpenicillin (Procaine)
Monohydrate
Hydrated Valaciclovir Hydrochloride Valaciclovir Hydrochloride Hydrate

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I-xxxviii -Introduction 2020

Formulated Preparations: Specific Monographs

Gastro-resistant Acamprosate Tablets Acamprosate Gastro-resistant Tablets

Dispersible Aciclovir Tablets Aciclovir Dispersible Tablets
Prolonged-release Alfuzosin Tablets Alfuzosin Prolonged-release Tablets
Chewable Aluminium Hydroxide Aluminium Hydroxide Chewable
Tablets Tablets
Prolonged-release Aminophylline Aminophylline Prolonged-release
Tablets Tablets
Sterile Arginine Hydrochloride Arginine Hydrochoride Sterile
Concentrate Concentrate
Chewable Ascorbic Acid Tablets Ascorbic Acid Chewable Tablets
Dispersible Aspirin Tablets Aspirin Dispersible Tablets
Effervescent Soluble Aspirin Tablets Aspirin Effervescent Soluble Tablets
Prolonged-release Bezafibrate TabletsBezafibrate Prolonged-release Tablets
Gastro-resistant Bisacodyl Tablets BisacodylGastro-resistant Tablets
Bumetanide and Prolonged-release Bumetanide and Potassium
Potassium Tablets Prolonged-release Tablets
Chewable Calcium and Colecalciferol Calcium and Colecalciferol Chewable
Tablets Tablets
Chewable Calcium and Ergocalciferol Calcium and Ergocalciferol Chewable
Tablets Tablets
Chewable Calcium Carbonate Calcium Carbonate Chewable
Tablets Tablets
Chewable Calcium Carbonate and Calcium Carbonate and Heavy
Heavy Magnesium Carbonate Tablets Magnesium Carbonate Chewable
Tablets
Chewable Calcium Gluconate Tablets Calcium Gluconate Chewable Tablets
Effervescent Calcium Gluconate Calcium Gluconate Effervescent
Tablets Tablets
Chewable Carbamazepine Tablets Carbamazepine Chewable Tablets
Prolonged-release Carbamazepine Carbamazepine Prolonged-release
Tablets Tablets
Prolonged-release Cefaclor Tablets Cefaclor Prolonged-release Tablets
Prolonged-release Clarithromycin Clarithromycin Prolonged-release
Tablets Tablets
Prolonged-release Clomipramine Clomipramine Prolonged-release
Tablets Tablets
Dispersible Co-amoxiclav Tablets Co-amoxiclav Dispersible Tablets
Prolonged-release Co-beneldopa Co-beneldopa Prolonged-release
Capsules Capsules
Dispersible Co-beneldopa Tablets Co-beneldopa Dispersible Tablets
Effervescent Co-codamol Tablets Co-codamol Effervescent Tablets
Dispersible Co-codaprin -Tablets Co-codaprin Dispersible Tablets
Dispersible Co-trimoxazole Tablets Co-trimoxazole Dispersible Tablets
Prolonged-release Diclofenac Diclofenac Prolonged-release
Capsules Capsules
Gastro-resistant Diclofenac Tablets Diclofenac Gastro-resistant Tablets
Prolonged-release Diclofenac Tablets Diclofenac Prolonged-release Tablets
Prolonged-release Diltiazem Tablets Diltiazem Prolonged-release Tablets
Prolonged-release Dipyridamole Dipyridamole Prolonged-release
Capsules Capsules

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 Introduction I-xxxix

Gastro-resistant Erythromycin Erythromycin Gastro-resistant

Capsules Capsules
Gastro-resistant Erythromycin Tablets Erythromycin Gastro-resistant Tablets
Prolonged-release Felodipine Tablets Felodipine Prolonged-release Tablets
Prolonged-release Ferrous Sulfate Ferrous Sulfate Prolonged-release
Tablets Tablets
Prolonged-release Fluvastatin Tablets Fluvastatin Prolonged-release Tablets
Prolonged-release Ibuprofen Capsules Ibuprofen Prolonged-release Capsules
Prolonged-release Ibuprofen Tablets Ibuprofen Prolonged-release Tablets
Prolonged-release Indapamide IndapamideProlonged-re1ease
Tablets Tablets
Ipratropium Pressurised Inhalation Ipratropium Pressurised Inhalation,
Solution
Prolonged-release Isosorbide Isosorbide Mononitrate Prolonged-
Mononitrate Capsules release Capsules
Prolonged-release Isosorbide Isosorbide Mononitrate Prolonged-
Mononitrate Tablets release Tablets
Dispersible Lamotrigine Tablets Lamotrigine Dispersible Tablets
Gastro-resistant Lansoprazole Lansoprazole Gastro-resistant
Capsules Capsules
Gastro-resistant Lansoprazole Tablets Lansoprazole Gastro-resistant Tablets
Prolonged-release Lithium Carbonate Lithium Carbonate Prolonged-release
Tablets Tablets
Orodispersible Loperamide Tablets Loperamide Orodispersible Tablets
Chewable Magnesium Magnesium Glycerophosphate
Glycerophosphate Tablets Chewable Tablets
Chewable .Compound Magnesium Compound Magnesium Trisilicate
Trisilicate Tablets Chewable Tablets
Prolonged-release Metformin Tablets Metformin Prolonged-release Tablets
Prolonged-release Metoprolol Metoprolol Tartrate Prolonged-
Tartrate Tablets release Tablets
Prolonged-release Minocyc1ine Minocyc1ine Prolonged-release
Capsules Capsules
Orodispersible Mirtazapine Tablets Mirtazapine Orodispersible Tablets
Chewable Montelukast Tablets Montelukast Chewable Tablets
Prolonged-release Morphine Capsules Morphine Prolonged-release Capsules
Prolonged-release Morphine Tablets Morphine Prolonged-release Tablets
Gastro-resistant Naproxen Tablets Naproxen Gastro-resistant Tablets
Prolonged-release Nifedipine Nifedipine Prolonged-release
Capsules Capsules
Prolonged-release Nifedipine Tablets Nifedipine Prolonged-release Tablets
Orodispersible Olanzapine Tablets Olanzapine Orodispersible Tablets
Prolonged-release Oxybutynin Tablets Oxybutynin Prolonged-release Tablets
Gastro-resistant Pancreatin Tablets Pancreatin Gastro-resistant Tablets
Soluble Paracetamol and Caffeine Paracetamol and Caffeine Soluble
Tablets Tablets
Dispersible Paracetamol Tablets Paracetamol Dispersible Tablets
Effervescent Paracetamol Tablets Paracetamol Effervescent Tablets
Soluble Paracetamol Tablets Paracetamol Soluble Tablets
Chewable Piperazine Phosphate Piperazine Phosphate Chewable
Tablets Tablets
Effervescent Potassium Chloride Potassium Chloride Effervescent
Tablets Tablets

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I-xl Introduction 2020

Prolonged-release Potassium Chloride Potassium Chloride Prolonged-release

Tablets Tablets
Prolonged-release Pramipexole . Pramipexole Prolonged-release
Tablets Tablets
Prolonged-release Propranolol Propranolol Prolonged-release
Capsules Capsules
Effervescent Ranitidine Tablets Ranitidine Effervescent Tablets
Prolonged-release Salbutamol Salbutamol Prolonged-release
Capsules Capsules
Salbutamol Pressurised Inhalation Salbutamol Pressurised Inhalation,
Suspension
Sterile Sodium Acetate Concentrate Sodium Acetate Sterile Concentrate
Sterile Sodium Benzoate Concentrate Sodium Benzoate Sterile Concentrate
Soluble Sodium Chloride Tablets Sodium Chloride Soluble Tablets
Prolonged-release Sodium Valproate Sodium Valproate Prolonged-release
Capsules Capsules
Prolonged-release Sodium Valproate Sodium Valproate Prolonged-release
Tablets Tablets
Sterile Silver Nitrate Solution Silver Nitrate Sterile Solution
Gastro-resistant Sulfasalazine Tablets Sulfasalazine Gastro-resistant Tablets
Prolonged-release Tamsulosin Tamsulosin Prolonged-release
Capsules Capsules
Prolonged-release TamsulosinTablets Tamsulosin Prolonged-release Tablets
Prolonged-release Theophylline Theophylline Prolonged-release
Tablets Tablets
Prolonged-release Tramadol Capsules Tramadol Prolonged-release Capsules
Prolonged-release Tramadol Tablets Tramadol Prolonged-release Tablets
Prolonged-release Trospium Chloride Trospium Chloride Prolonged-release
Capsules Capsules
Prolonged-released Venlafaxine Venlafaxine Prolonged-released
Capsules Capsules
Prolonged-released Venlafaxine Venlafaxine Prolonged-released
Tablets Tablets
Prolonged-released Verapamil Verapamil Prolonged-released
Capsules Capsules
Prolonged-released Verapamil Tablets Verapamil Prolonged-released Tablets

Herbal Drugs, Herbal Drug Preparations and Herbal Medicinal

Products

Gastro-resistant Peppermint Oil Peppermint Oil Gastro-resistant

Capsules Capsules

Radiopharmaceutical Preparations

Fluorodopa (18F) Ini ection et

Fluorodopa 8F) (Prepared by
Electrophilic Substitution) Injection

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Monographs

Medicinal and Pharmaceutical

Substances (A to I)

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2020 General Monographs 1-39

PRODUCTION
MEDICINAL AND PHARMACEUTICAL Substances for pharmaceutical use are manufactured by
SUBSTANCES procedures that are designed to ensure a consistent quality
and comply with the requirements of the individual
monograph or approved specification.
The manufacture of active substances must take place under
Substances for Pharmaceutical conditions of good manufacturing practice.
Use The provisions of general chapter 5.10 apply to the control of
impurities in substances for pharmaceutical use.
(Ph. Bur. monograph 2034)
Whether or not it is specificallystated in the individual
PhEur ~ _ _-:-- _ monograph that the substance for pharmaceutical use:
DEFINITION - is a recombinant protein or another substance obtained as
a direct gene product based on genetic modification,
Substances for pharmaceutical use are any organic or
where applicable, the substance also complies with the
inorganic substances that are used as active substances or
requirements of the general monograph Products of
excipients for the production of medicinal products for
recombinant DNA technology (0784);
human or veterinary use. They may be obtained from natural
- is obtained from animals susceptible to transmissible
sources or produced by extraction from raw materials,
spongiform encephalopathies other than by experimental
fermentation or synthesis.
challenge, where applicable, the substance also complies
This general monograph does not apply to herbal drugs, with the requirements of the general monograph Products
herbal drugs for homoeopathic preparations, herbal drug with risk of transmitting agents of animal spongiform
preparations, herbal drug extracts, or mother tinctures for encephalopathies (1483);
homoeopathic preparations, which are the subject of separate - is a substance derived from a fermentation process,
general monographs (Herbal drugs (1433), Herbaldrugs for whether or not the micro-organisms involved are modified
homoeopathic preparations (2045), Herbal drug by traditional procedures or recombinant DNA (rDNA)
preparations (1434), Herbal drug extracts (0765), Mother technology, where applicable, the substance also complies
tinctures for homoeopathic preparations (2029)). It does not with the requirements of the general monograph Products
apply to raw materials for homoeopathic preparations, exc~pt of fermentation (1468).
where there is an individual monograph for the substance m
If solvents are used during production, they are of suitable
the non-homoeopathic part of the Pharmacopoeia.
quality. In addition, their toxicity and their residual level are
Thi~ monograph does not apply to chemical precursors for taken into consideration (5.4). If water is used during
radiopharmaceutical preparations which are the subject of a production, it is of suitable quality.
separate monograph (Chemicalprecursors for
The identity of elemental impurities derived from
radiopharmaceutical preparations (2902)).
intentionally added catalysts and reagents is known, and
Where a substance for pharmaceutical use not described in strategies for controlling them should be established using the
an individual monograph of the Pharmacopoeia is used in a principles of risk management.
medicinal product prepared1for the special needs of
If substances are produced or processed to yield a certain
individual patients, the need for compliance with the present
form or grade,. that specific form or grade of the substance
general monograph is decided in the light of a risk
complies with the requirements of the monograph. Certain
assessment that takes account of the available quality of the
functionality-related tests may be described to control
substance and its intended use.
properties that may influence the suitability of the substance
Where medicinal products are manufactured using and subsequently the properties of dosage forms prepared
substances for pharmaceutical use of human or animal origin, from it.
the requirements of chapter 5.1.7. Viral safety apply.
Powdered substances May be processed to obtain a certain
Substances for pharmaceutical use may be used as such or as degree of fineness (2.9.35).
starting materials for subsequent formulation to prepare
Compactedsubstances Are processed to increase the particle
medicinal products. Depending on the formulation, certain
size or to obtain particles of a specific form and/or to obtain
substances may be used either as active substances or as
a substance with a higher bulk density.
excipients. Solid substances may be compacted, coated,
granulated, powdered to a certain fineness, or processed in Coated active substances Consist of particles of the active
other ways. A monograph is applicable to a substance substance coated with one or more suitable excipients.
processed with an excipient only where such processing is Granulatedactive substances Are particles of a specified size
mentioned in the definition section of the monograph. and/or form produced from the active substance by
Substancefor pharmaceutical use of special grade Unless granulation directly or with one or more suitable excipients.
otherwise indicated or restricted in the individual If substances are processed with excipients, these excipients
monographs, a substance for pharmaceutical use is intended comply with the requirements of the relevant monograph or,
for human and veterinary use, and is of appropriate quality where no such monograph exists, the approved specification.
for the manufacture of all dosage forms in which it can be Where active substances have been processed with excipients
used. to produce, for example, coated or granulated substances, the
Polymorphism Individual monographs do not usually specify processing is carried out under conditions of good
crystalline or amorphous forms, unless bioavailability is manufacturing practice and the processed substances are
affected. All fOnDS of a substance for pharmaceutical use regarded as intermediates in the manufacture of a medicinal
comply with the requirements of the monograph, unless product.
otherwise indicated.

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2020 Abacavir Sulfate 1-41

accordance with the recommendations of general chapter

5.1.10. Guidelines for using the testfor bacterial endotoxins.
Abacavir Sulfate *****
*<;»*
Pyrogens (2.6.8) (Ph. Bur. monograph 2589)
If the test for pyrogens is justified rather than the test for
bacterial endotoxins and if a pyrogen-free grade is offered,
the substance for pharmaceutical use complies with the test
for pyrogens. The limit and test method are stated in the
individual monograph or approved by the competent
authority. Based on appropriate test validation for bacterial
endotoxins and pyrogens, the test for bacterial endotoxins
may replace the test for pyrogens.
Additional properties
Control of additional properties (e.g. physical characteristics,
functionality-related characteristics) may be necessary for 671 188062-50-2
individual manufacturing processes or formulations. Grades
(suchas sterile, endotoxin-free, pyrogen-free) may be Action and use
produced with a view to manufacture of preparations for Nucleoside reverse transcriptase inhibitor; antiviral (lllV).
parenteral administration or other dosage forms and Preparations
appropriate requirements may be specified in an individual Abacavir Oral Solution
monograph. Abacavir Tablets
ASSAY Abacavir, Zidovudine and Lamivudine Tablets
Unless justified and authorised, contents of substances for Abacavir and Lamivudine Tablets
pharmaceutical use are determined. Suitable methods are
PhEur ~ _
used.
LABELLING DEFINITION
In general, labelling is subject to supranational and national Bis[[(lS,4R)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-
regulation and to international agreements. The statements yl]cyclopent-2-enyl]methanol] sulfate.
under the heading Labelling therefore are not comprehensive Content
and, moreover, for the purposes of the Pharmacopoeia only 99.0 per cent to 101.0 per cent (anhydrous substance).
those statements that are necessary to demonstrate
CHARACTERS
compliance or non-compliance with the monograph are
Appearance
mandatory. Any other labelling statements are included as
White or almost white powder.
recommendations. When the term 'label' is used in the
Pharmacopoeia, the labelling statements may appear on the Solubility
container, the package, a leaflet accompanying the package or Soluble in water, practically insoluble in ethanol
a certificate of analysis accompanying the article, as decided (96 per cent) and in methylene chloride.
by the competent authority.. IDENTIFICATION
Where appropriate, the label states that the substance is: A. Infrared absorption spectrophotometry (2.2.24).
- intended for a specific use; Comparison abacavir sulfate CRS.
- of a distinct crystalline form;
B. Enantiomeric purity (see Tests).
- of a specific degree of fineness;
- compacted; C. Solution S (see Tests) gives reaction (a) of sulfates
- coated; (2.3.1).
- granulated; TESTS
- sterile; Solution S
- free from bacterial endotoxins; Dissolve 0.250 g in water R and dilute to 25.0 mL with the
- free from pyrogens; same solvent.
- containing gliding agents.
Enantiomeric purity
Where applicable, the label states: Liquid chromatography (2.2.29).
- the degree of hydration;
Solution A Mix 0.5 mL of trifiuoroacetic acid Rand 100 mL
- the name and concentration of any excipi~~t.
of methanol R.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
Solution B Mix 30 volumes of methanol R, 30 volumes of
2-propanol Rand 40 volumes of heptane R.
Test solution Dissolve 40 mg of the substance to be
examined in 30 mL of solution A. Sonicate until dissolution
is complete. Add 30 mL of 2-propanol R and dilute to
100.0 mL with heptane R.
Reference solution (a) Dissolve 2 mg of abacavir for system
suitability CRS (containing impurities A and D) in 1.5 mL of
solution A. Sonicate until dissolution is complete.
Add 1.5 mL of 2-propanol R and dilute to 5.0 mL with
heptane R.

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2020 Acacia 1-43

A. [(lR,4S)-4- [2-amino-6-(cyclopropylamino)-9H-purin-9-yl]
cyclopent-2-enyl]methanol, F. 6-(cyclopropylamino)-9-[(lR,4S)-4-[[(1,1-dimethylethyl)
oxy]methyl]cyclopent-2-enyl]-9H-purine-2-amine.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur

Acacia ****
** *
(Ph. Bur. monograph 0307) *****
Action and use
Bulk-forming laxative;.excipient.
When Powdered Acacia is prescribed or demanded, material
B.6-{cyclopropylamino)-9-[(IR,4S)-4-[[(2,5-diamino-6- complying with the requirements below with the exception of
... chloropyrimidin-4-yl)oxy] methyl]cyc1opent-2-enyl] -9H- Identification test A shall be dispensed or supplied.
purine-2-amine, PhEur .,-.- .,-.-_ _

DEFINITION
Air-hardened, gummy exudate flowing naturally from or
obtained by incision of the trunk and branches of Acacia
senegal L. Willd. (syn. Senegalia senegal (L.) Britton), other
species of Acacia of African origin and Acacia seyal Delile.
C~CTERS
It is almost completely but very slowly soluble, after about
C. [(lS,4R)-4-(2,6-diamino-9H-purin-9-yl)cyclopent-2-enyl] 2·h, in twice its mass of water leaving only a very small
methanol, residue of vegetable particles; the liquid obtained is colourless
or yellowish, dense, viscous, adhesive, translucent and weakly
acid to blue litmus paper. It is practically insoluble in ethanol
(96 per cent).
IDENfIFICATION
A. It occurs as yellowish-white, yellow or pale amber,
sometimes with a pinkish tint, friable, opaque, spheroidal,
oval or reniform pieces (tears) of a diameter from about
1-3 em, frequently with a cracked surface, easily broken into
irregular, whitish or slightly yellowish angular fragments with
D. [(lR,4R)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl] a conchoidal fracture and a glassy and transparent
cyclopent-2-enyl]methanol, appearance. In the centre of an unbroken tear there is
sometimes a small cavity.
B. Microscopic examination (2.8.23). The powder is whiteor
yellowish-white. Examine under a microscope using ethanol
(96 per cent) R. The powder shows the following diagnostic
characters: angular, irregular, colourless, transparent
.fragments. Only traces of starch or plant tissues are visible.
No stratified membrane is apparent.
C. Examine the chromatograms obtained in the test for
glucose and fructose.
E. [(lR,3S)-3-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl] Results See below the sequence of zones present in the
cyclopentyl]methanol, chromatograms obtained with reference solution (a) and the
test solution.

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1-44 Acacia 2020

Top of the plate Reference solution (a) Dissolve 5 mg of arabinose R, 5.mg of

galactose R, 5 mg of glucose R, 5 mg of rhamnose Rand 5 mg
of xylose R in I· mL of water R and dilute to 10.0 mL with
methanol R.
3 blue zones, very faint Reference solution (b) Dilute 2.5 mL of reference solution (a)
to 10.0 mL with methanol R.
Reference solution (c) Dissolve 5 mg of galactose Rand 5 mg
Rhamnose: a greenish-brown zone A greenish-brown zone, very faint to of glucose R in 1 mL of water R and dilute to 10 mL with
equivalent (rhamnose) methanol R.
Intensity marker Galactose.
Xylose: a brownish-grey zone Plate TLC silica gel F 254 plate R (2-10 11m).
Mobile phase water R, acetonitrile R (15:85 VIV).
Application 4 ul, of the test solution and reference
solutions (a) and (b), and 2 ul, of reference solution (c), as
bands of 8 mm.
-- --
Development A 70 mm from the lower edge of the plate, in
an unsaturated tank.
Arabinose: a brownish-grey zone A brownish-grey zone, intense Drying A In air.
(arabinose)
DeuelopmentB 70 rom from the lower edge of the plate, in
an unsaturated tank, using freshly prepared mobile phase.
Glucose: a greyish-blue zone Drying B In air.
Detection Treat with a solution prepared as follows: dissolve
4 g of diphenylamine Rand 4 mL of aniline R in 160 mL of
Galactose: a greyish-blue zone A greyish-blue zone, intense
(galactose)
acetone R and add phosphoric acid R until the precipitate
formed dissolves again (about 30 ml.). Heat at 120 DC for
5-10 min and examine in daylight.
-- -- System suitability Reference solution (c):
I or 2 brownish-grey zones, very faint - the chromatogram shows in the middle third 2 distinct
to equivalent zones, which may be touching; the lower zone (galactose)
and the upper zone (glucose) are greyish-blue.
Results The chromatogram obtained with the test solution
I or 2 blue zones, faint to equivalent
shows no greyish-blue zone and no reddish zone between the
zones due to galactose and arabinose in the chromatogram
obtained with reference solution (a).
Reference solution (a) Test solution
Starch, dextrin and agar
To 10 mL of solution S, previously boiled and cooled, add
D. Dissolve 1 g of the powdered herbal drug (355) (2.9.12) 0.1 mL of 0.05 M iodine. No blue or reddish-brown colour
in 2 mL of water R by stirring frequently for 2 h. Add 2 mL develops.
of ethanol (96 per cent) R. After shaking, a white gelatinous
Sterculia gum
mucilage is formed that becomes fluid upon addition of
A. Place 0.2 g of the powdered herbal drug (355) (2.9.12) in
10 mL of water R.
a 10 mL ground-glass-stoppered cylinder graduated in
TESTS 0.1 mL. Add 10 mL of ethanol (60 per cent VIV) Rand
Solution S shake. Any gel formed occupies a maximum of 1.5 mL.
Dissolve 3.0 g of the powdered herbal drug (355) (2.9.12) in B. To 1.0 g of the powdered herbal drug (355) (2.9.12) add
25 mL of water R by stirring for 30 min. Allow to stand for 100 mL of water R and shake. Add 0.1 mL of methyl red
30 min and dilute to 30 mL with water R. solution R. Not more than 5.0 mL of 0.01 M sodium hydroxide
Insoluble matter is required to change the colour of the indicator.
Maximum 0.5 per cent. Tannins
To 5.0 g of the powdered herbal drug (355) (2.9.12) add To 10 mL of solution S add 0.1 mL offem'c chloride
100 mL of water R and 14 mL of dilute hydrochloric acid R, solution R1. A gelatinous precipitate is formed, but neither the
boil gently for 15 min, shaking frequently and filter while hot precipitate nor the liquid is dark blue.
through a tared sintered-glass filter (2.1.2). Wash with hot ~ Tragacanth
water R and dry at 100-105 DC. The residue weighs a
Examine the chromatograms obtained in the test for glucose
maximum of 25 mg. and fructose.
Glucose and fructose . Results The chromatogram obtained with the test solution
High-performance thin-layer chromatography (2.8.25). shows no faint to intense brownish-grey zone corresponding
Test solution To 0.1 g of the powdered herbal drug (355) to the zone due to xylose in the chromatogram obtained with
(2.9.12) in a thick-walled centrifuge tube, add 2 mL of a reference solution (a).
100 gIL solution of trifiuoroacetic acid R and shake vigorously. Loss on drying (2.2.32)
Stopper the tube and heat the mixture at 120 DC for 1 h. Maximum 15.0 per cent, determined on 1.000 g of the
Centrifuge, transfer 1 mL of the clear supernatant into a powdered herbal drug (355) (2.9.12) by drying in an oven at
10 mL flask and add 5 mL of methanol R. 105 DC.

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2020 Acacia, Dried Dispersion 1-45

Total ash (2.4.16) Top of the plate

Maximum 4.0 per cent.
Microbial contamination
TAMC: acceptance criterion 104 CFU/g (2.6.12).
TYMC: acceptance criterion 104 CFU/g (2.6.12). 3 blue zones, very faint
Absence of Escherichia coli (2.6.13).
Absence of Salmonella (2.6.13).
Rhamnose: a greenish-brown zone A greenish-brown zone, very faint to
FUNCTIONALITY-RELATED CHARACTERISTICS equivalent (rhamnose)
This section provides information on characteristics that are
recognised as beingrelevant control parametersfor one or more
Xylose: a brownish-grey zone
functions of the substance when used as an excipient (see chapter
5.15). Some of the characteristics described in the Functionality-
related characteristics section may also be presentin the mandatory
part of the monograph since they also represent mandatory quality
criteria. In such cases, a cross-reference to the tests described in the -- --
mandatory part is included in the Functionality-related
characteristics section. Controlof the characteristics can contribute
to the quality of a medicinalproductby improving the consistency Arabinose: a brownish-grey zone A brownish-grey zone, intense
of the.manufacturingprocess and the performance of the medicinal (arabinose)
product during use. W'here control methods are cited, they are
recognised as beingsuitable for the purpose, but othermethods can
Glucose: a greyish-blue zone
also be used. W'herever results for a particular characteristic are
reported, the control method must be indicated.
Thefollounng characteristic may be relevantfor acaciaused as a Galactose: a greyish-blue zone A greyish-blue zone, intense
viscosity-increasing agent and/or suspending agent in aqueous (galactose)
preparations.
Apparent viscosity
Determine the dynamic viscosity using a capillary viscometer -- --
(2.2.9) or a rotating viscometer (2.2.10) on a 100 gIL I or 2 brownish-grey zones, very faint
solution of acacia (dried substance). to equivalent

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _.-:- PhEur

1 or 2 blue zones, faint to' equivalent

Acacia, Dried Dispersion Reference solution (a) Test solution

Spray-dried Acacia
C. Dissolve 1 g of the preparation to be examined in 2 mL
(Ph. Eur. monograph 0308) of water R by stirring frequently for 20 min. Add 2 mL of
PhEur _ ethanol (96 per cent) R. After shaking, a white gelatinous
mucilage is formed that becomes fluid upon addition of
DEFINITION
10 mL of water R.
Powder obtained from a dispersion of Acacia (0307) after a
drying process. TESTS
Solution S
CHARACTERS
Dissolve 3.0 g of the preparation to be examined in 25 mL
It dissolves completely, after about 20 min, in twice its mass
of water R by stirring for 10 min. Allow to stand for 20 min
of water. The liquid obtained is colourless or yellowish,
and dilute to 30 mL with water R.
dense, viscous, adhesive, translucent and weakly acid to blue
litmus paper. It is practically insoluble in ethanol Glucose and fructose
(96 per cent). High-performance thin-layer chromatography (2.8.25)
Test solution To 0.1 g in a thick-walled centrifuge tube add
IDENTIFICATION
2 mL of a 100 gIL solution of trifluoroacetic acid R and shake
A. Examine under a microscope using ethanol (96 per cent) R
vigorously. Stopper the tube and heat the mixture at 120 DC
as the mounting medium. The preparation to be examined
for 1 h. Centrifuge, transfer 1 mL of the clear supernatant
consists of predominantly spheroidal or irregular and angular
into a 10 mL flask and add 5 mL of methanolR.
particles varying in size (4-500 urn), with 1 or more rounded
cavities containing 1 or several air bubbles; a few flat Reference solution (a) Dissolve 5 mg of arabinose R, 5 mg of
fragments are also present. Only traces of starch granules are galactose R, 5 mg of glucose R, .5 mg of rhamnose Rand 5 mg
visible and no plant tissue is observed. of xylose R in 1 mL of water R and dilute to 10.0 mL with
methanolR.
B. Examine the chromatograms obtained in the test for
glucose and fructose. Reference solution (b) Dilute 2.5 mL of reference solution (a)
to 10.0 mL with methanolR.
Results See below the sequence of zones present in the
chromatograms obtained with reference solution (a) and the Reference solution (c) Dissolve 5 mg of galactose Rand 5 mg
test solution. of glucose R in 1 mL of water R and dilute to 10 mL with
methanolR.

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1-46 Acamprosate Calcium 2020

Intensity marker Galactose. FUNCTIONALITY-RELATED CHARACTERISTICS

Plate TLC silica gel FZ54 plate R (2-10 urn). This section provides information on characteristics that are
Mobile phase water R, acetonitrile R (15:85 VIV)o recognised as being relevant control parameters for one or more
functions of the substance when used as an excipient (see chapter
Application 4,.tL of the test solution and reference
5.15). Some of the characteristics described in the Functionality-
solutions (a) and (b), and 2 ul, of reference solution (c), as
related characteristics section may also bepresent in the mandatory
bands of 8 mm.
part of the monograph since they also represent mandatory quality
Development A 70 mm from the lower edge of the plate, in criteria. In such cases, a cross-reference to the tests described in the
an unsaturated tank. mandatorypart is included in the Functionality-related
Drying A In air. characteristics section. Control of the characteristics can contribute
Development B 70 mm from the lower edge of the plate, in to the quality of a medicinal productby improving the consistency
an unsaturated tank, using freshly prepared mobile phase. of the manufacturing process and the performance of the medicinal
Drying B In air. product duringuse. Where control methods are cited, they are
recognised as being suitable for the purpose, but othermethods can
Detection Treat with a solution prepared as follows: dissolve
also be used. Wherever results for a particular characteristic are
4 g of diphenylamine Rand 4 mL of anilineR in 160 mL of reported) the control method must be indicated.
acetone R and add phosphoric acid R until the precipitate
formed dissolves again (about 30 mL). Heat at 120 DC for The following characteristic may be relevant for acacia dried
5-10 min and examine in daylight. dispersion used as a viscosity-increasing agent and/orsuspending
agent in.aqueous preparations.
System suitability Reference solution (c):
- the chromatogram shows in the middle third 2 distinct Apparent viscosity
zones, which may be touching; the lower zone (galactose) Determine the dynamic viscosity using a capillary viscometer"
and the upper zone (glucose) are greyish-blue. (2.2.9) or a rotating viscometer (2.2.10) on a 100 gIL
.soluti~:m of acacia, dried dispersion (dried substance).
Results The chromatogram obtained with the test solution
shows no greyish-blue zone and no reddish zone between the _ - - - - - - - - - - - - - - - - - - - - PhEur
zones due to galactose and arabinose in the chromatogram
obtained with reference solution (a).
Starch, dextrin and agar
To 10 mL ofsolution S, previously boiled and cooled, add Acamprosate Calcium
0.1 mL of 0.05 M iodine. No blue or reddish-brown colour
(Ph. Eur. monograph 1585)
develops.
Sterculia gum
A. Place 0.2 g in a 10 mL ground-glass-stoppered cylinder
graduated in 0.1 mL. Add 10 mL of ethanol
(60 per cent V/V? R and shake. Any gel formed occupies not
more than 1.5 mL.
B. To 1.0 g add 100 mL of water R and shake. Add 0.1 mL 400.5 77337-73-6
of methyl red solution R. Not more than 5.0 mL of
0.01 M sodium hydroxideis required to change the colour of Action and use
the indicator. Treatment of alcoholism.
Tannins Preparation
To 10 mL of solution S add 0.1 mL offerric chloride Acamprosate Gastro-resistant Tablets
solution R1. A gelatinous precipitate is formed, but neither the PhEur _
precipitate nor the liquid is dark blue.
DEFINITION
Tragacanth
Examine the chromatograms obtained in the test for glucose Calcium bis [3-(acetylamino)propane-1-sulfonate].
and fructose. Content
Results The chromatogram obtained with the test solution 98.0 per cent to 102.0 per cent (dried substance).
shows no faint to intense brownish-grey zone corresponding CHARACTERS
to the zone due to xylose in the chromatogram obtained with Appearance
reference solution (a). White or almost white powder.
Loss on drying (2.2.32) Solubility
Maximum 10.0 per cent, determined on 1.000 g by drying in Freely soluble in water, practically insoluble in ethanol
an oven at 105 DC. . (96 per cent) and in methylene chloride.
Total ash (2.4.16) IDENTIFICATION
Maximum 4.0 per cent. A. Infrared absorption spectrophotometry (22.24).
Microbial contamination Comparison Ph. Eur. reference spectrum of acamprosate calcium.
TAMC: acceptance criterion 104 CFU/g (2.6.12).
B. It gives reaction (a) of calcium (2.3.1).
TYMC: acceptance criterion 102 CFU/g (2.6.12).
TESTS
Absence of Escherichia coli (2.6.13).
Solution S
Absence of Salmonella (2.6.13). Dissolve 5.0 g in carbon dioxide-free waterR and dilute to
100 mL with the same solvent.

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2020 Acarbose 1-47

Appearance of solution IMPURITIES

Solution S is clear (2.2.1) and colourless (2.2.2, Method II).
pH (2.2.3)
5.5 to 7.0 for solution S.
A. 3-aminopropane-l-sulfonic acid (homo taurine ).
Impurity A _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
Liquid chromatography (2.2.29).
Test solution Dissolve 0.40 g of the substance to be
examined in distilled zoaterR and dilute to 20.0 mL with the
same solvent. Dilute 10.0mL of this solution to 100.0 mL
with borate buffer solution pH 10.4 R. Place 3.0 mL of the
Acarbose
solution obtained in a 25 mL ground-glass-stoppered tube. (Ph. Bur. monograph 2089)
Add 0.15 mL of a freshly prepared 5 gIL solution of

HO~OH ~:>H~O
fiuorescamine R in acetonitrile R. Shake immediately and 0
vigorously for 30 s. Place in a water-bath at 50°C for
30 min. Cool under a stream of cold water. Centrifuge and . OH 0 HOH
h 0 - OH
filter the supernatant through a suitable membrane filter
(nominal pore size 0.45 JlID.), 25 mm in diameter, HON 0 0
H
OH OH OH OH
Reference solution Dissolve 50 mg of acamprosate
impurity A CRS in distilled waterR and dilute to 200.0 mL
with the samesolvent. Dilute 0.4 mL of the solution to 646 56180-94-0
100.0 mL with borate buffersolution pH 10.4 R. Treat 3.0 mL
of this solution in the same way as the test solution Action and use
Alpha-glucosidase inhibitor; treatment of diabetes mellitus.
Column:
- size: l = 0.15 m, 0 = 4.6 rnm; PhEur _
- stationaryphase: spherical octadecylsilyl silica gelfor
DEFINITION
chromatography R (5 urn) with a specific surface area of
0-4,6-Dideoxy-4-[[(IS,4R,5S,6S)-4,5,6-trihydroxy-3-
170 m 2/g and a pore size of 12 nm.
(hydroxymethyl)cyclohex-2-enyl]amino] -c-n-glucopyranosyl-
Mobilephase acetonitrile R, methanol R, 0.1 M phosphate (l ~4)-O-ct-o-glucopyranosyl-(1~4)-p-glucopyranose,which
buffer solution pH 6.5 R (10:10:80 V/V/V). is produced by certain strains of Actinoplanes utahensis.
Flow rate 1 ml./min. Content
Detection Spectrophotometer at 261 nm. 95.0 per cent to 102.0 per cent (anhydrous substance).
Injection 20 J,tL. CHARACTERS
Run time 6 times the retention time of impurity A Appearance
Retention times Fluorescamine = about 4 min; White or yellowish, hygroscopic, amorphous powder.
impurity A= about 8 min; acamprosate is not detected by Solubility
this system. Very soluble in water, soluble in methanol, practically
Limits: insoluble in methylene chloride.
- impurity A: not more than the area of the corresponding
peak in the chromatogram obtained with the reference
IDENTIFICATION
solution (0.05 per cent). A. Infrared absorption spectrophotometry (2.2.24).
Comparison acarbose for identification CRS.
Loss on drying (2.2.32)
Maximum 0.4 per cent, determined on 1.000 g by drying in B. Examine the chromatograms obtained in the assay.
an oven at 105°C. Results The principal peak in the chromatogram obtained
with the test solution is similar in retention time and size to
ASSAY
the principal peak in the chromatogram obtained with
To 4 g of cation-exchange resin R (75-150 urn) add 20 mL of
reference solution (a).
distilled water R and stir magnetically for 10 min. Introduce
this suspension into a glass column, 45 em long and 2.2 ern TESTS
in internal diameter, equipped with a polytetrafluoroethylene Solution S
flow cap covered by a glass-wool plug. Allow a few millilitres Dissolve 1.00 g in carbon dioxide-free water R and dilute to
of this solution to flow, then place a plug of glass wool over 20.0 mL with the same solvent.
the resin. Pass 50 mL of 1 M hydrochloric acid through the pH (2.2.3)
column. The pH of the eluate is close to 1. Wash with 5.5 to 7.5 for solution S.
3 quantities, each of 200 mL, of distilled waterR to obtain an
Specific optical rotation (2.2.7)
eluate at pH 6. Dissolve 0.100 g of the substance to be
examined in 15 mL of distilled waterR. Pass through the
+ 168 to + 183 (anhydrous substance).
column and wash with 3 quantities, each of 25 mL, of Dilute 2.0 mL of solution S to 10.0 mL with waterR.
distilled toater.R, collecting the eluate. Allow to elute until an Absorbance (2.2.25)
eluate at pH 6 is obtained. Titrate the solution obtained with Maximum 0.15 at 425 nm for solution S.
0.1 M sodium hydroxide,'determining the end-point Related substances
potentiometrically (2.2.20). Liquid chromatography (2.2.29).
1 mL of 0.1 M sodium hydroxide corresponds to 20.02 mg of Testsolution Dissolve 0.200 g of the substance to be
ClOH20CaN20SS2' examined in water R and dilute to 10.0 mL with the same
solvent.

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 1-48 Acarbose 2020

Reference solution (a) Dissolve the contents of a vial of - any other impurity: for each impurity, not more than
acarbose CRS in s.o mL of water R. 0.2 times the area of the principal peak in the
Reference solution (b) Dissolve the contents of a vial of chromatogram obtained with reference solution (c)
acarbose for peak identification CRS (acarbose containing (0.2 per cent);
impurities A, B, C, D, E, F and G) in 1mL of waterR. - total: not more than 3 times the area of the principal peak
Reference solution (c) Dilute 1.0 mL of the test solution to in the chromatogram obtained with reference solution (c)
100.0 mL with water R. (3.0 per cent);
- disregard limit: 0.1 times the area of the principal peak in
Column: the chromatogram obtained with reference solution (c)
= =
- size: 1 0.25 m, 0 4 mrn; (0.1 per cent).
- stationary phase: aminopropylsilyl silica gelfor
chromatography R (5 J.lID); Water (2.5.12)
. - temperature: 35 DC. Maximum 4.0 per cent, determined on 0.300 g.
Mobile phase Mix 7S0 volumes of acetonitrile R1 and Sulfated ash (2.4.14)
250 volumes of a solution containing 0.60 gIL of potassium Maximum 0.2 per cent, determined on 1.0 g.
dihydrogen phosphate Rand 0.35 gIL of disodium hydrogen ASSAY
. phosphate dihydrate R. Liquid chromatography (2.2.29) as described in the test for
Flow rate 2.0 mL'min. related substances with the following modification.
Detection Spectrophotometer at 210 nrn. Injection Test solution and reference solution (a).
Injection 10 ul, of the test solution and reference Calculate the percentage content of C25H43N018 taking into
solutions (b) and (c). account the assigned content of acarbose CRS.
Run time 2.5 times the retention time of acarbose. STORAGE
Identification of impurities Use the chromatogram supplied In an airtight container.
with acarbose for peak identification CRS and the
IMPURITIES .
chromatogram obtained with reference solution (b) to
identify the peaks due to impurities A,B, C, D, E, F and G.
Specified impurities A, B, C, D, E, F, G.
Relative retention With reference to acarbose (retention Other detectable impurities (the following substances would, if
present at a sufficient level, be detected by one or otherof the tests
= =
time about 16 min): impurity D about 0.5;
in the monograph. They are limitedby the general acceptance
= =
impurity B about 0.8; impurity A about 0.9;
criterion for other/unspecified impurities. It is.therefore not
=
impurity C about 1.2; impurity E = about 1.7;
necessary to identifythese impurities for demonstration of
impurity F = about 1.9; impurity G = about 2.2.
compliance. See also 5.10. Control of impurities in substances for
System suitabz7ity Reference solution (b): pharmaceutical use) H.
- the chromatogram obtained is similar to the
chromatogram supplied with acarbose for peak
identification CRS; (Pr0H
- peak-to-valley ratio: minimum 1.2, where Hp =
height
above the baseline of the peak due to impurity A and
H; = height above the baseline of the lowest point of the
HO dH~~1 HO OH
A:>:>H:E>0
curve separating this peak from the peak due to acarbose. b OH
HO N 0 0
H
Limits: OH OH OH
- correction factors: for the calculation of content, multiply
the peak areas of the following impurities by the A. 0-4,6-dideoxy-4-[[(1S,4R,5S,6S)-4,S ,6-trihydroxy-3-
corresponding correction factor: impurity B = 0.63; (hydroxymethyl)cyc1ohex-2-enyl] amino] -rt-D-
= =
impurity D 0.7S; impurity E 1.25; impurity F =
1.2S; glu<;opyranosyl-(l-74)-O-rt-D-glucopyranosyl-(1-74)-n-
impurity G = 1.25; arabino-hex-2-ulopyranose,
- impurity C: not more than 1.5 times the area of the
principal peak in the chromatogram obtained with
reference solution (c) (1.5 per cent);
- impurity D: not more than the area of the principal peak
HO~ _
3
CH 0 H~O 0 Hh
_OOH

in the chromatogram obtained with reference solution (c) OH . A:>H • O.H OH

(1.0 per cent); HO N 0 0
H
- impurity A: not more than 0.6 times the area of the OH OH OH OH
principal peak in the chromatogram obtained with
reference solution (c) (0.6 per cent); B. (lR,4R,SS,6R)-4,S,6-trihydroxy-2-(hydroxymethyl)
- impurity B: not more than 0.5 times the area of the cyc1ohex-2-enyl 4-0-[4,6-dideoxy-4-[[(lS,4R,5S,6S)-
principal peak in the chromatogram obtained with 4,5,6-trihydroxy-3-(hydroxymethyl)cyclohex-2-enyn
reference solution (c) (0.5 per cent); amino]-ct-D-glucopyranosyl]-rt-n-glucopyranoside,
"'-- impurities F, G: for each impurity, not more than
o.s times the area of the principal peak in the
chromatogram obtained with reference solution (c)
(0.3 per cent);
- impurity E: not more than 0.2 times the area of the
principal peak in the chromatogram obtained with
reference solution (c) (0.2 per cent);

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2020 Acebutolol Hydrochloride 1-49

HO~ CHa CH
3
Hh-0
.

0-;- ~O" ~O" OH O. OH

HO HO ~~O~O
~O"HO~O:H
OH OH OH OH

HO:S
H. 0-4,6-dideoxy-4-[[(1S,4R,5S,6S)-4,5,6-trihydroxy-3-

H~~~O~O
(hydroxyrnethyl)cyclohex-2-enyl] amino] -a-o-
glucopyranosyl-(1-+4)-O-6-deoxy-a-o-glucopyranosyl-
OH OH OH (1-+4)-o-glucopyranose.
_ _ _ _ _ _~-------------PhEur
C. ce-n-glucopyranosyl 4-0- [4,6-dideoxy-4-[[(lS,4R, 5S, 6S)-
4,5,6-trihydroxy-3-(hydroxymethy1)cyclohex-2-enyl]
amino]-a-o-glucopyranosyl]-a-n-glucopyranoside,

Acebutolol Hydrochloride
HO~ 3
H~O
O~ ~O"
CH
(Ph. Bur. monograph 0871)
OH . 0 OH

HO .~ . " EOCH
a
H OH

I .~ O~~yCH3 ' HCI

OH OH OH
o
D.4-0-[4,6-dideoxy-4-[[(lS,4R,5S,6S)-4,5,6-trihydroxy-3-
(hydroxymethyljcyclohex-z-enyl] amino]-a-o-
H3C~N # CH3
H and enantiomer
glucopyranosyl] -n-giucopyranose,

r
OH 372.9 34381-68-5

~ OH ~:>hOHO~OH~O
HO HO HO HO
Action and use
Beta-adrenoceptor antagonist.
OH Preparations
HO N 0 0 0 Acebutolol Capsules
H ..
OH OH OH OH Acebutolol Tablets
E. 0-4,6-dideoxy-4-[[(lS,4R,5S,6S)-4,5,6-trihydroxy-3- PhEur _ _ ~- -_
(hydroxyrnethyl)cyclohex-2-enyl] amino]-a-o-
DEFINITION
glucopyranosyl-(1-+4)-0-a-o-glucopyranosyl-(1-+4)-0-a-
N-[3-Acetyl-4-[(2RS)-2-hydroxy-3-[(1-methylethyl)amino]
o-glucopyranosyl-(1-+4)-o-arabino-hex-2-ulopyranose
propoxy]phenyl] butanamide hydrochloride.
(4-0-a-acarbosyl-o-fructopyranose),
Content

HO~ . Eo" HO~O, HO~O, HO~.o 0, . OH

99.0 per cent to 101.0 per cent (dried substance).
CHARACTERS

J;~:~..I'~~l'~-(l'~-(lx ...(
Appearance
White or almost white, crystalline powder.
OH OH OH OH OH
Solubility
F. 0-4,6-dideoxy-4-[[(1S,4R,5S,6S)-4,S,6-trihydroxy-3-
Freely soluble in water and in ethanol (96 per cent), very
slightly soluble in acetone and in methylene chloride.
(hydroxyrnethyl)cyclohex-2-enyl] amino]-a-o-
glucopyranosyl-(l-+4)-0-a-D-glucopyranosyl.,.(1-+4)-0-a- mp
o-glucopyranosyl~(1-+4)-o-glucopyranose(4-O-rl- About 143°C.
acarbosyl-D-glucopyranose), IDENTIFICATION
First identification: B, D.
Second identification: A., C., D.
A. Ultraviolet and visible absorption spectrophotometry
(2.2.25).
HO
Test solution Dissolve 20.0 mg in a 0.1 per cent V/V

HO~.
OH ~c> HOHh OH~O
OH OOH 0
solution of hydrochloric acid R and dilute to 100.0 mL with
the same acid solution. Dilute 5.0 mL of this solution to
100.0 mL with a 0.1"per cent V/V solution of hydrochloric
HO N 0 0 0 acid R.
H
OH OH OH OH Spectral range 220-350 nrn.
Absorption maxima At 233 nm and 322 nrn.
G. o-n-glucopyranosyl 0-4,6-dideoxy-4-[[(1S,4R,5S,6S)-
Specific absorbance at the absorption maximum 555 to 605,;at
4,5,6-trihydroxy-3-(hydroxymethyl)cyclohex-2-enyl]
233 nrn.
amino] -a-n-glucopyranosyl-f1-+ 4)-0-a-o-glucopyranosyl-
(1-+4)-O-a-o-glucopyranoside (n-n-glucopyranosyl E. Infrared absorption spectrophotometry (2.2.24).
o-acarboside), Preparation Discs.

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 1...50 Acebutolol Hydrochloride 2020

Comparison acebutolol hydrochloride CRS. - stationary phase: end-capped octadecylsilyl silica gelfor_
C. Thin-layer chromatography (2.2.27). chromatography R (5 urn),
Test solution Dissolve 20 mg of the substance to be - temperature: ~O °C.
examined in methanol R and dilute to 20 mL with the same Mobilephase:
solvent. - mobile phaseA: mix 2.0 mL of phosphoric acid R, and
Reference solution (a) Dissolve 20 mg of acebutolol 3.0 mL of triethylamine R and dilute to 1000 mL with
hydrochloride CRS in methanolR and dilute to 20 mL with the water R;
same solvent. - mobile phaseB: mix equal volumes of aceionitrile Rand
mobile phase A;
Reference solution (b) Dissolve 20 mg of pindolol CRS in
methanolR and dilute to 20 mL with the same solvent. Time Mobile phase A Mobile phase B
To 1 mL of this solution add 1 mL of reference solution (a). (min) (per cent VIV) (per cent VIV)
Plate TLC silica gel F254 plate R. 0-2 98 2
Mobilephase perchloric acid R, methanolR, waterR 2 - 30.5 98 -> 10 2 -> 90
(5:395:600 V/V/V). 30.5 - 41 10 90
Application 10 JlL.
- Development Over 3/4 of the plate. Flow rate 1.2 mUmin.
Drying In air. Detection Spectrophotometer at 240 nm.
Detection Examine in ultraviolet light at 254 nm. Injection 25 j.tL.
System suitabz7ity The chromatogram obtained with System suitability Reference solution (c):
reference solution (b) shows 2 clearly separated principal _. resolution: minimum 7.0 between the peaks due to
spots. impurity I and acebutolol.
Results The principal spot in the chromatogram obtained Limits:
with the test solution is similar in position and size to the - impurityB: not more than the area of the principal peak in
principal spot in the chromatogram obtained with reference the chromatogram obtained with reference solution (e)
solution (a). (0.2 per cent);
- impurity C: not more than the area of the principal peak
D. It gives reaction (a) of chlorides (2.3.1).
in the chromatogram obtained with reference solution (d)
TESTS (0.1 per cent);
Appearance of solution - impurityI: not more than twice the area of the principal
The solution is not more opalescent than reference peak in the chromatogram obtained with reference
suspension II (2.2.1) and not more intensely coloured than solution (a) (0.2 per cent);
reference solution BYs (2.2.2, Method II). - any other impurity: for each impurity, not more than the
Dissolve 0.5 g in water R and dilute to 10 mL with the same area of the principal peak in the chromatogram obtained
solvent. with reference solution (a) (0;1 per cent);
- total: not more than 5 times the area of the principal peak
pH (2.2.3)
in the chromatogram obtained with reference solution (a)
5.0 to 7.0.
(0.5 per cent);
Dissolve 0.20 g in carbon dioxide-free waterR and dilute to - disregard limit: 0.5 times the area of the principal peak in
20 mL with the same solvent. the chromatogram obtained with reference solution (a)
Related substances (0.05 per cent).
Liquid chromatography (2.2.29). Loss on drying (2.2.32)
Test solution Dissolve 0.100 g of the substance to be Maximum 0.5 per cent, determined on 1.000 g by drying in
examined in mobile phase A and dilute to 50.0 mL with an oven at 105°C for 3 h.
mobile phase A.
Sulfated ash (2.4.14)
Reference solution (a) Dissolve 20.0 mg of the substance to Maximum 0.1 per cent, determined on 1.0 g.
be examined in mobile phase A and dilute to 100.0 mL with
mobile phase A. Dilute 0.5 mL of this solution to 50.0 mL ASSAY
with mobile phase A. Dissolve 0.300 gin 50 mL of ethanol (96 per cent) R and add
1 mL of 0.1 M hydrochloric acid. Carry out a potentiometric
Reference solution (b) Dissolve the contents of a vial of
titration (2.2.20), using 0.1 M sodium hydroxide. Read the
acebutolol impurity I CRS in 1.0 mL of mobile phase A.
volume added between the 2 points of inflexion.
Reference solution (c) Mix 2.0 mL of reference solution (a)
1 mL of 0.1 M sodium hydroxide is equivalent to 37.29 mg of
and 1.0 mL of reference solution (b) and dilute to 10.0 mL
ClsHz9ClNZ04.
with mobile phase A.
Reference solution (d) Dissolve 5.0 mg of acebutolol STORAGE
impurity C CRS in 10 mL of acetonitrile R and dilute to Protected from light.
25.0 mL with mobile phase A. Dilute 0.5 mL of this solution IMPURITIES
to 50.0 mL with mobile phase A. Specified impurities A, B, C, D, E, F, G, H, I, J, K
Reference solution (e) Dissolve 5.0 mg of acebutolol
impurity B CRS in 10.0 mL of acetonitnle R and dilute to
25.0 mL with mobile phase A. Dilute 1.0 mL of this solution
to 50.0 mL with mobile phase A.
Column:
- size: l = 0.125 m, 0 = 4 mID,

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2020 Aceclofenac I-51

C 3

o 0°X :
o~ and enantlorner

H3C~NH #

A. N- [3-acetyl-4-[(2RS)-oxiran-2-ylmethoxy]
phenyl] butanamide, H. N,N'- [(2-hydroxypropane-l ,3-diyl)bis [oxy(3-acetyl-l,4-
phenylene)]] dibutanamide,
o CH3 .

E
H pH H
o
O~N
.CH3

1:
E
'<:::::: CH3
~. .I . and enantiomer
'<::::::
H pH H
0~N~CH3
H C"...A-..N
3 H
# 3

H3C~N.
° I
,# andenantiomer
H
B. N-[3-acetyl-4- [(2RS)-2-hydroxy-3- [(l-methylethyl)amino]
propoxy]phenyl] acetamide (diacetolol), I. N-[3-acetyl-4- [(2RS)- 3-(ethylamino)-2-hydroxypropoxy]
phenyl]butanamide,
o CH3

° I
HC~N 3 H
E ~

#
OH

E
WI. .
H3C~N .
H
o

,#
·CH3

'<:::::: O~N
H pH H

. Y
CH a
CH 3
and enantiomer

C.··1V-(3-acetyl-4-hydroxyphenyl)butanamide,
J. N-[3-acetyl-4-[(2RS)-2-hydroxy-3-[(1-methylethyl)amino]
CH 3 propoxy]phenyl]propanamide,

E
O . H pH H

'<:::::: O~N. CH3

I·. .Y and enantiomer H3C=00 H pH H
O~N
,# CH3
H2N

HC~N
WI·
'<::::::

,#
1: CH 3

3
and enantlomer

D. 1-[5-amino-2- [(2RS)-2-hydroxy-3-[(l-methylethyl)amino] 3 H
propoxy]phenyl] ethanone,
K. N-[3-butanoyl-4-[(2RS)-2-hydroxy-3-[ (1-
H pH H
methylethyl) amino] propoxy]phenyl] butanamide.

O
O~ N CH3

~
W I·.
#
YCH3
and enantiomer
-'- PhEur

H3C ~.. .

E. N-[4-[(2RS)-2-hydroxy-3-[(1-methylethyl)amino]propoxy] Aceclofenac
phenyl] butanamide,
(ph. Eur. monograph 1281)
o CH3

E
H pH

° I '<::::::
°~.. OH
and enantiomer

H3C~NH #

F. N- [3-acetyl-4- [(2RS)-2,3-dihydroxypropoxy]
phenyl]butanamide,
354.2 89796-99-6

Action and use

Cyclo-oxygenase inhibitor; analgesic; anti-inflammatory.
PhEur --'--~--'----'----'-"'-- _

DEFINITION
[[[2-[(2,6-Dichlorophenyl)amino ]phenyl] acetyl]oxy]acetic
G. N,N'- [[ (l-methylethyl)imino]bis [(2-hydroxypropane-l ,3- acid.
diyl)oxy(3-acetyl-l ,4-phenylene)]]dibutanamide (biamine), Content
99.0 per cent to 101.0 per cent (dried substance).
CHARACTERS
Appearance
White 'or almost white, crystalline powder.

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I-52 Aceclofenac 2020

Solubility - stationary phase: spherical end-capped octadecylsilyl silica gel

Practically insoluble in water, freely soluble in acetone, for chromatography R (5 urn) with a pore size of 10 nm
soluble in ethanol (96 per cent). and a carbon loading of 19 per cent;
IDENTIFICATION - temperature: 40°C.
First identification: B. Mobilephase:
- mobile phase A: 1.12 gIL solution of phosphoric acidR
Second identification: A, C.
adjusted to pH 7.0 with a 42 gIL solution of sodium
A. Ultraviolet and visible absorption spectrophotometry hydroxide R;
(2.2.25). - mobile phase B: water R, acetonitrile R (10:90 VIV);
Test solution Dissolve 50.0 mg in methanol R and dilute to
100.0 mL with the same solvent. Dilute 2.0 rnL of the Time Mobile phase A Mobile phase B
solution to 50.0 rnL with methanolR. (min) (per cent VIP) (per cent VIP)

Spectra/range 220-370 nm. 0-25 70 -> 50 30 -> 50

Absorption maximum 275 nm. 25 - 30 50 -> 20 50 -> 80

30 - 50 20 80
Specific absorbance at the absorption maximum 320 to 350.
,B. Infrared absorption spectrophotometry (2.2.24).
Flow rate 1.0 mL/min.
Comparison: Ph, Bur. reference spectrum of aceclofenac.
Detection Spectrophotometer at 275 run.
C. Dissolve about 10 mg in 10 rnL of ethanol (96 per cent) R.
Injection 10 JlL of the test solution and reference
To 1 mL of the solution, add 0.2 mL of a mixture, prepared
solutions (c), (d), (e), (t), (g) and (h).
immediately before use, of equal volumes of a 6 gIL solution
of potassium ferricyanide R and a 9 gIL solution of ferric Identification of impurities Use the chromatogram obtained
chloride R. Allow to stand protected from light for 5 min. with reference solution (c) to identify the peak due to
Add 3 rnL of a 10.0 gIL solution of hydrochloric acid R. Allow impurity A; use the chromatogram supplied with acecZofenac
to stand protected from light for 15 min. A blue colour for peak identification CRS and the chromatogram obtained
develops and a precipitate is formed. with reference solution (h) to identify the peaks due to
impurities B, C, D, E and G; use the chromatogram
TESTS obtained with reference solution (d) to identify the peak due
Related substances to impurity F; use the chromatogram obtained with reference
Liquid chromatography (2.2.29). Prepare the solutions solution (e) to identify the peak due to impurity H; use the
immediately before use. chromatogram obtained with reference solution (g) to
Solvent mixture Mobile phase A, mobile phase B identify the peak due to impurity I.
(30:70 VIV). / Relative retention With reference to aceclofenac (retention
Test solution Dissolve 50.0 mg of the substance to be time = about 11 min): impurity A = about 0.8;
examined in the solvent mixture and dilute to 25.0 rnL with impurity G = about 1.3; impurity H = about 1.5;
the solvent mixture. impurity I = about 2.3; impurity D = about 3.1;
Reference solution (a) Dissolve 21.6 mg of diclofenac impurity B = about 3.2; impurity E = about3.3;
sodium CRS (impurity A) in the solvent mixture and dilute to impurity C = about 3.5; impurity F = about 3.7.
50.0 rnL with the solvent mixture. System suitabil£ty Reference solution (c):
Reference solution (b) Dilute 2.0 rnL of the test solution to - resolution: minimum 5.0 between the peaks due to
10.0 mL with the solvent mixture. impurity A and acec1ofenac.
Reference solution (c) Mix 1.0 rnL of reference solution (a) Limits:
and 1.0 rnL of reference solution (b) and dilute to 100.0 rnL - impurity A: not more than the area of the corresponding
with the solvent·mixture. peak in the chromatogram obtained with reference
Reference solution (d) Dissolve 4.0 mg ofaceclofenac solution (c) (0.2 per cent);
£mpurz"ty F CRS in the solvent mixture and dilute to 10.0 rnL - impurities B, C, D, B, G: for each impurity, not more than
the area of the peak due to aceclofenac in the
with the solvent mixture.
chromatogram obtained with reference solution (t)
Reference solution (e) Dissolve 2.0 mg of aceclofenac (0.2 per cent);
impurity H CRS in the solvent mixture and dilute to 10.0 rnL - impurity F: not more than the area of the corresponding
with the solvent mixture. peak in the chromatogram obtained with reference
Reference solution (j) Mix 1.0 mL of reference solution (b), solution (t) (0.2 per cent);
1.0 mL of reference solution (d) and 1.0 mL of reference - impurity H: not more than 1.5 times the area of the
solution (e) and dilute to 100.0 mL with the solvent mixture. corresponding peak in the chromatogram obtained with
Referencesolution (g) Dissolve 5.0 mg of aceclofenac reference solution (t) (0.15 per cent);
impurity I CRS in the solvent mixture and dilute to 50.0 mL - impurity I: not more than 1.5 times the area of the
with solvent mixture. Dilute 1.0 mL of the. solution to corresponding peak in the chromatogram obtained with
50.0 mL with the solvent mixture. reference solution (g) (0.15 per cent);
Reference solution (h) Dissolve 4 mg of aceclofenac for peak - unspecified impurities: for each impurity, not more than
identification CRS (containing impurities B, C, D, E and G) 0.5 times the area of the peak due to aceclofenac in the
in 2 rnL of the solvent mixture. chromatogram obtained with reference solution (t)
(0.10 per cent);
Column:
- total: maximum 0.7 per cent;
= =
- size: 1 0.25 m, 0 4.6 mm;
- disregard limit: 0.25 times the area of the peak due to
aceclofenac in the. chromatogram obtained with reference
solution (t) (0.05 per cent).

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2020 Acemetacin I-53

Loss on drying (2.2.32)

Maximum 0.5 per cent, determined on 1.000 g by drying in
an oven at 105°C.
Sulfated ash (2.4.14)
Maximum 0.1 per cent, determined on 1.0 g.
ASSAY
Dissolve 0.300 g in 40 rnL of methanol R. Titrate with 0.1 M
sodium hydroxide, determining the end-point F. benzyl [[[2-[(2,6-dichlorophenyl)amino]phenyl]acetyl]oxy]
potentiometrically (2.2.20). acetate (benzyl ester of aceclofenac),
I mL of 0.1 M sodium hydroxide is equivalent to 35.42 mg of
C16H13CI2N04'
STORAGE
Protected from light.
IMPURITIES
Specified impurities A, B, C, D, B, F, G, H, 1.

G. [[[[[2-[(2,6-dichlorophenyl)amino]phenyl]acetyl]oxy]
acetyl]oxy]acetic acid (acetic aceclofenac),

A. '; [2- [(2,6-dichlorophenyl)amino]phenyl]acetic acid

(diclofenac),

-,
ocr
~ I NH
0
OCH3

H. [[[[[[[2-[(2,6-dichlorophenyl)amino]phenyl]acetyl]oxy]
Cl~,
"" CI
acetyl].oxy]acetyl]oxy]acetk acid (diacetic aceclofenac),

V ~O
B. methyl [2- [(2~ 6-dichlorophenyl)amino]phenyl] acetate ~NrCI
(methyl ester of diclofenac),
C1-D
I. 1-(2,6-dichlorophenyl)-1,3-dihydro-2H-indol-2-one.
___ ....,--~ ~ PhEur

C . ethyl [2-[(2,6-dichlorophenyl)amino]phenyl] acetate (ethyl

ester of diclofenac), Acemetacin *****
** **
o (Ph. Bur. monograph 1686) ***
~O~OCH'
~NH6
CI~CI
V
D. methyl [[[2-[(2,6-dichlorophenyl)amino]phenyl]acetyl]oxy]
acetate (methyl ester of aceclofenac), 415.8 53164-05-9
o
Action and use
roO~O/'-.CH' Cyclo-oxygenase inhibitor; analgesic; anti-inflammatory.
PhEur _
NH

CI'&CI DEFINITION
[[[1-( 4-Chlorobenzoyl)-5-methoxy-2-methyl-IH-indol-3-yl]
acetyl]oxy]acetic acid.
E. ethyl [[[2-[(2, 6-dichlorophenyl)amino]phenyl] acetyl]oxy] Content
acetate (ethyl ester of acedofenac), 99.0 per cent to l Ol.O per cent (dried substance).

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I-54 Acemetacin 2020

CHARACTERS with reference solution (e) to identify the peaks due to

Appearance impurities C, D, E and F;
Yellow or greenish-yellow, crystalline powder. - use the chromatogram obtained with reference
Solubility solution (b) to identify the peak due to impurity B.
Practically insoluble in water, soluble in acetone, slightly Relative retention With reference to acemetacin (retention
soluble in anhydrous ethanol. time = about 15 min): impurity A = about 0.7;
It shows polymorphism (5.9). =
impurity B = about 0.9; impurity F about 1.2;
impurity C = about 1.3; impurity D = about 1.5;
IDENfIFICATION impurity E = about 2.2.
Infrared absorption spectrophotometry (2.2.24). System suitability Reference solution (d):
Comparison acemetacin CRS. - peak-to-valley ratio: minimum 15,. where Hp = height
If the spectra obtained in the solid state show differences, above the baseline of the peak due to impurity Band
dissolve the substance to be examined and the reference H; = height above the baseline of the lowest point of the
substance separately in acetone R, evaporate to dryness and curve separating this peak from the peak due to
record new spectra using the residues. acemetacin.
TESTS Limits:
Related substances - correction factors: for the calculation of content, multiply
Liquid chromatography (2.2.29). the peak areas of the following impurities by the
corresponding correction factor: impurity C = 1.3;
Test solution Dissolve 0.100 g of the substance to be
impurity D = 1.4; impurity F = 1.3;
examined in acetonitrile for chromatography R and dilute to
- impurity E: not more than 3 times the area of the principal
20.0 mL with the same solvent.
peak in the chromatogram obtained with reference
Reference solution (a) Dilute 5.0 mL of the test solution to solution (a) (0.3 per cent);
50.0 mL with acetonitrile for chromatography R. Dilute 1.0 mL - impurityB: not more than the area of the corresponding
of this solution to 100.0 mL with acetonitrile for peak in the chromatogram obtained with reference
chromatography R. solution (c) (0.2 per cent);
Reference solution (b) Dissolve 5.0 mg of acemetacin - impurityA: not more than the area of the corresponding
impurity A CRS and 10.0 mg ofindometacin CRS peak in the chromatogram obtained with reference
(impurity B) in acetonitrile for chromatography R, and dilute to solution (c) (0.1 per cent);
50.0 mL with the same solvent. - impurities C, D, F: for each impurity, not more than the
Reference solution (c) Dilute 1.0 mL of reference solution (b) area of the principal peak in the chromatogram obtained
to 20.0 mL with acetonitrile for chromatography R. . with reference solution (a) (0.1 per cent);
Reference solution (d) To 1 mL of reference solution (b), - unspecified impurities: for each impurity, not more than the
add 10 mL of the test solution and dilute to 20 mL with . area of the principal peak in the chromatogram obtained
acetonitrile for chromatography R. with reference solution (a) (0.10 per cent);
- total: not more than 4 times the area of the principal peak
Reference solution (e) Dissolve the contents of a vial of
in the chromatogram obtained with reference solution (a)
acemetacin impurity mixture CRS (containing impurities C, D,
(0.4 per cent);
E and F) in 1.0 mL of the test solution. .
- disregard limit: 0.5 times the area of the principal peak in
Column: the chromatogram obtained with reference solution (a)
- size: l = 0.25 m, 0 = 4 mm; (0.05 per cent).
- stationary phase: spherical end-capped octadecylsilyl silica gel
Loss on drying (2.2.32)
for chromatography R (5 urn);
Maximum 0.5 per cent, determined on 1.000 g by drying in
- temperature: 40°C.
an oven at 105°C.
Mobile phase:
- mobile phaseA: dissolve 1.0 g of potassium dihydrogen Sulfated ash (2.4.14)
phosphate R in 900 mL of waterR, adjust to pH 6.5 with Maximum 0.1 per cent, determined on 1.0 g.
1 M sodium hydroxide and dilute to 1000 mL with ASSAY
water R; Dissolve 0.350 gin 20 mLof acetone R and add 10 mL of
- mobile phase B: acetonimle for chromatography R; water R. Titrate with 0.1 M sodium hydroxide, determining the
end-point potentiometrically (2.2.20).
Time Mobile phase A Mobile phase B 1 mL of 0.1 M sodium hydroxide is equivalent to 41.58 mg
(min) (per cent VIJI) (per cent VIJI)
of C21HlSClN06'
0-5 95 5
5-9 95 -+ 65 5 -+ 35 STORAGE
9 - 16 65 35 Protected from light.
16 - 28 65 -+ 20 35 --+ 80 IMPURITIES
28 - 34 20 80 Specified impurities A, B, C, D, E, F.

Flow rate 1.0 mUmin.

Detection Spectrophotometer at 235 nm.
Injection 20 ilL.
Identification of impurities:
- use the chromatogram supplied with acemetacin
impurity mixture CRS and. the chromatogram obtained A. 4-chlorohenzoic acid,

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 2020 Acenocoumarol I-55

Acenocoumarol

B. [1-(4-cWorobenzoyl)-5-methoxy-2-methylindol-3-yl]acetic
acid (indometacin),
and enantiomer

353.3 152-72-7

Action and use

Vitamin K epoxide reductase inhibitor; oral anticoagulant.
Preparation
Acenocoumarol Tablets
C. [[[1-(3 A-:-dicWorobenzoyl)-5-methoxy-2-methyl-1H-indol-
DEFINITION
3-)71] acetYl]oxy]acetic acid,
Acenocoumarol is (RS)-4-hydroxy-3-(I-p-nitrophenyl-3-
oxobutyl) coumarin. It contains not less than 98.5% and not
more than 100.5% of C l gHlSN 0 6, calculated with reference
to the dried substance.
C~CTERISTICS
An almost white to buff powder.
Practically insoluble in water and in ether, slightly soluble in
ethanol (96%). It dissolves in aqueous solutions of the alkali
hydroxides. It exhibits polymorphism.
D. [[[1-(4-cWorobenzoyl)-6-(I,I-dimethylethyl)-5-methoxy-2- IDENTIFICATION
methyl-lH-indol-3-yl] acetyl]oxy]acetic acid, The infrared absorption spectrum, Appendix II A, is concordant
with the reference spectrum of acenocoumarol (RS 001). If the
spectra are not concordant, dissolve 0.1 g of the substance
being examined in 10 mL of acetone and add water drop wise
until the solution becomes turbid. Heat on a water bath until
the solution is clear and allow to stand. Filter, wash the
crystals with a mixture of equal volumes of acetone and water
and dry at 100° at a pressure of 2 kPa for 30 minutes.
Prepare a new spectrum of the residue.

E. 1, l-dimethylethyl [[[1-(4-chlorobenzoyl)-5-methoxy-2- TESTS

methyi-1H-indol-3-yl] acetyl]oxy]acetate, Clarity and colour of solution
A. A 2.0% w/v solution in acetone is clear, Appendix IV A.
B. The absorbance of a 4-cm layer of a 2.0% w/v solution in
acetone at 460 nm is not more than 0.12, Appendix II B.
C. A 2.0% w/v solution in O.IM sodium hydroxide is clear,
Appendix IV A, and yellow.
Light absorption
Absorbance of a 0.001 % w/v solution in a mixture of
1 volume of 1M hydrochloric acid and 9 volumes of methanol at
F. [[[[[I-(4-chlorobenzoyl)-5-methoxy-2-methyl-lH-indol-3- the maximum at 306 nm, 0.50 to 0.54, calculated with
yl]acetyl] oxy]acetyl]oxy]acetic acid. reference to the dried substance, Appendix II B.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur Related substances
Carry out the method for thin-layer chromatography,
Appendix ill A, using the following solutions in acetone.
(1) 2.0% w/v of the substance being examined.
(2) 0.0020% w/v of the substance being examined.
CHROMATOGRAPHIC CONDITIONS
(a) Use as the coating silica gel GF254 •
(b) Use the mobile phase as described below.
(c) Apply 20 J.LL of each solution.
(d) Develop the plate to 15 cm.
(e) After removal of the .plate, allow it to dry in air and
immediately examine under ultraviolet light (254 nm).

!
I
I

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I-56 Acesulfame Potassium 2020

MOBILE PHASE Reference solution (b) Dissolve 5 mg of acesulfame

20 volumes of glacial acetic acid, 50 volumes of cyclohexane potassium CRS· and 5 mg of saccharin sodium R in water Rand
and 50 volumes of dichloromethane. dilute to 5 mL with the same solvent.
LIMITS Plate cellulose for chromatography R as the coating substance.
Any secondary spot in the chromatogram obtained with Mobile phase. concentrated ammonia R, acetone R, ethyl
solution (1) is not more intense than the spot in the acetate R (10:60:60 ·V/V/V).
chromatogram obtained with solution (2) (0.1 %). Application 5 ul, as bands.
Loss on drying Development Twice over 2/3 of the plate.
When dried to constant weight at 105°, loses not more than Drying In a current of warm air.
0.5 % of its' weight. Use 1 g. Detection Examine in ultraviolet light at 254 nm.
Sulfated ash System suitability Reference solution (b):
Not more than 0.1 %, Appendix IX A. - the chromatogram shows 2 clearly separated zones.
ASSAY Results The principal zone in the chromatogram obtained
Dissolve 0.6 g in 50 mL of acetone and titrate with O.IM with the test solution is similar in position and size to the
sodium hydroxide VS using bromothymol blue solution R3 as principal zone in the chromatogram obtained with reference
indicator. Repeat the operation without the substance being solution (a).
examined. The difference between the titrations represents C. 0.5 mL of solution S (see Tests) gives reaction (b) of
the amount of sodium hydroxide required. Each mL of 0.1M potassium (2.3.1).
sodium hydroxide VS is equivalent to 35.33 mg of
TESTS
C19H15N06' Solution S
Dissolve 10.0 g in carbon dioxide-free water R and dilute to
50 mL with the same solvent.

Acesulfame Potassium Appearanceofsmution

Solution S is clear (2.2.1) and colourless (2.2.2, Method II).
(Ph. Bur. monograph 1282) Acidity or alkalinity
To 20 mL of solution S add 0.1 mL of bromothymol blue
solution Rl. Not more than 0.2 mL of 0.01 M hydrochloric
acid or 0.01 M sodium hydroxide is required to change the
colour of the indicator.
Impurity A
Thin-layer chromatography (2.2.27).
Test solution Dissolve 0.80 g of the substance to be
201.2 55589-62-3 examined in water R and dilute to 10 mL with the same
solvent.
Action and use
Reference solution (aJ Dissolve 50 mg of acetylacetamide R
Sweetening agent.
(impurity A) in water R and dilute to 25 mL with the same
PhEur _ solvent. To 5 mL of the solution add 45 mL of water Rand
dilute to 100 mL with methanol R.
DEFINITION
Reference solution (b) To 10 mL of reference solution (a)
Potassium 6-methyl-1,2,3-oxathiazin-4-olate 2,2-dioxide.
add 1 mL of the test solution and dilute to 20 mL with
Content methanol R.
99.0 per cent to 101.0 per cent (dried substance).
Plate TLC silica gel plate R.
CHARACTERS Mobile phase water R, ethanol (96 per cent) R, ethyl acetate R
Appearance (2:15:74 V/V/V).
White or almost white, crystalline powder or colourless
Application 5~.
crystals.
Development Over 2/3 of the plate.
Solubility
Drying In air until the solvents are completely removed.
Soluble in water, very slightly soluble in acetone and in
ethanol (96 per cent). Detection Spray with phosphoricvanillin solution R and heat
at 120°C for about 10 min; examine in daylight.
IDENTIFICATION
System suitability The chromatogram obtained with
First identification: A, C.
reference solution (a) shows a clearly visible spot and the
Second identification: B, C. chromatogram obtainedwith reference solution (b) shows
A. Infrared absorption spectrophotometry (2.2.24). 2 clearly separated spots.
Comparison acesulfame potassium CRS. Limit:
B. Thin-layer chromatography (2.2.27). - impurity A: any spot due to impurity A is not more
Test solution Dissolve 5 mg of the substance to be examined intense than the spot in the chromatogram obtained with
in water R and dilute to 5 mL with the same solvent. reference solution (a) (0.125 per cent).
Reference solution (a) Dissolve 5 mg of acesulfame ImpurityB
potassium CRS in water R and dilute to 5 mL with the same Liquid chromatography (2.2.29).
solvent.

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\
2020 Acetazolamide I-57

Test solution Dissolve 0.100 g of the substance to be

examined in waterR and dilute to 10.0 mL with the same
solvent.
Reference solution (a) Dissolve 4.0 mg of acesulfame potassium
impurity B CRS in water R and dilute to 100.0 mL with the
A. 3-oxobutanamide (acetylacetamide),
same solvent. Dilute 1.0 mL ofthe solution to 200.0 mL
with water R. o
Reference solution (b) Dissolve 0.100 g of the substance to
be examined in reference solution (a) and dilute to 10.0 mL
with the same solution.
:~X;i~o
o
Column:
- size: 1= 0.25 m, 0 = 4.6 mm; B. 5-chloro-6-methyl-l,2,3-oxathiazin-4(3H)-one 2,2-dioxide.
- stationary phase: octadecylsilyl silica gelfor chromatography R _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
(3 um).
Mobile phase Mix 40 volumes of acetonitrile R and
60 volumes of a 3.3 gIL solution of tetrabutylammonium
hydrogen sulfateR.
Flow rate 1·. mIJrnin.
Acetazolamide
Detection Spectrophotometer at 234 nm. (ph. Bur. monograph 0454)
Injection 20 J.lL.
Run time Twice the retention time of acesulfame.
Relative retention With reference to acesulfame (retention
time = about 5.3 min): impurity B = about 1.6.
System suitability:
222.2 59-66-5
- signal-to-noiseratio: minimum 10 for the peak due to
impurity B in the chromatogram obtained with reference Action and use
solution (a); Carbonic arihydrase inhibitor; diuretic; treatment of
- peak-to-valley ratio: minimum 1.2,·where Hp = height glaucoma and ocular hypertension; treatment of mountain
above the baseline of the peak due to impurity B and sickness.
=
H; height above the baseline of the lowest point of the
Preparation
curve separating this peak from the peak due to
Acetazolamide Tablets
acesulfame, in the chromatogram obtained with reference
solution (b). PhEur _

Limit:
DEFINITION
- impurity B: not more than the area ofthe principal peak in
N-(5-Sulfamoyl-l,3,4-thiadiazol-2-yl) acetamide.
the chromatogram obtained with reference solution (a)
(20 ppm). Content
98.5 per cent to 101.0 per cent (driedsubstance).
Fluorides
Maximum 3 ppm. CHARACTERS
Potentiometrytz.z.Je, Method 1). Appearance
White or almost white, crystalline powder.
Test solution Dissolve 3.000 g of the substance tobe
examined in distilled water R, add 15.0 mL of total-ionic- Solubility
strength-adjustment bufferRl and dilute to 50.0 mL with Very slightly soluble in water, slightly soluble in ethanol
distilled water R. (96 per cent). It dissolves in dilute solutions of alkali
Reference solutions To 0.5 mL, 1.0 mL, 1.5 mL and 3.0 mL hydroxides.
of fluoride standardsolution (10 ppm F) R add 15.0 mL of It shows polymorphism (5.9).
total-ionic-strength-adjustment buffer Rl and dilute to 50.0 mL IDENTIFICATION
with distilled waterR. First identification: A." B.
Indicator electrode Fluoride-selective. Secondidentification: A." C, D.
Reference electrode Silver-silver chloride. A. Ultraviolet and visible absorption spectrophotometry
Loss on drying (2.2.32) (2.2.25).
Maximum 1.0 per cent, determined on 1.000 g by drying in Solution A Dissolve 30;Omg in 0.01 M sodium hydroxide and
an oven at 105°C for 3 h. dilute to 100.0 mL with the same solvent. Dilute 10.0 mL of
ASSAY the solution to 100.0 mL with 0.01 M sodium hydroxide.
Dissolve 0.150 gin 50 mL of anhydrous acetic acid R. Titrate Solution B Dilute 25.0 mL of solution A to 100.0 mL with
with 0.1 M perchloric acid, determining the end-point 0.01 M sodium hydroxide.
potentiometrically (2.2.20). Spectral range 230-260 rim for solution A; 260-350 nm for
1 mL of 0.1 M perchloric acid is equivalent to 20.12 mg solution B.
of C 4 f4KN0 4S. Absorption maximum At 240 om for solution A; at 292 nm-
IMPURITIES for solution B. '
Specified impurities A."B. Specific absorbance at the absorption maximum' 162 to 176 for
solution A; 570 to 620 for solution B.

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I-58 Acetazolamide 2020

B. Infrared absorption spectrophotometry (2.2.24). chromatogram obtained with reference solution (a) -
Comparison acetazolamide CRS. (0.15 per cent);
If the spectra obtained in the solid state show differences,
- unspecified impurities: for each impurity, not more than the
area of the principal peak in the chromatogram obtained
dissolve the substance to be examined and the reference
with reference solution (a) (0.10 per cent);
substance separately in ethanol (96 per cent) R, evaporate to
dryness and record new spectra using the residues. - total: not more than 6 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
C. Introduce about 20 mg into a test-tube and add 4 mL of (0.6 per cent);
dilute hydrochloric acid Rand 0.2 g of zinc powder R. - disregard limit: 0.5 times the area of the principal peak in
Immediately place a piece of lead acetate paperR over the the chromatogram obtained with reference solution (a)
mouth of the tube. The paper shows a brownish-black (0.05 per cent).
colour.
Sulfates (2.4.13)
D. Dissolve about 25 mg in a mixture of 0.1 mL of dilute
Maximum 500 ppm.
sodium hydroxide solution Rand 5 mL of waterR. Add 0.1 mL
of copper sulfatesolution R. A greenish-blue precipitate is To 0.4 g add 20 mL of distilled waterR and dissolve by
formed. heating to boiling. Allow to cool with frequent shaking and
filter.
TESTS
Loss on drying (2.2.32)
Appearance of solution
Maximum 0.5 per cent, determined on 1.000 g by drying in
The solution is not more opalescent than reference
an oven at 105°C.
suspension II (2.2.1) and not more intensely coloured than
reference solution v, or BYs (2.2.2~ Method II). Sulfated ash (2.4.14)
Maximum 0.1 per cent, determined on 1.0 g.
Dissolve 1.0 g in 10 mL of 1 M sodium hydroxide.
Related substances ASSAY
Liquid chromatography (2.2.29). Dissolve 0.200 gin 25 mL of dimethylformamide R. Titrate
with 0.1 M ethanolic sodium hydroxide, determining the
Test solution Dissolve 40 mg of the substance to be
end-point potentiometrically (2.2.20).
examined in the mobile phase and dilute to 100.0 mL with
the mobile phase. 1 mL of 0.1 M ethanolic sodium hydroxide is equivalent to
Reference solution (a) Dilute 1.0 mL of the test solution to 22.22 mg of C4H6N403S2'
100.0 mL with the mobile phase. Dilute 1.0 mL of this IMPURITIES
solution to 10.0 mL with the mobile phase. Specified impurities A~ B~ C~ D~ E~ F.
Reference solution (b) Dissolve the contents of a vial of Otherdetectable impurities (the following substances toould; .if
acetazolamide for system suitability CRS (containing present at a sufficient leoel, be detected by one or otherof the tests
impurities A, B, C, D~ E and F) in 1.0 mL of the mobile in the monograph. They are limitedby the general acceptance
phase. criterion for other/unspecified impurities and/orby the general
Column: monograph Substances for pharmaceutical use (2034). It is
- size: 1= 0.15 m, 0 = 4.6 mID; therefore not necessary to identify these impurities for
- stationary phase: end-capped propoxybenzene silica gelfor demonstration of compliance. See also 5.10. Control of impurities
chromatography R (4 urn). in substances for pharmaceutical use) G.
Mobzle phase acetonitrile for chromatography R, 6.8 gIL
solution of potassium dihydrogen phosphate R (10:90 VIV)o
Flow rate 1.0 mL/min.
Detection Spectrophotometer at 265 om.
Injection 25 t-tl. A. N-(5-chloro-1,3,4-thiadiazol-2-yl)acetamide,
Run time 3.5 times the retention time of acetazolamide.
Identification of impurities Use the chromatogram supplied
with acetazolamide for systemsuitability CRS and the
chromatogram obtained with reference solution (b) to
identify the peaks due to impurities A~ B, C, D, E and F.
B. N-(1,3,4-thiadiazol-2-y1)acetamide,
Relativeretention With reference to acetazolamide (retention
time = about 8 min): impurity E = about 0.3;
impurity D = about 0.4; impurity B = about 0.6;
impurity C = about 1.4; impurity A = about 2.1;
impurity F = about 2.6.
System suitability Reference solution (b):
C. N-(5-sulfanyl-1,3,4:-thiadiazol-2-yl)acetamide,
- resolution: minimum 2.0 between the peaks due to
impurities E and D.
Limits:
- correction factors: for the calculation of content, multiply
the peak areas of the following impurities by the
corresponding correction factor: impurity B = 2.3;
impurity C = 2.6; impurity D = 1.6; D. 5-amino-1 ,3,4-thiadiazole-2-sulfonamide,
- impurities A~ B~ C~ D~ E~ F: for each impurity, not more
than 1.5 times the area of the principal peak in the

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2020 Acetic Acid I-59

Iron (2.4.9)
Maximum 5 ppm.
Dissolve the residue obtained in the test for residue on
evaporation by heating with 2 quantities, each of 15 mL, of
E. 5-acetamido-1,3,4-thiadiazole-2-sulfonic acid, waterR and dilute to 50.0 mL with waterR. Dilute 5.0 mL
of the solution to 10.0 mL with waterR.
Residue on evaporation
Maximum 0.01 per cent.
Evaporate 20 g to dryness on a water-bath and dry at
F. N- [5-[ (5-acetamido-1,3,4-thiadiazol-2-yl) 100-105 "C. The residue weighs a maximum of 2.0 mg.
sulfonyl] sulfamoyl-1,3,4-thiadiazol-2-yl] acetamide, ASSAY
Weigh accurately a conical flask with a ground-glass stopper
containing 25 mL of water R. Add 1.0 mL of the substance
to be examined and weigh again accurately. Add 0.5 mL of
phenolphthalein solution R and titrate with 1 M sodium
G. 5-amino-1,3,4-thiadiazole-2-thiol. hydroxide.
_-'-- Phfur 1 mL of 1 M sodium hydroxide is equivalent to 60.1 mg
ofC2~02'
STORAGE
In an airtight container.
Glacial Acetic Acid _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur

(Ph~ Bur. monograph 0590)

Acetic Acid (6 per cent)

Dilute Acetic Acid
DEFINITION
CZH40Z 60.1 64-19-7 Acetic Acid (6 per cent) contains not less than 5.7% and not
PhEur --:;- _
more than 6.3% w/w of acetic acid, C ZH40Z ' It may be
DEFINITION prepared by mixing 182 g of Acetic Acid (33 per cent) with
Content 818 g of Purified Water.
99.0 per cent mlm to 100.5 per cent mlm. IDENTIFICATION
CHARACTERS A. Strongly acidic.
Appearance B. When neutralised, yields the reactions characteristic of
Crystalline mass or clear, colourless, volatile liquid. acetates, Appendix VI.
Solubility TESTS
Miscible with water, with ethanol (96 per cent) and with Weight per mL
methylene chloride. About 1.005 g, Appendix V G.
IDENTIFICATION. Chloride
A. A 100 gIL solution is strongly acid (2.2.4). Dilute 5.0 mL with sufficient water to produce 100 mL.
B. To 0.03 mL add 3 mL of waterR and neutralise with 15 mL of the resulting solution complies with the limit testfor
dilute sodium hydroxide solution R. The solution gives chlorides, Appendix VII (70 ppm).
reaction (b) of acetates (2.3.1). Sulfate
12.5 mL of the solution used in the testfor Chloride, diluted
TESTS
to 15 mL with water, complies with the limit testfor sulfates,
Solution S Appendix VII (240 ppm).
Dilute 20 mL to 100 mL with distilled waterR..
Aldehydes
Appearance
Distil 75 mL. To the first 5 mL of the distillate add 10 mL
The substance to be examined is clear (2.2.1) and colourless
of a 5% w/v solution of mercury(n) chloride, make alkaline
(2.2.2, Method II).
with 5M sodium hydroxide, allow to stand for 5 minutes and
Freezing point (2.2.18) acidify with 1M sulfuric acid. The .solution shows not more
Minimum 14.8 -c. than a faint turbidity.
Reducing substances Formic acid and oxidisable impurities
Dilute 2.0 mL to 10.0 mL with waterR. Add 0.1 mL of Mix 5 mL with 6 mL of sulfuric acid and cool to 20 e •
0.02 M potassium permanganate. Heat on a water-bath for Add 0.4 mL ofO.0167Mpotassium dichromate VS, allow to
1 min, the colour remains pink. stand for 1 minute, add 25 mL of water and 1 mL of freshly
Chlorides (2.4.4) prepared dilute potassium iodide solution and titrate the
Maximum 25 mgIL. liberated iodine with O.IM sodium thiosulfate VS using starch
Dilute 10 mL of solution S to 15 mL with waterR. mucilage as indicator. Not less than 0,2 mL of 0.1M sodium
thiosulfate VS is required.
Sulfates (2.4.13)
Maximum 50 mgIL, determined on solution S.

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1-60 Acetic Acid 2020

Readily oxidisable impurities ASSAY

To 25 mL add 0.2 mL of 0.02M potassium permanganate VS Weigh 5 g into a stopper flask containing 50 mL of water and
and allow to stand for 1 minute. The pink colour is not titrate with 1M sodium hydroxide VS using phenolphthalein
entirely discharged. solution R1 as indicator. Each mL of 1M sodium hydroxide VS
Non-volatile matter is equivalent to 60.05 mg of C ZH40Z '
When evaporated to dryness and dried at 105°, leaves not
more than 0.01% w/w of residue.
ASSAY
Add 30 mL of water to 20 g in a stopper flask and titrate
Acetone
with 1M sodium hydroxide VS using phenolphthalein solution R1 (Ph. Bur. monograph 0872)
as indicator. Each mL of 1M sodium hydroxide VS is
equivalent to 60.05 mg of C ZH40Z'

Acetic Acid (33 per cent) 58.08 67-64-1

Acetic Acid PhEur ~ _

Preparation DEFINITION
Acetic Acid (6 per cent) Propanone.
DEFINITION CHARACTERS
Acetic Acid (33 per cent) contains not less than 32.5% and Appearance
not more than 33.5% w/w of acetic acid, C ZH40Z ' Volatile, clear, colourless liquid.
CHARACTERISTICS Solubility
A clear, colourless liquid. Miscible with water and with ethanol (96 per cent).
Miscible with water, with ethanol (96%) and with glycerol. The vapour is flammable.
IDENTIFICATION IDENTIFICATION
A. Strongly acidic, even when diluted freely. A. Relative density (see Tests).
B. When neutralised, yields the reactions characteristic of B. To 1 mL, add 3 mL of dilute sodium hydroxide solution R
acetates, Appendix VI. and 0.3 mL of a 25 gIL solution of sodium nitroprusside R.
An intense red colour is produced which becomes violet with
TESTS
the addition of 3.5 mL of acetic acid R.
Weight per mL
1.040 to 1.042 g, Appendix V G. C. To 10 mL of a 0.1 per cent V/V solution of the substance
to be examined in ethanol (50 per cent V/V? R, add 1 mL of a
Chloride
10 gIL solution of nitrobenzaldehyde R in ethanol
Dilute 5.0 mL with sufficient water to produce 100 mL.
(50 per cent V/V? Rand 0.5 mL of strong sodium hydroxide
15 mL of the resulting solution complies with the limit testfor
solution R. Allow to stand for about 2 min and acidify with
chlorides, Appendix VII (70 ppm).
acetic acid R. A greenish-blue colour is produced.
Sulfate
TESTS
12.5 mL of the solution used in the test for Chloride, diluted
to 15 mL with water, complies with the limit testfor sulfates, Appearance of solution
Appendix VII (240ppm). To 10 mL add 10 mL of water R. The solution is clear
(2.2.1) and colourless (2.2.2~ Method II).
Aldehydes
DistillS mL. To the first 5 mL of the distillate add 10 mL Acidity or alkalinity
of a 5% w/v solution of mercury (II) chloride, make alkaline To 5 mL add 5 mL of carbon dioxide-free water R, 0.15·mL of
with 5M sodium hydroxide, allow to stand for 5 minutes and phenolphthalein solution Rand 0.5 mL of 0.01 M sodium
make acidic with 1M sulfuric acid. The solution shows not hydroxide. The solution is pink. Add 0.7 mL of 0.01 M
more than a faint turbidity. hydrochloric acid and 0.05 mL of methyl red solution R.
The solution is red or orange.
Formic acid and oxidisable impurities
Mix 5 mL with () mL of sulfuric acid and cool to 20°. Relative density (2.2.5)
Add 2 mL of 0.0167M potassium dichromate VS, allow to 0.790 to 0.793.
stand for 1 minute, add 25 mL of water and 1 mL of freshly Reducing substances
prepared dilute potassium iodide solution and titrate the To 30 mL add 0.1 mL of 0.02 M potassiumpermanganate
liberated iodine with O.IM sodium thiosulfate VSusing starch and allow to stand in the dark for 2 h. The mixture is not
mucilage as indicator. Not less than 1.0 mL of O.IM sodium completely decolourised.
thiosulfate VS is required. Related substances
Readily oxidisable impurities Gas chromatography (2.2.28).
To 5.0 mL add 20 mL of water and 0.2 mL of Test solution The substance to be examined.
0.02M potassium permanganate VS and allow to stand for Reference solution (a) To O.5mL of methanolR add 0.5 mL
1 minute. The pink colour is not entirely discharged. of 2-propanol R and dilute to 100.0 mL with the test solution.
Non-volatile matter Dilute 1.0 mL of this solution to 10.0 mL with the test
When evaporated to dryness and dried at 105°, leaves not solution.
more than 0.01 % w/w of residue.

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2020 Acetylcholine Chloride 1-61

Reference solution (b) .Dilute 100 ,.tL of benzene R to

100.0 mL with the test solution. Dilute 0.20 mL of this
solution to 100.0 mL with the test solution. A. methanol;
Column:
- material: fused silica,
- size: l = 50 m, 0 = 0.3 mm,
- stationary phase: macrogol20 OOOR (film thickness 1 lJID).
Carrier gas helium for chromatography R. B. propan-Z-ol (isopropanol),
Linear velocity 21 cm/s.
Split ratio 1:50.
Temperature:
C. benzene.
o
Time Temperature
(min) CC) PhEur
Column 0-11 45 -> 100
11 - 20 100
Injection port 150
Detector 250 Acetylcholine Chloride
Detection Flame ionisation. (Ph. Eur. monograph 1485)
Injection 1 ut,
=
Retentiontime Impurity C about 7.5 min,
System suitability:
- resolution: minimum 5.0 between the peak due to
impurity A (2n d peak) and the peak due to impurity B 181.7 60-31-1
(3 rd peak) in the chromatogram obtained with reference
Action and use
solution (a);
Cholinoceptor agonist.
- signal-to-noise ratio: minimum 5 for the peak due to
impurity C in the chromatogram obtained with reference PhEur _
solution (b).
DEFINITION
Limits:
2-(Acetyloxy)-N,N,N-trimethylethanaminium chloride.
- impurities A., B: for each impurity; not more than the
difference between the areas of the corresponding peaks in Content
the chromatogram obtained with reference solution (a) 98.5 per cent to 101.5 per cent (dried substance).
and the areas of the corresponding peaks in the CHARACTERS
chromatogram obtained with the test solution Appearance
(0.05 per cent VIV), White or almost white crystalline powder or colourless
- impurity C: not more than the difference between the area crystals, very hygroscopic.
of the peak due to impurity C in the chromatogram
Solubility
obtained with reference solution (b) and the area of the
Very soluble in water, freely soluble in alcohol, slightly
corresponding peak in the chromatogram obtained with
soluble in methylene chloride.
the test solution (2 ppm VIV),
- any other impurity: for each impurity, not more than the IDENTIFICATION
difference between the area of the peak due to impurity A Firstidentification: B., E.
in the chromatogram obtained with reference solution (a) Second identification: A., C, D, E.
and the area of the corresponding peak in the
A. Melting point (2.2.14): 149°C to 152 °C.
chromatogram obtained with the test solution
(0.05 per cent VIV). Introduce the substance to be examined into a capillary tube.
Dry in an oven at 100-105 °C for 3 h. Seal the tube and
Matter insoluble in water determine the melting point.
Dilute 1.0 mL to 20 mL with waterR. The solution is clear
B. Infrared absorption spectrophotometry (2.2.24).
(2.2.1).
Comparison acetylcholine chloride CRS.
Residue on evaporation
Maximum 50 ppm. C. Examine the chromatograms obtained in the test for
related substances.
Evaporate 20.0 g to dryness on a water-bath and dry at
100-105 "C. The residue weighs a maximum of 1 mg. Results The principal zone in the chromatogram obtained
with test solution (b) is similar in position, colour and size to
Water (2.5.12) the principal zone in the chromatogram obtained with
Maximum 3 gIL, determined on 10.0 mL. reference solution (b).
STORAGE D. To 15 mg add 10 mL of dilute sodium hydroxide solution R,
Protected from light. 2 mL of 0.02 M potassium permanganate and heat.
IMPURITIES The vapours formed change the colour of redlitmuspaperR
Specifiedimpurities A., B., C. to blue.
E. 0.5 mL of solution S (see Tests) gives reaction (a) of
chlorides (2.3.1).

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1-62 Acetylcysteine 2020

TESTS STORAGE
Solution S In ampoules, protected from light.
Dissolve 5.0 g in carbon dioxide-free waterR and dilute to IMPURITIES
50 mL with the same solvent.
Appearance of solution H3C,+,cH3
Solution S is clear (2.2.1) and not more intensely coloured HO~N'CH3 CI
than reference solution Y6 or BY6 (2.2.2) Method II).
Acidity A. 2-hydroxy-N,N,N-trimethylethanaminium chloride
Dilute 1 mL of solution S to 10 mL with carbon dioxide-free (choline chloride),
Water R. Add 0.05 mL of phenolphthalein solution R. Not more
than 0.4 mL of 0.01 M sodium hydroxide is required to
change the colour of the indicator to pink.
Related substances
Thin-layer chromatography (2.2.27). Prepare the solutions B. 2-(acetyloxy)-N,N-dimethylethanaminium chloride,
immediately before use.
Testsolution (a) Dissolve 0.30 g of the substance to be
examined in methanol R and dilute to 3.0 mL with the same
solvent.
Testsolution (b) Dilute 1 mL oftest solution (a) to 10 mL
C. N,N-dimethyhnethanamine.
with methanolR.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
Reference solution (a) Dilute 1 mL of test solution (a) to
100 mL with methanol R.
Reference solution (b) Dissolve 20.0 mg of acetylcholine
chloride CRS in methanol R and dilute to 2.0 mL with the
same solvent.
Acetylcysteine
Reference solution (c) Dissolve 20 mg of choline chloride R in (Ph. Bur. monograph 0967)
methanolR, add 0.4 mL of test solution (a) and dilute to
2.0 mL with methanol R.
Plate TLC silica gel plate R.
Mobilephase Mix 20 volumes of a 40 gIL solution of
ammonium nitrate R, 20 volumes of methanol Rand
60 volumes of acetonitrile R.
Application 5 ~ as bands of 10 rom by 2 rom. 163.2 616-91-1
Development Over 2/3 of the plate.
Action and use
Detection Spray with potassium iodobismuthate solution R3.
Sulfydryl donor; antidote to paracetamol poisoning;
System suitability The chromatogram obtained with . mucolytic.
reference solution (c) shows 2 clearly separated zones.
Preparations
Limits:
Acetylcysteine eye drops
- any impurity: any zones in the chromatogram obtained
with test solution (a), apart from the principal zone, are Acetylcysteine Injection
not more intense than the principal zone in the PhEur ~ __
chromatogram obtained with reference solution (a)
(1 per cent). DEFINITION
(2R)-2-(Acetylamino)-3-sulfanylpropanoic acid.
Trimethylamine
Dissolve 0.1 g in 10 mL of sodium carbonate solution R and Content
heat to boiling. No vapours appear which turn red litmus 98.0 per cent to 101.0 per cent (dried substance).
paper R blue. CHARACTERS
Loss on drying (2.2.32) Appearance
M.aximum 1.0 per cent, determined on 1.000 g by drying in White or almost white, crystalline powder or colourless
an oven at 105 DC for 3 h. crystals. .
Sulfated ash (2.4.14) Solubility
Maximum 0.1 per cent, determined on the residue obtained Freely soluble in water and in ethanol (96 per cent),
in the test for loss on drying. practically insoluble in methylene chloride.
ASSAY IDENTIFICATION
Dissolve 0.200 gin 20 mL of carbon dioxide-free water R. First identification: A) C.
Neutralise with 0.01 M sodium hydroxide using 0.15 mL of / Secondidentification: A) B) D) B.
phenolphthalein solution R as indicator. Add 20.0 mL of 0.1 M A. Specific optical rotation (see Tests).
sodium hydroxide and allow to stand for 30 min. Titrate with
B. Melting point (2.2.14): 104 DC to 110 DC.
0.1 M hydrochloric acid.
C. Infrared absorption spectrophotometry (2.2.24).
1 mL of 0.1 M sodium hydroxide is equivalent to 18.17 mg of
C 7H16ClNOZ' Preparation Discs of potassium bromide R.
Comparison acetylcysteine CRS.

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2020 Acetylcysteine 1-63

D. Examine the chromatograms obtained in the test for System suitability Reference solution (a):
related substances. - resolution: minimum 1.5 between the peaks due to
Results The principal peak in the chromatogram obtained impurities A and B and minimum 2.0 between the peaks
with test solution (b) is similar in retention time and size to due to impurities C and D.
the principal peak in the chromatogram obtained with From the chromatogram obtained with test solution (a),
reference solution (b). calculate the percentage content of the known impurities
E. To 0.5 mL of solution S (see Tests) add 0.05 mL of a (T 1) and the unknown impurities (T2) using the following
50 gIL solution of sodium nitroprusside Rand 0.05 mL of equations:
concentrated ammonia R. A dark violet colour develops.
T 1 = A 1 X m2 x 100
TESTS A2 x ml
Solution S
Dissolve 1.0 g in carbon dioxide-free water R and dilute to
T = A 3 x m3 x 100
20 mL with the same solvent. z
A4 x ml
Appearance of solution
Al peak area of individual impurity (impurity A, impurity B,
Solution S is clear (2.2.1) and colourless (2.2.2, Method II).
impurity C and impurity D) in the chromatogram obtained with
pH (2.2.3) test solution (a);
2.0 to 2.8. A2 peak area of the corresponding individual impurity (impurity A,
impurity B, impurity C and impurity D) in the chromatogram
To 2 mL of solution S add 8 mL of carbon dioxide-free obtained with reference solution (a);
waterR and. mix. A3 peak area of unknown impurity in the chromatogram obtained
with test solution (a);
Specific optical rotation (2.2.7) A4 peak area of acetylcysteine in the chromatogram obtained with
+21.0 to + 27.0 (dried substance). reference solution (b);
In a 25 mL volumetric flask, mix 1.25 g with 1 mL of a mI Plass of the substance to be examined in test solution (a);
m2 mass of the individual impurity in reference solution (a);
10 gIL solution of sodium edetate R. Add 7.5 mL of a 40 gIL m3 mass of acetylcysteine in reference solution (b).
solution of sodium hydroxide R, mix and dissolve. Dilute to
25.0 mL with phosphate buffer solution pH 7.0 R2. Limits:
Related substances - impurities A, B, C, D: for each impurity, maximum
Liquid chromatography (2.2.29). Exceptwhere otherwise 0.5 per cent;
prescribed, prepare the solutions. immediately before use. - any otherimpurity: for each impurity, maximum
Test solution (a) Suspend 0.80 g of the substance to be 0.5 per cent;
examined in 1 mL of 1 M hydrochloric acid and dilute to - total: maximum 0.5 per cent;
100.0 mL with water R. - disregard limit: 0.1 times the area of the principal peak in
Test solution (b) Dilute 5.0 mL oftest solution (a) to the chromatogram obtained with reference solution (b)
100.0 mL with water R. Dilute 5.0 mL of this solution to (0.05 per cent); disregard any peak with a retention time
of about 3.3 min due to 2-methyl-2-thiazoline-4-
50.0 mL with water R.
carboxylic acid.
Test solution (c) Use test solution (a) after storage for at
least 1 h. Zinc
Maximum 10 ppm.
Reference solution (a) Suspend 4.0 mg of acetylcysteine CRS,
4.0 mg of L-cystine R (impurity A), 4.0 mg of L-cysteine R Atomic absorption spectrometry (2.2.23, Method II).
(impurity B), 4.0 mg of acetylcysteine impurity C CRS and Test solution Dissolve 1.00 gin 0.001 M hydrochloric acid
4.0 mgof acetylcysteine impurityD CRS in 1 mL of 1M and dilute to 50.0 mL with the same acid.
hydrochloric acid and dilute to 100.0 mL with water R. Reference solutions Prepare the reference solutions using zinc
Reference solution (b) Suspend 4.0 mg of acetylcysteineCRS standardsolution (5 mg/mL Zn) R, diluting with 0.001 M
in 1 mL of 1 M hydrochloric acid and dilute to 100.0 mL with hydrochloric acid.
waterR. Source Zinc hollow-cathode lamp.
Column: Wavelength 213.8 nm.
- size: 1 = 0.25 m, 0 = 4 mm; Atomisation device Air-acetylene flame.
- stationary phase: octadecylsilyl silica gelfor chromatography R
Use a correction procedure for non-specific absorption.
(5 urn).
Loss on drying (2.2.32)
Mobile phase Stir 3 volumes of acetonitrile Rand 97 volumes
Maximum 1.0 per cent, determined on 1.000 g by drying in
of waterR in a beaker; adjust to pH 3.0 with phosphoric
an oven in vacuo at 70°C for 3 h.
acid R.
Flow rate 1.0 mUmin. Sulfated ash (2.4.14)
Maximum 0.2 per cent, determined on 1.0 g.
Detection Spectrophotometer at 220 nm.
Injection 20 IlL, 3 times; inject O. 01 M hydrochloric acid as a ASSAY
blank. Dissolve 0.140 gin 60 mL of water R and add 10 mL of
dilute hydrochloric acid R. After cooling in iced water, add
Run time 5 times the retention time of acetylcysteine (about
10 mL of potassium iodide solution R and titrate with O. 05 M
30 min).
iodine, using 1 mL of starch solution R as indicator.
Retention time Impurity A = about 2.2 min; impurity B =
1 mL of 0.05 M iodine is equivalent to 16.32 mg of
about 2.4 min; 2-methyl-2-thiazoline-4-carboxylic acid,
CsHgN03S.
=
originating in test solution (c) about 3.3 min;
acetylcysteine = about 6.4 min; impurity C = about 12 min; STORAGE
impurity D· = about 14 min. Protected from light.

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1-64 Acetyldigoxin 2020

IMPURITIES PhEur ---,. _

Specified impurities A J B J C D.
J DEFINITION
3p-[(4-0-Acetyl-2,6-dideoxy-p-n-ribo-hexopyranosyl-(l ~ 4)-
2,6-dideoxy-p-n-ribo-hexopyranosyl-(1 ~4)-2,6-dideoxy-p-n­
ribo-hexopyranosyl)oxy]-12P,14-dihydroxy-5p-card-20(22)-
enolide.
Content
A. 3,3'-disulfanediylbis[(2R)-2-aminopropanoic acid] (L- 97.0 per cent to 102.0 per cent (dried substance).
cystine),
CHARACTERS
H NH2 Appearance
HS0 C02H
White or almost white powder.
Solubility
Practically insoluble in water, sparingly soluble in methylene
B. (2R)-2-amino-3-sulfanylpropanoic acid (t-cysteine), chloride, slightly soluble in ethanol (96 per cent).
IDENTIFICATION
Infrared absorption spectrophotometry (2.2.24).
Comparison fJ-acetyldigoxin CRS.
TESTS
Specific optical rotation (2.2.7)
+ 26.2 to + 28.2 (dried substance).
Dissolve 0.50 g in a mixture of equal volumes of methanol R
and methylene chloride R and dilute to 25.0 mL with the same
C. (2R,2'R)- 3,3'-disulfanediylbis[2-(acetylamino)propanoic
mixture of solvents.
acid] (N,N'-diacetyl-L-cystine),
Related substances
Liquid chromatography (2.2.29). Prepare the solutions
immediately before use.
Solvent mixture Mix equal volumes of methanol R2 and
acetonitrile for chromatography R.
Test solution Dissolve 50.0 mg of the substance to be
examined in the solvent mixture and dilute to 100.0 mL with
D. (2R)-2-(acetylamino)-3-(acetylsulfanyl)propanoic acid (N, the solvent mixture.
S-diacetyl-L-cysteine). Reference solution (a) Dissolve 10.0 mg of
_ _ _ _ _ _ _~ - - - - - - - - - - - -PhEur
fJ-acetyldigoxin CRS in the solvent mixture and dilute to
20.0 mL with the solvent mixture.
Reference solution (b) Dilute 1.0 mL of the test solution to
20.0 mL with the solvent mixture. Dilute 1.0 mL of this
Acetyldigoxin solution to 10.0 mL with the solvent mixture.
Reference solution (c) Dissolve 5 mg of gitoxin CRS
(fJ-Acetyldigoxin, Ph. Bur. monograph 2168) (impurity D) in the solvent mixture and dilute to 100.0 mL
with the solvent mixture. To 5.0 mL of this solution, add
° 0.5 mL of reference solution (a) and dilute to 100.0 mL with
the solvent mixture.
Reference solution (d) Dissolve 5.0 mg of f3-acetyldigoxin for
peak identification CRS (containing impurities A and B) in .
10.0 mL of the solvent mixture.
Column:
- size: 1 = 0.125 m, 0 = 4.0 mm;
- stationary phase: octadecylsilyl silica gelfor chromatography R
(4 urn).
Mobilephase:
- mobile phase A: waterfor chromatography R;
- mobile phase B: acetonitrile for chromatography R;

Time Mobile phase A Mobile phase B

(min) (per cent V/Ii') (per cent VIJI)
0-10 70 30
10 - 29 70 -+ 35 30 -+ 65
823 5355-48-6 20 - 20.1 3S -+ 70 6S -+ 30
20.1 - 25 70 30
Action and use
Cardiac Glycoside.
Flow rate 1.5 mUmin.

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2020 Acetyldigoxin 1-65

o
Detection Spectrophotometer at 225 DIn.
Injection 10 ~ of the test solution and reference
solutions (b), (c) and (d).
Identification of impurities Use the chromatograms obtained
with reference solutions (c) and (d) to identify the peaks due
to impurities A, B and D.
Relative retention With reference to ~-acetyldigoxin
(retention time = about 9 min): impurity B = about 0.3;
impurity A= about 0.7; impurity D= about 1.2.
System suitability Reference solution (c):
- resolution: minimum 1.5 between the peaks due to
~-acetyldigoxin and impurity D;
- symmetry factor. maximum 2.5 for the peak due to
~-acetyldigoxin.
Limits:
- impurities A, B: for each impurity, not more than the area
of the principal peak in the chromatogram obtained with
reference solution (b) (0.5 per cent); A. 3 ~- [(3-0-acetyl-2,6-dideoxy"'~-D-ribo-hexopyranosyl-
- impurity D: not more than 0.6 times the area of the (l ~4)-2,6-dideoXY-~-D-n'bo-hexopyranosyl-(1 ~4)-2,6­
principal peak in the chromatogram obtained with dideoXY-~-D-ribo-hexopyranosyl)oxy] -12~, 14-dihydroxy-

reference solution (b) (0.3 per cent); 5 ~-card- 20(22) -enolide (c-acetyldigoxin),
- any other impurity: for each impurity, not more than
o
OA times the area of the principal peak in the
chromatogram obtained with reference solution (b)
(0.2 per cent);
- sum of impuritiesotherthan A, Band D: not more than
1.2 times the area of the principal peak in the
chromatogram obtained with reference solution (b)
(0.6 per cent);
- total: not more than 3 times the area of the principal peak
in the chromatogram obtained with reference solution (b)
(1.5 per cent);
~ disregard limit: 0.1 times the area of the principal peak in
the chromatogram obtained with reference solution (b)
(0.05 per cent).
The thresholds indicated under Related substances
(Table 2034.-1) in the general monograph Substances for \
pharmaceutical use (2034) do not apply.
Eo 3~-[(2,6-dideoXY-~-D-ribo-hexopyranosyl-(l ~4)-2,6­
Loss on drying (2.2.32) dideoXY-~-D-ribo-hexopyranosyl-( 1-+4)-2, 6-dideoXY-~-D­
Maximum 1.5 per cent, determined on 1.000 g by drying in
ribo-hexopyranosyl)oxy]-12~,14-dihydroxy-5~-card-20(22)­
an oven at 105°C. enolide (digoxin),
Sulfated ash (2.4.14)
Maximum 0.1 per cent, determined on the residue obtained o
in the test for loss on drying.
ASSAY
Liquid chromatography (2.2.29) as described in the test for
related substances with the following modification.
Injection Test solution and reference solution (a).
Calculate the percentage content of C43H66015 from the
declared content of p-acetyldigoxin CRS.
STORAGE C. 3~, 12~, 14-trihydroxy-5 ~-card-20(22)-enolide
Protected from light. (digoxigenin),

IMPURITIES
Specifiedimpurities .A, B, D.
Other detectable impurities (the following substances would, if
present at a sufficient level, be detected by one or other of the tests
in the monograph. They are limited by the general acceptance
criterion for other/unspecified impurities. It is therefore not
necessary to identify these impurities for demonstration of
compliance. See also 5.10. Control of impurities in substances for
pharmflceutical use) C, E, F, G, H.

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1-66 Acetylene Intermix (1 per cent) in Nitrogen 2020

O. 3~-[(2,6-dideoxY-~-D-ribo-hexopyranosyl-(1-+4)-2,6­
dideoXY-~-D-ribo-hexopyranosyl-(1-+4)-2,6-dideoxy-~-D­
ribo-hexopyranosyl)oxy]-14,16~-dihydroxy-5~-card-20(22)­
G. 3P- [(3-0-acetyl-2,6-dideoxy-~-D-ribo-hexopyranosyl­
(1-+4)-2,6-dideoxy-~-D-ribo-hexopyranosyl-(1-+4)-2,6­
enolide (gitoxin),
dideoxy-Bsn-reo-hexopyranosyl) oxy]-14-hydroxy-5 ~-card­
o 20(22)-enolide (ce-acetyldigitoxin),

E. 3~-[(2,6-dideoxy-~-D-n'bo-hexopyranosyl-(1-+4)-2,6­
dideoXY-~-D~noo-hexopyranosy~(1-+4)-2,6-dideoXY-~-D­
ribo-hexopyranosyl)oxy]-14-hydroxy-5~-card-20(22)­
H. 3 ~- [(4-0-acetyl-2,6-dideoxy-~-D-ribo-hexopyranosyl­
enolide (digitoxin), (1-+4)-2,6-dideoxy-~-D-ribo-hexopyranosyl-(1-+4)-2,6­
dideoxy-~:D-ribo-hexopyranosynoxy]-14-hydroxy-5~-card­
o
20(22)-enolide (~-acetyldigitoxin).
_ _ _ _ _ _ _ _'-- :----_ PhEur

Acetylene Intermix (1 per cent) in

"Nitrogen
(Ph. Bur. monograph 2903)
PhEur _

DEFINITION
A mixture containing 1 per cent VIV of acetylene in Low-
oxygen nitrogen (1685).
Content
0.95 per cent VIV to 1.05 per cent VIV of acetylene (CzHz)
in nitrogenCNz).
F. 3 ~- [(3,4-0-diacetyl-2, 6-dideoXY-~-D-ribo-hexopyranosyl­ This monograph applies to acetylene intermix (1 per cent) in
(1-+4)-2,6-dideoxy-~-D-ribo-hexopyranosyl-(1-+4)-2,6­ nitrogen used in the preparation of lung function test gas
dideoXY-~-D-ribo-hexopyranosynoxy]-12~,14-dihydrpxy­ mixtures for medicinal use.
5 ~-card-20(22)-enolide (diacetyldigoxin),
PRODUCTION
The acetylene used in the manufacturing process is produced
by hydrolysis of calcium carbide.

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~--

2020 Acetylene Intermix (1 per cent) in Nitrogen 1-67

Prior to using the gas in the manufacturing process, the Phosphine

acetylene may be passed through an activated charcoal filter. Maximum 0.2 ppm VIl7, determined using a phosphine
The acetylene is stored in cylinders, which may be filled with detector tube (2.1.6).
a porous mass with acetone the only solvent permitted. Hydrogen sulfide
CHARACTERS Maximum 0.2 ppm VIl7, determined using a hydrogen
sulfide detector tube (2.1.6).
Appearance
Colourless gas. Water (2.5.28)
Maximum 10 ppm VIV.
IDENTIFICATION
A. Examine the chromatograms obtained in the assay. ASSAY
Results The peak due to acetylene in the chromatogram Gas chromatography (2.2.28).
obtained with the gas to be examined is similar in retention Gas to beexamined The substance to be examined.
time and size to the peak due to acetylene in the Reference gas Mixture containing 1.0 per cent VIVof
chromatogram obtained with the reference gas. acetylene R in nitrogen Rl.
E. Gas chromatography (2.2.28). Column:
Gas to be examined The substance to be examined. - material: stainless steel;
Reference gas -Nitrogen Rl. = =
- size: I 2 m, (2) 2 mID;
- stationary phase: 3 per cent squalane R on alumina.
Column:
- material: stainless steel; - Carrier gas helium for chromatography R.
- size: I = 2 m, (2) = 2 mm; Flow rate 20 mUmin.
- stationary phase: molecular sieve for chromatography R Temperature:
(0.5 nm). - column: 100°C;
Carrier gas helium for chromatography R. - detector:-250 "C.
Flow rate 20 mUmin. Detection Flame ionisation.
Temperature: Injection 100 ~L.
- column: 80°C; Retention time Acetylene = about 6 min.
-detector. 130°C. Calculate the percentage content of C 2H z.
Detection Thermal conductivity.
STORAGE
Injection 10 J,tL. As a compressed gas, in appropriate high-pressure cylinders
=
Retention time Nitrogen about 2 min. complying with the legal regulations.
Results The principal peak in the chromatogram obtained LABELLING
with the gas to be examined is similar in retention time to The label states the nominal content, in per cent. VIV, of
the principal peak in the chromatogram obtained with the acetylene in nitrogen.
reference gas.
IMPURITIES
TESTS Specified impurities A, B, C, D, E.
Acetone
Gas chromatography (2.2.28).
Gas to be examined The substance to be examined.
Reference gas Mixture containing 250 ppm VIV of acetone R
in nitrogen R.
A. propan-z-one (acetone),
Column:
- material: fused silica;
- size: 1= 10 m, (2) = 0.53 IDm;
- stationary phase: polyorganosiloxane for oxygen-containing
compounds R (film thickness 10 urn).
Carrier gas helium for chromatography R. B. arsane (arsine),
Flow rate 50 mUmin.
Temperature:
- column: 200°C;
- injection port: 240 DC;
- detector. 250°C. C. phosphane (phosphine),
Detection Flame ionisation.
Injection 25 J,tL.
Retention time Acetone = about 1 min.
Calculation of percentage content: D. hydrogen sulfide,
- use the concentration of acetone in the reference gas.
Limit:
- acetone: maximum 250 ppm VIV.
Arsine E. water.
Maximum 0.25 ppm VIl7,determined using an arsine _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur

detector tube (2.1.6).

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1-68 Acetyltryptophan 2020

E ..It gives the reaction of acetyl (2.3.1). Proceed as described

Acetyltryptophan for substances hydrolysable only with difficulty.
(Ni-Acetyltryptophan, Ph. Bur. monograph 1383) TESTS
Appearance of solution
The solution is clear (2.2.1) and not more intensely coloured
and enantiomer than reference solution Y7 or GY7 (2.2.2~ Method II).
Dissolve 1.0 g in a 40 gIL solution of sodium hydroxide Rand
dilute to 100 mL with the same alkaline solution.
Optical rotation (2.2.7)
C13Hl~203 246.3 87-32-1
-0.1 D to + 0.1 D.
PhEur ...:.- _
Dissolve 2.50 g in a 40 gIL solution of sodium hydroxide R
DEFINITION and dilute to 25.0 rnL with the same alkaline solution.
(RS)-2-Acetylamino-3-( 1H-indol-3-yl)propanoic acid. Related substances
Content Liquid chromatography (2.2.29). Prepare the test and reference
99.0 per cent to 101.0 per cent (dried substance). solutions immediately beforeuse.
PRODUCTION Buffer solution pH 2.3 Dissolve 3.90 g of sodium dihydrogen
Tryptophan used for the production of N-acetyltryptophan phosphate R in 1000 mL of water R. Add about 700 mL of a
complies with the test for impurity A and other related 2.9 gIL solution of phosphoric acid R and adjust to pH 2.3
substances in the monograph on Tryptophan (1272). with the same acid solution.
Solvent mixture acetonitrile R, water R (10:90 VIV).
CHARACTERS
Appearance Test solution Dissolve 0.10 g of the substance to be
White or almost white, crystalline powder, or colourless examined in a mixture of 50 volumes of acetonitrile R and
crystals. 50 volumes of water R and dilute to 20.0 mL with the same
mixture of solvents.
Solubility
Reference solution (a) Dilute 1.0 mL of the test solution to
Slightly soluble in water, very soluble in ethanol
100.0 mL with the solvent mixture.
(96 per cent); It dissolves in dilute solutions of alkali
hydroxides. Reference solution (b) Dilute 4.0 mL of reference solution (a)
to 100.0 mL with the solvent mixture.
mp
About 205 DC. Reference solution (c) Dissolve the contents of a vial of 1,1'-
ethylidenebistryptophan CRS in 1 mL of reference solution (b).
IDENTIFICATION
Column:
First identification: A~ B.
=
- size: 1 0.25 m, 0 =4.6 mrn;
Second identification: A~ C, D, E. - stationary phase: octadecylsilylsilica gelfor chromatography R
A. Optical rotation (see Tests). (5 um);
B. Infrared absorption spectrophotometry (2.2.24). - temperature: 40 DC.
Comparison Nsacetyltryptophan CRS. Mobile phase:
- mobile phase A: acetonitrileR, buffer solution pH 2.3
C. Thin-layer chromatography (2.2.27).
(115:885 VIV);
Test solution Dissolve 50 mg of the substance to be - mobile phase B: acetonitrileR, buffer solution pH 2.3
examined in 0.2 mL of concentrated ammonia R and dilute to (350:650 VIV);
10 mL with water R.
Reference solution (a) Dissolve 50 mg of N- Time Mobile phase A Mobile phase B
acetyltryptophan CRS in 0.2 mL of concentrated ammonia R (min) (per cent VIJl) (per cent VIJl)
and dilute to 10 mL with water R. 0-10 100 o
Reference solution (b) Dissolve 10 mg of tryptophan R in the 10 - 45 100 -> 0 a -> 100
test solution and dilute to 2 mL with the test solution. 45 - 65 o 100
Plate TLC silica gel F 254 plate R.
Mobile phase glacial acetic acid R, water R, butanol R Flow rate 0.7 mUmin.
(25 :25:40 VIVIV). Detection Spectrophotometer at 220 nm.
Application 2 ilL. Injection 20 ul, of the test solution and reference
Development Over a path of 10 em. solutions (a) and (c). ,-
Drying In an oven at 100-105 DC. for 15 min. Retention time N-acetyltryptophan = about 29 min;
1,I'-ethylidenebis(tryptophan) = about 34 min.
Detection Examine in ultraviolet light at 254 nm.
System suitability Reference solution (c):
System suitability Reference solution (b):
- resolution: minimum 8.0 between the peaks due to
- the chromatogram shows 2 clearly separated spots.
N-acetyltryptophan and 1,l'-ethylidenebis(tryptophan);
Results The principal spot in the chromatogram obtained if necessary, adjust the time programme for the elution
with the test solution is similar in position and size to the gradient (an increase in the duration of elution with
principal spot in the chromatogram obtained with reference mobile phase A produces longer retention times and a
solution (a). better resolution);
D. Dissolve about 2 mg in 2 rnL of water R. Add 2 rnL of
dimethylaminobenzaldehyde solution R6. Heat on a water-bath.
A blue or greenish-blue colour develops.

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2020 Acetyltryptophan 1-69

~
- symmetry factor: maximum 3.5 for the peak due to HN H NH
~ ... 2
I,I'-ethylidenebistryptophan in the chromatogram
~ '\ -" C0 2H
obtained with reference solution (c). ~

Limits:
HO
- impurities A, B, C, D, E, F, G, H, I, J, K, L: for each
impurity, not more than 0.25 times the area of the
principal peak in the chromatogram obtained with D. (S)-2-amino-3-(5-hydroxy-IH-indol-3-yl)propanoic acid
(5-hydroxytryptophan),
reference solution (a) (0.25 per cent);
- total: not more than 0.5 times the area of the principal
OHC,
peak in the chromatogram obtained with reference NH 0 H NH2

~Co,H
solution (a) (0.5 per cent);
--'- disregard limit: 0.01 times the area of the principal peak
the chromatogram obtained with reference solution (a)
(0.01 per cent).
Ammonium (2.4.1, Method B) E. (S)-2-amino-4-[2-(fonnylamino)phenyl] -4-oxobutanoic
Maximum 200 ppm, determined on 0.10 g. acid. (N-fonnylkynurenine),
Prepare the standard using 0.2 mL of ammonium standard
solution (100 ppm NH4J R.
Iron (2.4.9)
Maximum 10 'ppm.
Dissolve 1.0 gin 50 mL of hydrochloric acid R1, with heating
at 50°C. Allow to cool. In a separating funnel, shake with F. (S)-2-amino-3-(phenylamino)propanoic acid
3 quantities, each of 10 mL, of methyl isobutyl ketone R1, (3-phenylaminoalanine),
-shaking for 3 min each time. To the combined organic layers
add 10 mL of water R and shake for 3 min. Examine the
aqueous layer.
Loss on, drying (2.2.32)
Maximum 0.5 per cent, determined on 1.000 g by drying in
an oven at 105 "C.
Sulfated ash (2.4.14) G. (S)-2-amino-3-(2-hydroxy-lH-indol-3-yl)propanoic acid
Maximum 0.1 per cent, determined on 1.0 g. (2-hydroxytryptophan),

ASSAY
Dissolve 0.200 ginS mL of methanol R. Add 50 mL of
ri~Y'*7H
anhydrous ethanol R. Titrate with 0.1 M sodium hydroxide,
determining the end-point potentiometrically (2.2.20). ~co,H
I mL of 0.1 M sodium hydroxide is equivalent to 24.63 mg of
e13Hl~203' H. (3RS)-1,2,3,4-tetrahydro-9H-/}-carboline-3-carboxylic
STORAGE acid,
Protected from light.
IMPURITIES
SPecified impurities A, B, C, D, E, F, G, H, I, J, K, L.

1. l-methyl-I,2,3,4-tetrahydro-9H-t}-carboline-3-carboxylic
acid,
A. (S)-2-amino-3-(lH-indol-3-yl)propanoic acid
HO
(tryptophan),

and epimer at C*

B. (S)-2-amino-3-[(3RS)-3-hydroxy-2-oxo-2,3-dihydro-IH-
indol-3-yl]propanoic acid (dioxyindolylalanine),
J. (S)-2-amino-3- [2- [2,3-dihydroxy-I-( IH-indol-3-yl)
propyl] -I H-indol-3-yl]propanoic acid,

C. (S)-2-amino-4-(2-aminophenyl)-4-oxobutanoic acid
(kynurenine),

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1-70 Acetyltyrosine 2020

J.,1obile phase water R, glacialacetic acid R, ethyl acetate R

(10:15:75 V/V/V).
Application 5 ilL.
Development Over 2/3 of the plate.
Drying In air.
Detection Examine in ultraviolet light at 254 nm.
Results The principal spot in the chromatogram obtained
K. (S)- 2-amino-3-[2-(IH-indol-3-ylmethyl)-IH-indol-3-yl]
with the test solution is similar in position and size to the
propanoic acid,
principal spot in the chromatogram obtained with the
reference solution.
D. Solution S (see Tests) is strongly acid (2.2.4).
TESTS
Solution S
Dissolve 2.50 g in water R and dilute to 100.0 mL with the
same solvent.
Appearance of solution
Solution S is clear (2.2.1) and colourless (2.2.2, Method II).
L. 1-( IH-indol-3-ylmethyl)-1,2,3,4-tetrahydro-9H-~­ Specific optical rotation (2.2.7)
carboline-3-carboxylic acid. + 46 to + 49 (dried substance).
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur Dilute 10.0 mL of solution S to 25.0 mL with waterR.
Related substances
Liquid chromatography (2.2.29). Carry out the testprotected
from light.
Acetyltyrosine Test solution Dissolve 50.0 mg of the substance to be
examined in mobile phase A and dilute to 50.0 mL with
(N-Acetyltyrosine, Ph. Bur. monograph 1384) mobile phase A.
Reference solution (a) Dilute 1.0 mL of the test solution to
100.0 mL with mobile phase A. Dilute 1.0 mL of this
solution to 10.0 mL with mobile phase A.
Reference solution (b) Dissolve 20.0 mg of tyrosine CRS
(impurity A) in 2 mL of a 40 gIL solution of sodium
hydroxide R and dilute to 20.0 mL with waterR. Dilute
CllH13N04 223.2 537-55-3 1.0 mL of this solution to 10.0 mL with waterR.
PhEur ~ _
Reference solution (c) Dilute 1.0 mL of reference solution (b)
DEFINITION to 10.0 mL with mobile phase A.
(2S)-2-(Acerylamino)-3-(4,-hydroxyphenyl)propanoic acid. Reference solution (d) Dilute 1.0 mL of reference
Content solution (b) to 20.0 mL with the test solution.
98.5 per cent to 101.0 per cent (dried substance). Column:
- size: l = 0.15 m, 0 = 3 mm;
CHARACTERS
- stationary phase: spherical octadecylsl.7yl silica gelfor
Appearance
chromatography R (3 urn);
White or almost white, crystalline powder or colourless
- temperature: 40 "C.
crystals.
Mobile phase:
Solubility - mobile phase A: mix 1.0 mL of phosphoric acid Rand
Freely soluble in water, practically insoluble in cyclohexane. 1000 mL of waterfor chromatography R;
IDENT;lFICATION - mobile phase B: acetonitrile R1;
First identification: A, B.
Time Mobile phase A Mobile phase B
Second identification: A, C, D.
(min) (per cent VJJl) (per cent VJJl)
A. Specific optical rotation (see Tests).
0-2 97 3
B. Infrared absorption spectrophotometry (2.2.24). 2 ... 15 97 --+ 62 3....,. 38
Comparison N-acetyltyrosine CRS.
C. Thin-layer chromatography (2.2.27). Flow rate 0.7 mUmin.
Test solution Dissolve 80 mg of the substance to be Detection Spectrophotometer at 219 nm.
examined in a mixture of 3 volumes of glacial acetic acid R, Injection 2!J.L of the test solution and reference
3 volumes of water Rand 94 volumes of anhydrous ethanol R, solutions (a), (c) and (d).
and dilute to 10 mL with the same mixture of solvents.
Relative retention With reference to·N-acetyltyrosine
Reference solution Dissolve 80 mg of N-acetyltyrosine CRS in (retention time = about 6 min): impurity A = about 0.5.
a mixture of 3 volumes of glacial acetic acid R, 3 volumes of
System suitability Reference solution (d):
water Rand 94 volumes of anhydrous ethanolR, and dilute to
- resolution: minimum 5.0 between the principal peak and
10 mL with the same mixture of solvents.
the peak due to impurity A.
Plate TLC silica gelF254 plate R.

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2020 Aciclovir 1-71

Limits:
- impurity A: not more than 0.8 times the area of the
corresponding peak in the chromatogram obtained with
reference solution (c) (0.8 per cent);
- unspecified impurities: for each impurity, not more than the A. (2S)-2-amino-3-(4-hydroxyphenyl) prop anoic acid
area of the principal peak in the chromatogram obtained (tyrosine) ,
with reference solution (a) (0.10 per cent);
- total: maximum 1.0 per cent;
- disregard limit: 0.5 times the area of the principal peak in
the chromatogram obtained with reference solution .(a)
(0.05 per cent).
Chlorides (2.4.4)
Maximum 200 ppm. B. (2S)-2-( acetylamino)-3- [4-(acetoxy)phenyl] propanoic acid
Dilute 10 mL of solution S to 15 mL with water R. (diacetyltyrosine) .

Sulfates (2.4.13) PhEur

Maximum 200 ppm.

Dissolve 1.0 g in distilled water R and dilute to 20 mL with
the same solvent.
Ammonium (2.4.1, Method B)
Aciclovir
Maximum zoo ppm, determined on 0.100 g. (Ph. Bur. monograph 0968)
Prepare the standard using 0.2 mL of ammonium standard
solution (1 00 ppm NH4J R.
Iron (2.4.9)
Maximum 20 ppm.
In a separating funnel, dissolve 0.5 g in 10 mL of dilute
hydrochloric acid R. Shake with 3 quantities, each of 10 mL,
of methyl isobutyl ketone R1, shaking for 3 min each time.
To the combined organic layers add 10 mL of water Rand 225.2 59277-89-3
shake for 3 min. The aqueous layer complies with the test.
Action and use
Loss on drying (2.2.32) Purine nucleoside analogue; antiviral (herpesviruses).
Maximum 0.5 per cent, determined on 1.000 g by drying in
an oven at 105°C.
Preparations
.
Aciclovir Cream
Sulfated ash (2.4.14)
Aciclovir Eye Ointment
Maximum 0.1 per cent, determined on 1.0 g.
Aciclovir Infusion
Bacterial endotoxins (2.6.14)
Aciclovir Oral Suspension
Less than 25 illig, if intended for use in the manufacture of
parenteral preparations without a further appropriate Aciclovir Tablets
procedure for the removal of bacterial endotoxins. Aciclovir Dispersible Tablets
ASSAY PhEur _
Dissolve 0.180 gin 50 mL of carbon dioxide-free water R.
Titrate with 0.1 M sodium hydroxide, determining the
DEFINITION
end-point potentiometrically (2.2.20). 2-Amino-9-[(2-hydroxyethoxy) methyl] -1,9-dihydro-6H-purin-
6-one.
1 mL of 0.1 M sodium hydroxide is equivalent to 22.32 mg of
C llH13N04 · Content
98.5 per cent to 101.0 per cent (anhydrous substance).
STORAGE
Protected from light. If the substance is sterile, store in a CHARACTERS
sterile, airtight, tamper-proof container. Appearance
White or almost white, crystalline powder.
IMPURITIES
Solubility
Specified impurities A.
Slightly soluble in water, very slightly soluble in ethanol
Other detectable impurities (the following substances would, if (96 per cent), practically insoluble in heptane. It dissolves in
present at a sufficient level, be detected by one or other of the tests dilute solutions of mineral acids and alkali hydroxides.
in the monograph. They are limited by the general acceptance
criterion for other/unspecified impurities and/or by the general IDENTIFICATION
monograph Substances for pharmaceutical use (2034). It is Infrared absorption spectrophotometry (2.2.24).
therefore not necessary to identify these impurities for Comparison aciclooir CRS.
demonstration of compliance. See also 5.10. Control of impurities
TESTS
in substances for pharmaceutical use) B.
Appearance of solution
The solution is clear (2.2.1) and not more intensely coloured
than reference solution Y7 (2.2.2, Method II).
Dissolve 0.25 g in a 4 gIL solution of sodium hydroxide Rand
dilute to 25 mL with the same solvent.

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1-72 Aciclovir 2020

Related substances System suitability:

Liquid chromatography (2.2.29). Prepare the solutions - resolution: minimum 1.5 between the peaks due to
immediately before use. impurity C and aciclovir in the chromatogram obtained
Solvent mixture dimethJ7l sulfoxide R, water R (20:80 V/V). with reference solution (c); minimum 1.5 between the
Phosphate buffersolutionpH 2.5 Dissolve 3.48 g of peaks due to impurities F and A and minimum 1.5
dipotassium hydrogen phosphate R in 1000 mL of water Rand between the peaks due to impurities K and G in the
adjust to pH 2.5 with plwsphoric acid R. chromatogram obtained with reference solution (d).
Limits:
Phosphate buffersolutionpH 3.1 Dissolve 3.48 g of
- correction factor. for the calculation of content, multiply the
dipotassium hydrogen plwsphateR in 1000 mL of waterR and
peak area of impurity I by 1.5;
adjust to pH 3.1 with phosphoric acid R.
- impurity B: not more than 7 times the area of the
Test solution Dissolve 25 mg of the substance to be principal peak in the chromatogram obtained with
examined in 5.0 mL of dimethyl sulfoxide R and dilute to reference solution (b) (0.7 per cent);
25.0 mL with water R. - sum of impurities 0 and Q: not more than 3 times the area
Reference solution (a) DissolveS rng of aciclovir for system of the principal peak in the chromatogram obtained with
suitability CRS (containing impurities A, B, J, K, N, 0 and reference solution (b) (0.3 per cent);
P) in 1 mL of dimethyl sulfoxide R and dilute to 5.0 mL with - sum of impuritiesK and R: not more than twice the area of
'water R. the principal peak in the chromatogram obtained with
Reference solution (b) Dilute 1.0 mL of the test solution to reference solution (b) (0.2 per cent); .
100.0 mL with the solvent mixture. Dilute 1.0 mL of this - impurities A~ G~ J~ N~ P. for each impurity, not more than
solution to 10.0 mL with the solvent mixture. twice the area of the principal peak in the chromatogram
Reference solution (c) Dissolve the contents of a vial of obtained with reference solution (b) (0.2 per cent);
aciclovir for peak identification 1 CRS (containing impurities C - impurities C~ F~ I: for each impurity, not more than the
and I) in 200 J.L1... of dimethyl sulfoxide R and dilute to 1.0 mL area of the principal peak in the chromatogram obtained
with water R. with reference solution (b) (0.1 per cent);
- unspecified impurities: for each impurity, not more than
Reference solution (d) .Dissolve the contents of a vial of
0.5 times the area of the principal peak in the
aciclovir for peak identification 2 CRS (containing impurities F
chromatogram obtained with reference solution (b)
and G) in 1.0 mL of reference solution (a).
(0.05 per cent);
Column: ~ total: not more than 15 times the area of the principal
- size:·l = 0.25 m, 0 = 4.6 mID; peak in the chromatogram obtained with reference
- stationary phase: end-capped octadecylsilyl szIica gelfor solution (b) (1.5 per cent);
chromatography R (5 um), . - disregard limit: 0.3 times the area of the principal peak in
Mobile phase: the chromatogram obtained with reference solution (b)
- mobile phase A: acetonitrile R, phosphate buffer solution (0.03 per cent).
pH 3.1 (1:99 V/V);
Water (2.5.12)
- mobile phase B: acetonitrlle R, phosphate buffer solution Maximum 6.0 per cent, determined on 0.500 g.
pH 2.5 (50:50 VIV);
Sulfated ash (2.4,14)
Tim.e Mobile phase A Mobile phase B Maximum 0.1 per cent, determined on 1.0 g.
(min) (per cent VIJ') (per cent VIJ') Bacterial endotoxins (2. 6.14~ Method D)
0-5 100 o Less than 0.50 ill/mg, if intended for use in the manufacture
5 - 27 - 100 -> 80 0-+20 of parenteral preparations without a further appropriate
27 - 40 80 20 procedure for the removal of bacterial endotoxins.
ASSAY
Flow rate 1.0 mUmin. Dissolve 0.150 gin 60 mL of anhydrous acetic add R. Titrate
Detection Spectrophotometer at 254 om. with 0.1 M perchloric add, determining the end-point
Injection 10 J.1L of the test solution and reference potentiometrically (2.2.20). Carry out a blank titration.
solutions (b), (c) and (d). 1 rnL of 0.1 M perchloric acid is equivalent to 22.52 mg
Identification of impurities Use the chromatogram supplied of C SH llNs0 3 .
with aciclovir for peak identification 1 CRS and the IMPURITIES
chromatogram obtained with reference solutiori (c) to identify Specified impurities A~ B~ C~ F~ G~ I~ J~ K~ N~ O~ P~ Q, R.
the peaks due to impurities C and I; use the chromatogram
Other detectable impurities (thefollowing substances would, if
supplied with aciclovir for peak identification 2 CRS and the
present at a sufficient leoel, bedetected by one or otherof the tests
chromatogram obtained with reference solution (d) to
in the monograph. They are limitedby the general acceptance
identify the peaks due to impurities A, B, F, G, J, K, N, 0
criterion for other/unspecified impurities and/or by the general
and P.
monograph Substances for pharmaceutical use (2034). It is
Relative retention With reference to aciclovir (retention therefore not necessary to identify these impurities for
time = about 13 min): impurity B = about 0.4; demonstration of compliance. See also 5.10. Control of impurities
impurity P = about 0.7; impurity C = about 0.9; in substances for pharmaceutical use) L~ M.
impurity N = about 1.37; impurities 0 and Q= about 1.42;
impurity I = about 1.57; impurity J = about 1.62;
impurity F = about 1.7; impurity A = about 1.8;
impurities K and R = about 2.5; impurity G = about 2.6.

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2020 Aciclovir 1-73

A. 2- [(2-amino-6-oxo-1 ,6-dihydro-9H-purin-9-yl)methoxy]
ethyl acetate, L. N-(9-acetyl-6-oxo-6,9-dihydro-1H-purin-2-y1)acetamide
(N2 , 9-diacetylguanine),

o
o 0 oJ
/ "-.J \
o HN. N CH3
II I~: \
I )
HC,-A....NAN N
B. 2-amino-1,7-dihydro-6H-purin-6-one (guanine), 3 H

M. 2- [[2-(acetylamino)-6-oxo-l,6-dihydro-7H-purin- 7-y1]
methoxy]ethyl acetate, .
N. unknown structure,
O.unknownstructur~

C. 2-amino-7- [(2-hydroxyethoxy)methyl]-1,7 -dihydro-6H-

purin..6-one,

P. 2-amino-9-(2-hydroxyethy1)-1,9-dihydro-6H-purin-6-one,

F. N-[9-[(2-hydroxyethoxy)methyl]-6-oxo-6,9-dihydro-1H-
purin-2-yl] acetamide,

G. 2- [[2-( acetylamino)-6-oxo-1, 6-dihydro-9H-purin-9-yl]

methoxy] ethyl acetate,
Q. mixture of 2-amino-9-[[2-(hydroxymethoxy)
ethoxy] methyl] -1, 9-dihydro-6H-purin-6-one and 2-amino-
9- [[2-(hydroxyethoxy)methoxy] methyl] -1, 9-dihydro-6H-'
purin-6-one,

1. 2-amino-7-[[2-[(2-amino-6-oxo-1,6-dihydro-9H-purin-9-
yl)methoxy] ethoxy]methy1]-1,7-dihydro-6H-purin-6-one,

R. 9,9'- [methy1enebis(oxyethy1eneoxymethy1ene)]bis (2-

amino-I,9-dihydro-6H-purin-6-one).
_ _ _ _ _ _ _ _----'-----'- PhEur

J. 9,9'-[ethy1enebis(oxymethy1ene)]bis(2-amino-1,9-dihydro-
6H-purin-6-one),

K. 2,2'-(methy1enediimino)bis [9-[ (2-hydroxyethoxy)methy1]-

1,9-dihydro-6H-purin-6-onel.

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1-74 Acitretin 2020

Acitretin *** Reference solution (c) Dilute 1.0 mL of the test solution {a)
*** *** to 100.0 mL with anhydrous ethanol R. Dilute 1.0 mL of this
(Ph. Bur. monograph 1385) *** solution to 10.0 ml, with anhydrous ethanol R.
Reference solution (d) Dissolve 2.5 mg of acitretin for
impurity A identification CRS in 0.5 mL of tetrahydrofuran R
and dilute to 10.0 mL with anhydrous ethanol R.
Column:
- size: 1 = 0.25m, 0 = 4 mm;
- stationary phase: octadecylsilyl silica gel for
chromatography for separation of polycyclic aromatic
hydrocarbons R (5 11m);
326.4 55079-83-9 - temperature: 25 o~.
Action and use Mobile phase 0.3 per cent VIV solution of glacial acetic
Vitamin A analogue (retinoid); treatment of psoriasis; acid R in a mixture of 8 volumes of water for
ichthyosis; Darier's disease. chromatography Rand 92 volumes of anhydrous ethanol R.
Preparation Flow rate 0.6 mLImin.
Acitretin Capsules Detection Spectrophotometer at 360 nm.
PhEur _
Autosampler Set at 4°C.
Injection 10 ilL oftest solution (a) and reference
DEFINITION solutions (b), (c) and (d).
(2B,4E,6B,8E)-9-( 4-Methoxy-2,3,6-trimethylphenyl)-3,7- Run time 2.5 times the retention time of acitretin.
dimethylnona-2,4,6,8-tetraenoic acid
Identification of impurities. Use the chromatogram supplied
Content with acitretin for impurity A identification CRS and the
98.0 per cent to 102.0 per cent (dried substance). chromatogram obtained with reference solution (d) to
CHARACTERS identify the peak due to impurity A.
Appearance Relative retention With reference to acitretin (retention
Yellow or greenish-yellow, crystalline powder. time = about 6 min): impurity A = about 0.8;
Solubility =
tretinoin about 0.85.
Practically insoluble in water, sparingly soluble in System suitability Reference solution (b):
tetrahydrofuran, slightly soluble in acetone and in ethanol - resolution: minimum 2.0 between the peaks due to
(96 per cent), very slightly soluble in cyclohexane. tretinoin and acitretin.
It is sensitive to air, heat and light, especially in solution. Calculation of percentage contents:
It shows polymorphism (5.9). - for each impurity, use the concentration of acitretin in
reference solution (c).
Carry out all operations as rapidly as possible and avoid exposure
to actinic light; use freshly prepared solutions. Limits:
- impurity A: maximum 0.2 per cent;
IDENTIFICATION - unspecified impurities: .for each impurity, maximum
Infrared absorption spectrophotometry (2.2.24). 0.10 per cent;
Comparison acitretin CRS. - total: maximum 0.5 per cent;
If thespectra obtained in the solid state show differences, - reporting threshold: 0.05 per cent.
dissolve the substance to be examined and the reference Loss on drying (2.2.32)
substance separately in 2-propanol R by heating under reflux; Maximum 0.5 per cent, determined on 1.000 g by drying in
filter, evaporate to dryness and record new spectra using the vacuo at 100°C for 4 h.
residues. Sulfated ash (2.4.14)
TESTS Maximum 0.1 per cent, determined on 1.0 g.
Related substances ASSAY
Liquid chromatography (2.2.29). Carry out the test protected Liquid chromatography (2.2.29) as described in the test for
from light and prepare the solutions immediately before use. related substances with the following modification.
Test solution (a) Dissolve 25.0 mg of the substance to be Injection Test solution (b) and reference solution (a).
examined in 5 mL of tetrahydrofuran R and dilute to
Calculate the percentage content of CZIHz603 taking into
100.0mL with anhydrous ethanol R.
account the assigned content of acitretin CRS.
Test solution (b) Dilute 10.0 mL of test solution (a) to
25.0 mL with anhydrous ethanol R. STORAGE
Reference solution (a) Dissolve 25.0 mg of acitretin CRS in In an airtight container, protected from light, at a
5 mL of tetrahydrofuran R and dilute to 100.0 mL with temperature of 2 °C to 8°C. .
anhydrous ethanol R. Dilute 10.0 mL of the solution to It is recommended that the contents of an opened container
25.0 mL with anhydrous ethanol R. be used as soon as possible and any unused part be protected
Reference solution (b) Dissolve 1 mg of tretinoin CRS in by an atmosphere of inert gas.
anhydrous ethanol R and dilute to 20.0·mL with the same IMPURITIES
solvent. Mix '5.0 mL of the solution with 2.5 mL of reference Specified. impurities A.
solution (a) and dilute to 100.0 mL with anhydrous ethanol R. Other detectable impurities (the following substances would) if
present at a sufficient level, be detected by one or other of the tests

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2020 Adapalene 1-75

in the monograph. They are limited by the general acceptance TESTS

criterion for other/unspecified impurities and/or by the general Appearance of solution
monograph Substancesfor pharmaceutical use (2034). It is The solution is clear (2.2.1) and not more intensely coloured
therefore not necessary to identify these impurities for than reference solution BY6 (2.2.2~ Method 11).
demonstration of compliance. See also 5.10. Control of impurities Dissolve 0.2 gin tetrahydrofuran R and dilute to 20 mL with
in substances for pharmaceuticaluse) B. the same solvent.
CH3 CH3 'CH3
Related substances

H3co~o,H
Liquid chromatography (2.2.29).
Solvent mixture tetrahydrofuran R, acetonitrile R, water R
(20:37:43 V/V/V).
CH3 Test solution (a) Dissolve 40.0 mg of the substance to be
examined in 10 mL of tetrahydrofuran. R, add 7 mL of the
solvent mixture and dilute to 20.0mL with tetrahydrofuran R.
A. (2Z,4E,6E,8E)-9-( 4-methoxy:-2,3,6-triInethylphenyl)-3,7-
dimethylnona-2,4,6,8-tetraenoic acid, Test solution (b) Dissolve 20.0 mg of the substance to be
examined in 50 mL of tetrahydrofuran R, add 35 mL of the
CH3 CH3 CH3 0 solvent mixture and dilute to 100.0 mL with

~O/'-.CH'
tetrahydrofuran R. Dilute 5.0 mL of the solution to 50.0 mL
with the solvent mixture.
H3 C O Y C H3 Reference solution (a) Dilute 1.0 mL of test solution (a) to
CH3 10.0 mLwith tetrahydrofuran R. Dilute 1.0 mL of this
solution to 100.0 mL with the solvent mixture.
Biethyl (2E,4E,6E,8E)-9-(4-methoxy-2,3,6-trimethylphenyl)- Reference solution (b) Dissolve 2.4 mg of adapalene
'3,7 -dimethylnona-2,4,6,8-tetraenoate. impurity C CRS in 2 mL of tetrahydrofuran R and dilute to
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _~ PhEur
20.0 mL with the same solvent. Dilute 2.0 mL of the
solution to 20.0 mL with the solvent mixture. To 2.0 mL of
this solution add 2.0 mL of reference solution (a) and dilute
to 20.0 mL with the solvent mixture.
Reference solution (c) Dissolve the contents of a vial of
Adapalene adapalenefor peak identification CRS (containing impurities A,
C and D) in 0.5 mL of tetrahydrofuran R and dilute to
(Ph. Eur. monograph 2445)
1.0 mL with the solvent mixture.
Reference solution (d) Dissolve 20.0 mg of adapalene CRS in
. 50 mL of tetrahydrofuran R, add 35 mL of the solvent
mixture and dilute to 100.0 mL with tetrahydrofuran R.
Dilute 5.0 mL of the solution to 50.0 mL with the solvent
mixture.
Column:
- size: 1= 0.25 m, (2) = 4.6 mm;
- stationaryphase: end-capped phenylsilyl silica gelfor
412.5 106685-40-9
chromatography R (5 urn):with a carbon loading of
Action and use 7.5 per cent;
~ temperature: 30°C.
Vitamin A analogue (retinoid); treatment of acne.
Mobile phase:
Preparations
- mobilephase A: glacialacetic acid R, water R (0.1: 100 V/V);
Adapalene Cream
- mobilephase B: tetrahydrofuran R, acetonitrile R
Adapalene Gel (35:65 V/V);
PhEur ~ _
Time Mobile phase A Mobile phase B
DEFINITION (min) (per cent V/V) (per cent V/V)
6-(4-Methoxy-3-tricyclo[3.3.1.1 3,7]dec-1-ylphenyl) 0-2.5 50 50
naphthalene-2-carboxylic acid. 2.5 - 40 50 -> 28 50 -> 72
Content 40 - 42 28 72
98.0 per cent to 102.0 per cent (dried substance).
CHARACTERS Flow rate 1.2 mUmin.
Appearance Detection Spectrophotometer at 270 nm.
White or almost white powder. Injection 25 JlL of test solution (a) and reference
Solubility solutions (a), (b) and (c).
Practically insoluble in water, sparingly soluble in Identification of impurities Use the chromatogram supplied
tetrahydrofuran, practically insoluble in ethanol with adapalene for peak identification CRS and the
(96 per cent). chromatogram obtained with reference solution (c) to identify
the peaks due to impurities A, C and D.
IDENTIFICATION
Infrared absorption spectrophotometry (2.2.24).
Comparison adapaleneCRS.

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 1-76 Adenine 2020

Relative retention With reference to adapalene (retention

time = about 20 min): impurity A = about 0.3;
impurity C = about 0.9; impurity D = about 1.9.
System suitability Reference solution (b):
- resolution: minimum 4.5 between the peaks due to
impurity C and adapalene;
- signal-to-noise ratio: minimum 10 for the peak due to
B. 6-[3-(3-hydroxytricyclo[3.3.1.1 3,7] dec-1-yl)-4-
impurity C.
methoxyphenyl]naphthalene-2-carboxylic acid,
Limits:
- correction factors: for the calculation of content, multiply
the peak areas of the following impurities by the
corresponding correction factor: impurity A = 0.7;
impurity C = 7; impurity D = 1.4;
- impurity A: not more than 3 times the area of the
principal peak in the chromatogram obtained with C. 1-(2-methoxyPhenyl)tricyclo[3.3.1.1 3,7] decane,
reference solution (a) (0.3 per cent);
-- impurity D: not more than twice the area of the principal
peak in the chromatogram obtained with reference
solution (a) (0.2 per cent);
- impun'ty C: not more than 1.5 times the area of the
principal peak in the chromatogram obtained with
reference solution (a) (0.15 per cent);
D. 1,1'-[4,4/-bis(methoxy)biphenyl-3,3/-diyl]bis(tricyclo
- unspecified impurities: for each impurity, not more than the
[3.3.1.13,7]decane).
area of the principal peak in the chromatogram obtained
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
with reference solution (a) (0.10 per cent);
-,...- total: not more than 5 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.5 per cent);
- disregard limit: 0.5 times the area of the principal peak in Adenine
the chromatogram obtained with reference solution (a)
(0.05 per cent). (Ph. Bur. monograph 0800)
Loss on drying (2.2.32)
Maximum 0.5 per cent, determined on 1.000 g by drying in
an oven at 105°C for 4 h.
Sulfated ash (2.4.14)
Maximum 0.1 per cent, determined on 1.0 g.
ASSAY 135.1 73-24-5
Liquid chromatography (2.2.29) as described in the test for
Action and use
related substances with the following modification.
Constituent of anticoagulant and preservative solutions for
. Injection Test solution (b) and reference solution (d). blood.
Calculate the percentage content of adapalene from the
declared content of adapalene CRS. PhEur ---------'-----------

IMPURITIES DEFINITION
Specifiedimpurities A~ C~ D.
I
Adenine contains not less than 98.5 per cent and not more
than the equivalent of 101.0 per cent of 7H...,purin-6-amine,
Other detectable impurities (the following substances would, if
present at a sufficient leuel, be detected by one or otherof the tests calculated with reference to the dried substance.
in the monograph. They are limited by the general acceptance CHARACTERS
criterion for other/unspecified impurities and/orby the general A white or almost white powder, very slightly soluble in
monograph Substances for pharmaceutical use (2034). It is water and in alcohol. It dissolves in dilute mineral acids and
therefore not necessary to identify these impurities for in dilute solutions of alkali hydroxides.
demonstration of compliance-.See also 5.10. Control of impurities
IDENTIFICATION
in substances for pharmaceutical use) B.
First identification: A.
Second identification: B, C.
A. Examine by infrared absorption spectrophotometry
(2.2.24), comparing with the spectrum obtained with
adenine CRS. Examine the substances prepared as discs.
B. Examine the chromatograms obtained inthe test for
related substances. The principal spot in the chromatogram
obtained with test solution (b) is similar in position and size
A. 2,2'-binaphthalene-6,6 /-dicarboxylic acid, to the principal spot in the chromatogram obtained with
reference solution (a).
C. To 1 g add 3.5 mL of propionic anhydride R and boil for
15 min with stirring. Cool. To the resulting crystalline mass

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2020 Adenosine 1-77

add 15 mL of light petroleum R and heat to boiling with watch-glass stick a piece of red litmus paper R 5 mm square
vigorous stirring. Cool and filter. Wash the precipitate with and wetted with a few drops of water R. Finely powder the
two quantities, each of 5 mL, of light petroleum R. Dissolve substance to be examined, place 0.5 g in the lower watch-
the precipitate in 10 mL of water R and boil for 1 min. Filter glass and suspend in 0.5 mL of water R. To the suspension
the mixture at 30°C to 40 "C. Allow to cool. Filter, and dry add 0.30 g of heavy magnesium oxide R. Briefly triturate with
the precipitate at 100 "C to 105°C for 1 h. The melting a glass rod. Immediately close the cell by putting the two
point (2.2.14) of the precipitate is 237°C to 241 "C. watch-glasses together. Heat at 40°C for 15 min. The litmus
TESTS paper is not more intensely blue coloured than a standard
prepared at the same time and in the same manner using
Solution S
0.05 mL of ammonium standard solution (100 ppm NH4J R,
Suspend 2.5 gin 50 mL of distilledwater R and boil for
0.5 mL of water Rand 0.30 g of heavy magnesium oxide R
3 min. Cool and dilute to 50 mL with distilledwater R. Filter.
(10 ppm).
Use the filtrate as solution S.
Loss on drying (2.2.32)
Appearance of solution
Not more than 0.5 per cent, determined on 1.000 g by
Dissolve 0.5 g in dilute hydrochloric acid R and dilute to
drying in an oven at 105°C.
50 mL with the same acid. The solution is clear (2.2.1) and
colourless (2.2.2~ Method II). Sulfated ash (2.4.14)
Not more than 0.1 per cent, determined on 1.0 g.
Acidity or alkalinity
To lQ mL of solution S add 0.1 mL of bromothymol blue ASSAY
solution Rl and 0.2 mL of 0.01 M sodium hydroxide. Dissolve 0.100 g in a mixture of 20 mL of acetic anhydride R
The solution is blue. Add 0.4 mL of 0.01 M hydrochloric acid. and 30 mL of anhydrous acetic acid R. Titrate with 0.1 M
The solution is yellow. perchloric acid, determining the end-point potentiometrically
Related substances (2.2.20);
Examine by thin-layer chromatography (2.2.27), using silica 1 mL of 0.1 M perchloric acid is equivalent to 13.51 mg of
gel GF254 R as the coating substance. CsHsNs·
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
Test solution (a) Dissolve 0.10 g of the substance to be
examined in dilute acetic acid R, with heating if necessary, and
dilute to1 0 mL with the same acid.
Test solution (b) Dilute 1 mL oftest solution (a) to 10 mL
with dilute acetic acid R. Adenosine
Reference solution (a) Dissolve 10.mg of adenine CRS in
dilute acetic acid R, with heating if necessary, and dilute to (Ph. Bur. monograph 1486)
10 mL with the same acid.
Reference solution (b) Dilute 1 mL of test solution (b). to
20 mL with dilute acetic acid R.
Reference solution (c) Dissolve 10 mg of adenine CRS and
10 mg of adenosine R in dilute acetic acid R, with heating if
necessary, and dilute to 10 mL with the same acid.
Apply to the plate 5 J1L of each solution. Develop over a
path of 12 em using a mixture of 20 volumes of concentrated
ammonia R, 40 volumes of ethyl acetate Rand 40 volumes of
propanol R. Dry the plate in a current of warm air and 267.2 58-61-7
examine in ultraviolet light at 254 nm. Any spot in the
chromatogram obtained with test solution (a), apart from the Action and use
principal spot, is not more intense than the spot in the Antiarrhythmic.
chromatogram obtained with reference solution (b) PhEur _
(0.5 per cent). The test is not valid unless the chromatogram
obtained with reference solution (c) shows two clearly DEFINITION
separated spots. 9-~-D-Ribofuranosyl-9H-purin-6-amine.

Chlorides (2.4.4) Content

To 10 mL of solution S add 1 mL of concentrated ammonia R 99.0 per cent to 101.0 per cent (dried substance).
and 3 mL of siloer nitrate solution R2. Filter. Wash the CHARACTERS
precipitate with a little water R and dilute the filtrate to
Appearance
15 mL with water R.. The solution complies with the limit
White or almost white, crystalline powder.
test for chlorides (100 ppm). When carrying out the test, add
2 mL of dilute nitric acid R instead of 1 mL of dilute nitric Solubility
acid R. Slightly soluble in water, soluble in hot water, practically
insoluble in ethanol (96 per cent) and in methylene chloride.
Sulfates (2.4.13)
It dissolves in dilute mineral acids.
Dilute 10 mL of solution S to 15 mL with distilled water R.
The solution complies with the limit test for sulfates mp
(300 ppm). About 234 -c.
Ammonium IDENTIFICATION
Prepare a cell consisting of two watch-glasses 60 mm in Infrared absorption spectrophotometry (2.2.24).
diameter placed edge to edge. To the inner wall of the upper Comparison adenosine CRS.

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1-78 Adenosine 2020

TESTS - unspecified impurities: for each impurity, not more than the
Solution S area of the principal peak in the chromatogram obtained
Suspend 5.0 gin 100 rnL of distilled water R and heat to with reference solution (a) (0.10 per cent);
boiling. Allow to cool, filter with the aid of vacuum and - total: not more than 5 times the area of the principal peak
dilute to 100 mL with distilled water R. in the chromatogram obtained with reference solution (a)
i\ppearanceofsolution (0.5 per cent);
Solution S is colourless (2.2.2, Method II). - disregard limit: 0.5 times the area of the principal peak in
the chromatogram obtained with reference solution (a)
Acidity or alkalinity (0.05 per cent).
To 10 mL of solution S, add 0.1 mL of bromocresol purple
solution Rand 0.1 mL of 0.01 M hydrochloric acid. Chlorides (2.4.4)
The solution is yellow. Add 0.4 mL of 0.01 M sodium Maximum 100 ppm.
hydroxide. The solution is violet-blue. Dilute 10 mL of solution S to 15 mL with waterR.
Specific optical rotation (2.2. 7) Sulfates (2.4.13)
-45 to -49 (dried substance). Maximum 200 ppm, determined on solution S.
Dissolve 1.25 g in 1 M hydrochloric acid and dilute to .Ammonium (2.4.1, Method B)
50.0 mL with the same acid. Examine within 10 min of Maximum 10 ppm, determined on 0.5 g.
preparing the solution. Prepare the standard using 5 mL of ammonium standard
Related substances solution (1 ppm NH.J R.
Liquid chromatography (2.2.29). Loss on drying (2.2.32)
Solvent mixture Dissolve 6.8 g of potassium hydrogen sulfate R Maximum 0.5 per cent, determined on 1.000 g by drying in
and 3.4 g of tetrabutylammomum hydrogen sulfate R in waterR, an ovenat 105 °e.
adjust to pH 6.5 with a 60 gIL solution of potassium Sulfated ash (2.4.14)
hydroxide R and dilute to 1000 mLwith the same solvent. Maximum 0.1 per cent, determined on 1.0 g.
Use a freshly prepared solvent mixture.
i\SSi\Y
Test solution Dissolve 20 mg of the substance to be
Dissolve 0.200 g, warming slightly if necessary, in a mixture
examined in the mobile phase and dilute to 20 mL with the
of 20 mL of acetic anhydride Rand 30 mL of anhydrous acetic
mobile phase.
acid R. Titrate with 0.1 M perchloric acid, determining the
Reference solution (a) Dilute 1.0 mL of the test solution to end-point potentiometrically (2.2.20).
100.0 mL with the mobile phase. Dilute 1.0 mL of this
1 mL of 0.1 M perchloric acid is equivalent to 26.72 mg
solution to 10.0 mL with the mobile phase.
of C lOH13Ns0 4 •
Reference solution (b) Dissolve 5 mg of adenine R
(impurity A) and 5 mg of inosine R (impurity G) in the IMPURITIES
mobile phase and dilute to 50 mL with the mobile phase. Specified impurities A, G.
Dilute 4 mL of this solution to 100 mL with the mobile Other detectable impurities (the following substances would, if
phase. present at a sufficient level, be detected by one or other of the tests
Column: in the monograph. They are limited by the general acceptance
=
- size: l = 0.25 m, 0 4.6 mm; criterion for other/unspecified impurities and/orby.thegeneral
- stationary phase: end-capped octadecylsilyl silica gelfor monograph Substances for pharmaceutical use (2034). It is
chromatography R (5 um). therefore not necessary to identify these impurities for
Mobilephase water R, solvent mixture (40:60 VIV)o demonstration of compliance. See also 5.10. Control of impurities
in substances for pharmaceutical use) F, H.
Flow rate 1.5 mLlmin.
Detection Spectrophotometer at 254 nm.
Injection 20 ~L.
Run time 1.5 times the retention time of adenosine,
Relative retention With reference to adenosine (retention
time = about 13 min): impurity A = about 0.3;
impurity G = about 0.4. A. 7H-purin-6-amine (adenine),
System suitability Reference solution (b):
- resolution: minimum 1.5 between the peaks due to o
impurities A and G.
Limits:
HO O~N
HN:J
- correction factors: for the calculation of content, multiply
the peak areas of the following impurities by the
corresponding correction factor: impurity A = 0.6;
impurity G = 1.4;
\:)
OH OH
- impurityA: not more than twice the area of the principal
peak in the chromatogram obtained with reference
F. 1-~-D-ribofuranosylpyrimidine-2,4(lH,3H)-dione
solution (a) (0.2 per cent);
(uridin~, .
- impurity G: not more than the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.1 per cent);

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2020 Adipic Acid 1-79

o Dissolve 1.0 g in methanolR and dilute to 20 mL with the

HNJYN>
lJ-
same solvent.
Related substances
N

HO~
Liquid chromatography (2.2.29).
Test solution Dissolve 0.20 g of the substance to be
examined in the mobile phase and dilute to 10.0 mL with
the mobile phase.
OH OH Reference solution (a) Dissolve 20 mg of glutaric acid R in
1.0 mL of the test solution and dilute to 10.0 mL with the
G. 9-~-D-ribofuranosyl-l, 9-dihydro-6H-purin-6-one (inosine), mobile phase.
o Reference solution (b) Dilute 1.0 mL of the test solution to

HN:e N>
100.0 mL with the mobile phase, dilute 1.0 mL of the
solution to 10.0 mL with the mobile phase.

H2N~AN
HO
N
Column:
=
- size: 1 = 0.125 m, 0 4.0 mm,
o - stationaryphase: spherical octadecylsilyl silica gelfor
chromatography R (5 urn) with a specific surface area of
350 m 2/g and a pore size of 10 nm,
OH OH - temperature: 30°C.
H! 2-a:rTIin0-9-~-D-ribofuranosyl-l, 9-dihydro-6H-purin-6-one Mobile phase Mix 3 volumes of acetonitrile Rand
'(guanosine). 97 volumes of a 24.5 gIL solution of dilute phosphoric add R.
_______ ~ PhEur Flow rate 1 mUmin.
Detection Spectrophotometer at 209 nm.
Injection 20 j.lL.
Run time 3 times the retention time of adipic acid.
Adipic Acid System suitability Reference solution (a):
- resolution: minimum 9.0 between the peaks due to glutaric
(Ph. Bur. monograph 1586) acid and adipic acid.
Limits:
- any impurity: not more than the area of the principal peak
in the chromatogram obtained with reference solution. (b)
146.1 124-04-9 (0.1 per cent),
- total: not more than 5 times the area of the principal peak
Action and use in the chromatogram obtained with reference solution (b)
Excipient. (0.5 per cent),
PhEur --'- _ - disregard limit: 0.5 times the area of the principal peak in
the chromatogram obtained with reference solution (b)
DEFINITION (0.05 per cent).
Hexanedioic acid.
Chlorides (2.4.4)
Content Maximum 200 ppm.
99.0 per cent to 101.0 per cent (dried substance).
Dilute 2.5 mL of solution S to 15 mL with waterR.
CHARACTERS Nitrates
Appearance Maximum 30 ppm.
White or almost white, crystalline powder.
To 1 mL of solution S add 2 mL of concentrated ammonia R,
Solubility 0.5 mL of a 10 gIL solution of manganese sulfate R, 1 mL of
Sparingly soluble in water, soluble in boiling water, freely a 10 gIL solution of sulfanilamide R and dilute to 20 mL with
soluble in ethanol (96 per cent) and in methanol, soluble in water R. Add 0.10 g of zinc powder R and cool in iced water
acetone. for 30 min; shake from time to time. Filter and cool 10 mL
IDENTIFICATION of the filtrate in iced water. Add 2.5 mL of hydrochloric
A. Melting point (2.2.14): 151°C to 154 °C. acid R1 and 1 mL of a 10 gIL solution of
naphthylethylenediamine dihydrochloride R. Allow to stand at
B. Infrared absorption spectrophotometry (2.2.24).
room temperature. After 15 min the mixture is not more
Comparison adipic acid CRS. intensely coloured thana standard prepared at the same time
TESTS and in the same manner, using 1.5 mL of nitrate standard
Solution S solution (2 ppm NOJJ R instead of 1 mL of solution S.
Dissolve 5.0 g with heating in distilled water R and dilute to The test is invalid if a blank solution prepared at the same
50 mL with the same solvent. Allow to cool and to time and in the same manner, using 1 mL of waterR instead
crystallise. Filter through a sintered-glass filter (40) (2.1.2). of 1 mL of solution S, is more intensely coloured than a
Wash the filter with distilled waterR. Collect the filtrate and 2 mgIL solution of potassium permanganate R.
the washings until a volume of 50 mL is obtained. Sulfates (2.4.13)
Appearance of solution Maximum 500 ppm.
The solution is clear (2.2.1) and colourless (2.2.2, Dilute 3 mL of solution S to 15 mL with distilled waterR.
Method 11).

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1-80 Adrenaline 2020

Iron (2.4.9) Solubility

Maximum 10 ppm, determined on solution S. Practically insoluble in water, in ethanol (96 per cent) and in
Loss on drying (2.2.32) . methylene chloride. It dissolves in hydrochloric acid.
Maximum 0.2 per cent, determined on 1.000 g by drying in IDENTIFICATION
an oven at 105°C. A. Infrared absorption spectrophotometry (2.2.24).
Sulfated ash (2.4.14) Comparison adrenaline CRS.
Maximum 0.1 per cent. B. Specific optical rotation (see Tests).
Melt 1.0 g completely over a gas burner, then ignite the
TESTS
melted substance with the burner. After ignition, lower or
remove the flame in order to prevent the substance from
Solution S
boiling and keep it burning until completely carbonised. Dissolve 1.000 g in a 25.75 gIL solution of hydrochloric acid R
and dilute to 50.0 mL with the same solvent. Examine the
Carry out the test for sulfated ash using the residue.
solution immediately.
ASSAY
Appearance of solution
Dissolve 60.0 mg in 50 mL of water R. Add 0.2 mL of Solution S is not more opalescent than reference
phenolphthalein solution R and titrate with 0.1 M sodium suspension II (2.2.1) and not more intensely coloured than
hydroxide. reference solution BYs (2.2.2, Method II).
1 mL of 0.1 M sodium hydroxide is equivalent to 7.31 mg of
Specific optical rotation (2.2.7)
C 6H IQ0 4· -50.0 to -54.0 (dried substance), determined on solution S.
IMPURITIES Related substances
Liquid chromatography (2.2.29). Prepare the solutions protected
from light.
A. pentanedioic acid (glutaric acid), Solvent mixtureA Dissolve 5.0 g of potassium dihydrogen
phosphate Rand 2.6 g of sodium octanesulfonate R in waterfor
~C02H chromatography R and dilute to 1000 mL with the same
H02C
solvent (it is usually necessary to stir for at least 30 min to
achieve complete dissolution). Adjust to pH 2.8 with
B. butanedioic acid (succinic acid), phosphoric acid R.
Solvent mixtureB acetonitrile Rl, solvent mixture A
(13:87 VIV).
C. heptanedioic acid (pimelic acid). Test solution Dissolve 40 mg of the substance to be
examined in 5 mL of 0.1 M hydrochloric acid and dilute to
_ _ _ _ _ _~_-'----~ PhEur
50.0 mL with solvent mixture B.
Reference solution (a) Dilute 1.0 mL of the test solution to
100.0 mL with solvent mixture B. Dilute 1.0 mL of this
solution to 10.0 mL with solvent mixture B.
Adrenaline I Epinephrine Reference solution (b) Dissolve 1.5 mg of noradrenaline
(Ph. Bur. monograph 2303) tartrate CRS (impurity B) and .1.5 mg of adrenalone
hydrochloride R (impurity C) in solvent mixture B, add
1.0 mL of the test solution and dilute to 100 mL with
solvent mixture B.
Reference solution (c) Dissolve the contents of a vial of
adrenaline impurity mixture CRS (containing impurities' D
and E) in 1.0 mL of the blank solution.
183.2 51-43-4 Reference solution (d) Dissolve 4 mg of adrenaline with
impurity F CRS in 0.5 mL of 0.1 M hydrochloric acid and
Action and use dilute' to 5 mL with solvent mixture B.
Adrenoceptor agonist.
Blank solution 0.1 M hydrochloric acid, solvent mixture B
Preparations (1:9 VIV).
Adrenaline Eye Drops/Epinephrine Eye Drops Column:
Dilute Adrenaline Injection (1 in 10,000)lDilute Epinephrine - size: 1= 0.10 m, (2) = 4.6 mm;
Injection (1 in 10,000) - stationary phase: end-capped octadecylsilyl silica gelfor
chromatographyR (3 urn);
PhEur ----------------
- temperature: 50°C.
DEFINITION Mobilephase: .
4-[(1 R)-I-Hydroxy-2-(methylamino) ethyl]benzene-1,2-diol. - mobile phaseA: acetonitrile Rl, solvent mixture A
Synthetic product. (5:95 VIV);
Content ~ mobile phase B: acetonitrile Rl, solvent mixture A
99.0 per cent to 101.0 per cent (dried substance). (45:55 VIV);

CHARACTERS I

Appearance
White or almost white crystalline powder, becoming coloured
on exposure to air and light.

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2020 Adrenaline Acid Tartrate 1-81

Time Mobile phase A Mobile phase B H OH

(min) (per cent Vfll) (per cent VIV) HO~NH2
0-15 92 50 8 50
15 - 20 50 92 50 8 Ho)l)
20 - 25 92 8
B. (lR)- 2-amino-1-(3,4-dihydroxyphenyl)ethanol
(noradrenaline),
Flow rate 2.0 mUmin.
Detection Spectrophotometer at 210 run.
Injection 20~.
Identification of impurities Use the chromatogram supplied
with adrenaline impuritymixture CRS and the chromatogram
obtained with reference solution (c) to identify the peaks due
to impurities D and E; use the chromatogram supplied with C. 1-(3,4-dihydroxyphenyl)-2-(methylamino)ethanone
adrenaline with impurityF CRS and the chromatogram (adrenalone),
obtained with reference solution (d) to identify the peak due
to impurity F. ~
-Relative retention .With reference to adrenaline (retention H.. OH~
time = about 4 min): impurity F = about 0.2;
impurityB = about 0.8; impurity C = about 1.3;
Hoif·~··
I. . N, CH 3

impurity D = about 3.3; impurity E = about 3.7. HO .#

System suitability Reference solution (b):
- resolution: minimum 3.0 between the peaks due to D. 4- [(1R)-2..{benzylmethylamino)-l-hydroxyethyl]benzene-
impurity B and adrenaline. 1,2-diol,
Limits:
- correction factors: for the calculation of content, multiply ('ll~
the peak areas of the following impurities by the
° .
~
corresponding correction factor: impurity D = 0.7;
=
impurity E 0.6; HOm~ I • N, CH3

- impurities B, C, F: for each impurity, not more than twice HO .#

the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.2 per cent); E. 2-(benzylmethylamino)-1-(3,4-dihydroxyphenyl)ethanone,
- impurities D, B: for each impurity, not more than the area
of the principal peak in the chromatogram obtained with
reference solution (a) (0.1 per cent);
~ unspecified impurities: for each impurity, not more than the
area of the principal peak in the chromatogram obtained
with reference solution (a) (0.10 per cent);
- total: not more than 5 times the area of the principal peak F. (lR)-1-(3,4-dihydroxyphenyl)-2-(methylamino)
in the chromatogram obtained with reference solution (a) ethanesulfonic acid.
(0.5 per cent); --- ~ .:.--_ _ PhEur
- disregard limit: 0.5 times the area of the principal peak in
the chromatogram obtained with reference solution (a)
(0.05 per cent).
Loss on drying (2.2.32) Adrenaline Acid Tartrate I
Maximum 0.5 per cent, determined on 1.000 g by drying
over diphosphorus pentoxide R at a pressure not exceeding
Epinephrine Acid Tartrate
0.7 kPa for 18 h. (Adrenaline Tartrate, Ph. Bur. monograph 0254)
Sulfated ash (2.4.14) H\ OH H H OH

if Xy', C0
Maximum 0.1 per cent, determined on 1.0 g. HO ~ N, CH3 ' 2H.
ASSAY
HO
I .#
H0 2C
H OH
Dissolve 0.150 gin 50 mL of anhydrous acetic acid R. Titrate
with 0.1 M perchloric acid, determining the end-point
potentiometrically (2.2.20). 333.3 51-42-3
1mL of 0.1 M perchloric acid is equivalent to 18.32 mg Action and use
of C9 H 13N03 • Adrenoceptor agonist.
STORAGE Preparations
Under nitrogen, protected from light. Adrenaline InjectionlEpinephrine Injection
IMPURITIES Dilute Adrenaline Injection (1 inI O,OOO)/Dilute Epinephrine
Specified impurities B, C, D, E, F. Injection (l in 10,000)
Adrenaline Solution/Epinephrine Solution
Adrenaline and Cocaine Intranasal Solution
Bupivacaine and Adrenaline InjectionlBupivacaine and
Epinephrine Injection

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1-82 Adrenaline Acid Tartrate 2020

Lidocaine and Adrenaline Injection/Lidocaine and 1.0 mL of the test solution and dilute to 100.0 mL with-
Epinephrine Injection solvent mixture B.
PhEur _ Reference solution (c) Dissolve the contents of a vial of
adrenaline impurity mixture CRS (impurities D and E) in
DEFINITION 0.1 mL of 0.1 M hydrochloric acid and 0.9 mL of solvent
(lR)-1-(3~4-Dihydroxyphenyl)-2-(methylamino)ethanol mixture B.
hydrogen (2R,3R)-2~3-dihydroxybutanedioate. Reference solution (d) Dissolve 7.5 mg of adrenaline tartrate
Content with impurity A CRS in 0.5 mL of 0.1 M hydrochloric acid and
98.5 per cent to 101.0 per cent (dried substance). dilute to 5.0 mL with solvent mixture B.
CHARACTERS Blank solution 0.1 M hydrochloric acid, solvent mixture B
Appearance (1:9 VIV).
White or greyish-white, crystalline powder. Column:
Solubility - size: 1 = 0.10 m, 0 = 4.6 mm;
Freely soluble in water, slightly soluble in ethanol - stationaryphase: end-capped octadecylsilyl silica gelfor
(96 per cent). chromatography R (3 urn);
- temperature: 50 "C.
IDENTIFICATION Mobz7e phase:
A. Dissolve 5 gin 50 mL of a 5 gIL solution of sodium - mobilephaseA: acetonitrile R1, solvent mixture A
metabisulfite R and make alkaline by addition of ammonia R. (5:95 VIV);
Keep the mixture at room temperature for at least 15 min - mobile phase B: acetonitrile R1 ~ solvent mixture A .
and filter. Reserve the filtrate for identification test C. Wash (45:55 VIV);
the precipitate with 3 quantities, each of 10 mL, of
methanol R. Dry at 80 "C. The specific optical rotation Time Mobile phase A Mobile phase B
(2.2.7) of the residue (adrenaline base) is -53.5 to -50, (min) (per cent V/JI) (per cent V/JI)
determined using a 20.0 gIL solution in 0.5 M hydrochloric 0-15 92 -> 50 8 -+ 50
acid. 15 - 20 50 -> 92 50 -+ 8
B. Infrared absorption spectrophotometry (2.2.24). 20 - 25 92 8
Preparation Discs of adrenaline base prepared as described
under identification test A. Flow rate 2.0 mUmin.
Comparison Use adrenaline base prepared as described Detection Spectrophotometer at 210 nm.
under identification test A from 50 mg of adrenaline Injection 20 j.tL.
tartrate CRS dissolved in 5 mL of a 5 gIL solution of sodium
Identification of impurities Use the chromatogram supplied
metabisulfite R. Keep the mixture at room temperature for at
with adrenaline impurity mixture CRS and the chromatogram
least 30 min. Filter through a sintered-glass filter (2.1.2).
obtained with reference solution (c) to identify the peaks due
e. 0.2 mL of the filtrate obtained in identification test A to impurities D and E; use the chromatogram supplied with
gives reaction (b) of tartrates (2.3.1). adrenaline tartrate with impurity A CRS and the chromatogram
TESTS obtained with reference solution (d) to identify the peak due
Appearance of solution to impurity A.
The solution is not more opalescent than reference Relative retention With reference to adrenaline (retention
suspension II (2.2.1) and not more intensely coloured than time = about 4 min): impurity B = about 0.8;
reference solution BYs (2.2.2~ Method II). impurity e = about 1.3; impurity A = about 3.2;
Dissolve 0.5 g in water R and dilute to 10 mL with the same impurity D = about 3.3; impurity E = about 3.7.
solvent. Examine the solution immediately. System suitability Reference solution (b):
Related substances - resolution: minimum 3.0 between the peaks due to
Liquid chromatography (2.2.29). Prepare the solutions protected impurity B and adrenaline.
from light. Limits:
Solvent mixture A Dissolve 5.0 g of potassium dihydrogen - correction factors: for the calculation of content, multiply
phosphate R and then 2.6 g of sodium octanesulfonate R in the peak areas of the following impurities by the
waterfor chromatography R, and dilute to 1000 mL with the corresponding correction factor: impurity D = 0.7;
same solvent (it is usually necessary to stir for at least 3.0 min =
impurity E 0.6; .
to achieve complete dissolution). Adjust to pH 2.8 with - impurity A: not more than 3 times the area of the
phosphoric acid R. principal peak in the chromatogram obtained with
reference solution (a) (0.3 per cent);
Solvent mixture B acetonitrile R1, solvent mixture A
(130:870 VIV).
- impurities B~ C: for each impurity, not more than twice the
area of the principal peak in the chromatogram obtained
Test solution Dissolve 75 mg of the substance to be with reference solution (a) (0.2 per cent);
examined in 5 mL of 0.1 M hydrochloric acid and dilute to - impurities D~ E: for each impurity, not more than the area
50 mL with solvent mixture B. of the principal peak in the chromatogram obtained with
Reference solution (a) Dilute 1.0 mL of the test solution to reference solution (a) (0.1 per cent);
100.0 mL with solvent mixture B. Dilute 1.0 mL of this - unspecified impurities: for each impurity, not more than the
solution to 10.0 mL with solvent mixture B. area of the principal peak in the chromatogram obtained
Reference solution (b) Dissolve 1.5 mg of noradrenaline with reference solution (a) (0.10 per cent);
tartrate CRS (impurity B) and 1.5 mg of adrenalone
hydrochloride R (impurity C) insolvent mixture B,add

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2020 Agar 1-83

- total: not more than 6 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
Agar
(0.6 per cent); (Ph. Eur. monograph 0310)
- disregard limit: 0.5 times the area of the principal peak in
the chromatogram obtained with reference solution (a) Action and use
(0.05 per cent). Excipient.
Loss on drying (2.2.32) PhEur _ _- - - - - _
Maximum 0.5 per cent, determined on 1.000 g by drying in
DEFINITION
vacuo for 18 h.
Polysaccharides from various species of Rhodophyceae
Sulfated ash (2.4.14) mainly belonging to the genus Gelidium. It is prepared by
Maximum 0.1 per cent, determined on 1.0 g. treating the algae with boiling water; the extract is filtered
ASSAY whilst hot, concentrated and dried.
Dissolve 0.300 g in 50 mL of anhydrous acetic acid R, heating CHARACTERS
gently if necessary. Titrate with 0.1 M perchloric acid until a Appearance
bluish-green colour is obtained, using 0.1 mL of crystal violet Powder or crumpled strips 2-5 mm wide or sometimes flakes,
solution R as indicator. colourless or pale yellow, translucent, somewhat tough and
1 mL of 0.1 M perchloric acid is equivalent to 33.33 mg difficult to break, becoming more brittle on drying.
of C 13H19NOg • Mucilaginous taste.
STORAGE IDENTIFICATION
In an airtight container, or preferably in a sealed tube under A. Examine under a microscope. When mounted in 0.005 M
vacuum or under an inert gas, protected from light. iodine, the strips or flakes are partly stained brownish-violet.
IMPURITIES Magnified 100 times, they show the following diagnostic
Specified impurities A, B, C, D, E. characters: numerous minute, colourless, ovoid or rounded
A. unknown structure, grains on an amorphous background; occasional brown,
round or ovoid spores with a reticulated surface, measuring
H OH up to 60 urn, may be present. Reduce to a powder, if
HO~NH, necessary. The powder is yellowish-white. Examine under a
microscope using 0.005 M iodine. The powder presents
HoN angular fragments with numerous grains similar to those seen
in the strips and flakes; some of the fragments are stained
brownish-violet.
B. (IR)-2-amino-I-(3,4-dihydroxyphenyl)ethanol
(noradrenaline), B. Dissolve 0.1 g with heating in 50 rnL of water R. Cool.
To 1 mL ofthe mucilage carefully add 3 mL of water R so as
o to form 2 separate layers. Add 0.1 ml, of 0.05 M iodine.

HO~,~.~,
A dark brownish-violet colour appears at the interface. Mix.
I CH3 The liquid becomes pale yellow.
HO # C. Heat 5 ml..of the mucilage prepared for identification
test B on a water-bath with 0.5 mL of hydrochloric acid R for
C. 1-(3,4-dihydroxyphenyl)-2-(methylamino)ethanone 30 min. Add 1 mL of barium chloride solution Rl. A white
(adrenalone), turbidity develops within 30 min.
D. Heat 0.5 g with 50 mL of water R on a water-bath until
dissolved. Only a few fragments remain insoluble. During
(') cooling, the solution gels between 35 DC and 30 DC. Heat the
Hv OH·~ gel thus obtained on a water-bath; it does not liquefy below
Hoif~"
I N....
CH 3
80 DC.
TESTS
HO #
Swelling index (2.8.4)
Minimum 10 and within 10 per cent of the value stated on
D.4-[(IR)-2-(be~lmethylamino)-1-hydroxyethyl]benzene­ the label, determined on the powdered herbal drug (355)
1,2-diol, (2.9.12).
Insoluble matter
("I., ~ Maximum 1.0 per cent.
o ~ To 5.00 g ofthe powdered herbal drug (355) (2.9.12) add

HO~~.
I . ,N .... CH 3
100 mL of water Rand 14 mL of dilute hydrochloric acid R.
Boil gently for 15 min with frequent stirring. Filter the hot
HO # liquid through a tared, sintered-glass filter (160) (2.1.2), rinse
the filter with hot water R and dry at 100-105 DC.
The residue weighs a maximum of 50 mg.
E. 2-(benzylmethylamino)-1-(3,4-dihydroxyphenyl)ethanone.
_---'- ---'----'- PhEur Gelatin
To 1.00 gadd 100 mL of waterR and heat on a water-bath
until dissolved. Allow to cool to 50 DC. To 5 mL of this

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1-84 Air 2020

solution add 5 mL of picric acid solution R. No turbidity CHARACTERS

appears within 10 min. Appearance
Loss on drying (2.2.32) Colourless gas.
Maximum 20.0 per cent, determined on 1.000 g of the Solubility
powdered herbal drug (355) (2.9.12) by drying in an oven at At 20°C at a pressure of 101 kPa, 1 volume dissolves in
105°C. about 50 volumes of water.
Total ash (2.4.16) PRODUCTION
Maximum 5.0 per cent. Carbon dioxide
Microbial contamination Maximum 500 ppm V/~ determined using an infrared
TAMC: acceptance criterion 103 CFU/g (2.6.12). analyser (2.5.24).
TYMC: acceptance criterion 102 CFU/g (2.6.12). Gas to be examined Filter the substance to be examined to
Absence of Escherichia coli (2.6.13). avoid stray light phenomena.
Absence of Salmonella (2.6.13). Reference gas (a) Use a mixture of 21 per cent V/Vof
oxygen Rand 79 per cent V/Vof nitrogen Rl, containing less
LABELLING than 1 ppm V/V of carbon dioxide Rl.
The label states the swelling index.
Reference gas (b) Use a mixture of 21 per cent V/Vof
_ _ _ _ _ _ _ _ _ _ _ _- - - - - - - - PhEur oxygen Rand 79 per cent V/V of nitrogen Rl, containing
500 ppm V/Vof carbon dioxide Rl.
Calibrate the apparatus and set the sensitivity using reference
gases (a) and (b). Measure the content of carbon dioxide in
Medical Air the gas to be examined.
(Medicinal Air, Ph. Eur. monograph 1238)
Carbon monoxide
Maximum 5 ppm V/V, determined using an infrared analyser
When Medical Air is intended for use in a room in which (2.5.25).
magnetic resonance im,aging (MRl) is being performed, the Gas to be examined Filter the substance to be examined to
cylinder and fittings should be made from suitable non- avoid stray light phenomena.
ferromagnetic materials and labelled accordingly.
Reference gas (a) Use a mixture of 21 per cent V/Vof
PhEur _
oxygen Rand 79 per cent V/Vof nitrogen Rl, containing less
DEFINITION than 1 ppm V/V of carbon monoxide R.
Compressed ambient air. Reference gas (b) Use a mixture of 21 per cent V/Vof
Content oxygen Rand 79 per cent V/Vof nitrogen Rl, containing
20.4 per cent V/Vto 21.4 per cent V/Vof oxygen (0 2) , 5 ppm V/Vof carbon monoxide R.
Calibrate the apparatus and set the sensitivity using reference
gases (a) and (b). Measure the content of carbon monoxide
in the gas to be examined.

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2020 Air 1-85

Reference gas (b) Use a mixture of 21 per cent VIVof

oxygen R and 79 per cent VIVof nitrogen Rl, containing
0.5 ppm VITI to 2 ppm VIV of sulfurdioxide Rl,
Calibrate the apparatus and set the sensitivity using reference
gases (a) and (b). Measure the content of sulfur dioxide in
the gas to be examined.
Oil
1+---25mm
Maximum 0.1 mg/rrr', determined using an oil detector tube
(2.1.6), when an oil-lubricated compressor is used for the
production.
Nitrogen monoxide and nitrogen dioxide
Maximum 2 ppm VIV in total, determined using a
chemiluminescence analyser (2.5.26).
Gas to be examined The substance to be examined.
Reference gas (a) Use a mixture of 21 per cent VIVof
oxygen Rand 79 per cent VIVof nitrogen Rl, containing less
than 0.05 ppm VIV of nitrogen monoxide and nitrogen
dioxide.
Reference gas (b) Use a mixture of 2 ppm VIV of nitrogen
monoxide R in nitrogen Rl.
Calibrate the apparatus and set the sensitivity using reference
gases (a) and (b). Measure the content of nitrogen monoxide
23.0 % and nitrogen dioxide in the ,gas to be examined.
Water
Maximum 67 ppm VIll, determined using an electrolytic
hygrometer (2.5.28), except where the competent authority
210mm decides that the following limit applies to medicinal air
21.0 % generated on-site and distributed in pipe-line systems
operating at a pressure not greater than lObar and a
temperature not less than 5°C: maximum 870 ppm VIll;
determined using an electrolytic hygrometer (2.5.28).
Assay
Determine the concentration of oxygen in air using a
paramagnetic analyser (2.5.27).
IDENTIFICATION
First identification: C.
Second identification: A, B.
A. In a conical flask containing the substance to be
examined, place a glowing wood splinter. The splinter
remains glowing.
B. Use a gas burette (Figure 1238.-2) of 25 mL capacity in
the form of a chamber in the middle of which is a tube
graduated in 0.2 per cent between 19.0 per cent and
23.0 per cent, and isolated at each end by a tap with a
conical barrel. The lower tap is joined to a tube with an
Figure 1238.-2. - Gas burette olive-shaped nozzle and is used to introduce the gas into the
apparatus. A cylindrical funnel above the upper tap is used to
Sulfur dioxide introduce the absorbent solution. Wash the burette with
Maximum 1 ppm VIV, determined using an ultraviolet water R and dry. Open the 2 taps. Connect the nozzle to the
fluorescence analyser (Figure 1238.-1). source of the gas to be examined and set the flow rate to
The apparatus consists of the following: 1 Umin. Flush the burette by passing the gas to be examined
- a system generating ultraviolet radiation with a wavelength through it for l min. Close the lower tap of the burette and
of 210 nm, made up of an ultraviolet lamp, a collimator, immediately afterwards the upper tap. Rapidly disconnect the
and a selective filter; the beam is blocked periodically by a burette from the source of the gas to be examined. Rapidly
chopper rotating at high speeds; give a half turn to the upper tap to eliminate any excess
- a reaction chamber, through which flows the.gas to be pressure in the burette. Keeping the burette vertical, fill the
examined; funnel with a freshly prepared mixture of 21 mL of a 560 gIL
- a system that detects radiation emitted at a wavelength of solution of potassium hydroxide Rand 130 mL of a 200 gIL
350 nm, made up of a selective filter, a photomultiplier solution of sodium dithionite R. Open the upper tap slowly.
tube and an amplifier. The solution absorbs the oxygen and enters the burette.
Allow to stand. for 10 min without shaking. Read the level of
Gas to be examined Filter the substance to be examined.
the liquid meniscus on the graduated part of the burette.
Reference gas (a) Use a mixture of 21 per cent VIVof
oxygen Rand 79 per cent VIV of nitrogen Rl . .

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1-86 Air 2020

This figure represents the percentage VIV of oxygen.

The value read is 20.4 to 21.4.
C. It complies with the limits of the assay.
TESTS
Carbon dioxide
Maximum 500 ppm VI1/, determined using a carbon dioxide
detector tube (2.1.6). K---25mm
Sulfur dioxide
Maximum 1 ppm VIl1, determined using a sulfur dioxide
detector tube (2.1.6).
Oil
Maximum 0.1 mg/rrr', determined using an oil detector tube
(2.1.6), when an oil-lubricated compressor is used for the
production.
Nitrogen monoxide and nitrogen dioxide
Maximum 2 ppm VIl1, determined using a nitrogen
monoxide and nitrogen dioxide detector tube (2.1.6).
Carbon monoxide
Maximum 5 ppm VIr; determined using a carbon monoxide 370mm
detector tube (2.1. 6).
Water vapour
Maximum 67 ppm VIV; determined using a water vapour 23.0%
detector tube (2.1.6), except where the competent authority
decides that the following limit applies to medicinal air
generated on-site and distributed in pipe-line systems
operating at a pressure not greater than 10 bar and a
temperature not less than 5°C: maximum 870 ppm VITl, 210 mm
determined using a water vapour detector tube (2.1.6). 21.0 %

STORAGE
---+it!"tl~-- 8 mm
As a gas, in suitable containers complying with the legal
regulations or as a gas supplied by a pipe network.
LABELLING
Where applicable, the label states the production method, as 19.0 %
regards to the use of an oil - lubricated compression.
IMPURITIES
A. CO 2 : carbon dioxide,
B. S02: sulfur dioxide,
C. NO: nitrogen monoxide,
D. N02 : nitrogen dioxide,
E. oil,
F. CO: carbon monoxide,
G. H 2 0 : water.
___ ~ PhEur

Figure 1684.-1.- Gas burette

CHARACTERS
Synthetic Air Colourless and odourless gas.
(Synthetic Medicinal Air) Ph. Bur. monograph 1684) Solubility
At a temperature of 20°C and a pressure of 101 kPa,
When Synthetic Air is intended for use in a room in which
1 volume dissolves in about 50 volumes of water.
magnetic resonance imaging (MRl) is being performed; the
cylinder and fittings should be made from suitable non- , PRODUCTION
ferromagnetic materials and labelled accordingly. Water (2.5.28)
PhEur -'- _ Maximum 67 ppm VIV.
Assay (2.5.27)
DEFINITION
Carry out the determination of oxygen in gases.
Mixture of Nitrogen (1247) and Oxygen (0417).
IDENTIFICATION
Content
First identification: C.
95.0 per cent to 105.0 per cent of the nominal value which is
between 21.0 per cent VIVto 22.5 per cent VIVof oxygen Second identification: A) B.
(0 2 ) .

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2020 Alanine 1-87

A. In a conical flask containing the substance to be CHARACTERS

examined, place a glowing splinter of wood. The splinter Appearance
remains glowing. White or almost white, crystalline powder or colourless
B. Use a gas burette (Figure 1684.-1) of 25 mL capacity in crystals.
the form of a chamber, in the middle of which is a tube Solubility
graduated in 0.2 per cent between 19.0 per cent and Freely soluble in water, very slightly soluble in ethanol
23.0 per cent, and isolated at each end by a tap with a (96 per cent).
conical barrel. The lower tap is joined to a tube with an
IDENfIFICATION
olive-shaped nozzle and is used to introduce the gas into the
apparatus. A cylindrical funnel above the upper tap is used to Firstidentification: A, B.
introduce the absorbent solution. Wash the burette with Second identification: A,C, D.
waterR and dry. Open both taps. Connect the nozzle to the A. Specific optical rotation (see Tests).
source of the substance to be examined and set the flow rate B. Infrared absorption spectrophotometry (2.2.24).
to 1 Umin. Flush the burette by passing the substance to be
Comparison alanine CRS.
examined through it for 1 min. Close the lower tap of the
burette and immediately afterwards the upper tap. Rapidly C. Thin-layer chromatography (2.2.27).
disconnect the burette from the source of the substance to be Test solution Dissolve 10 mg of the substance to be
examined. Rapidly give a half turn of the upper tap to examined in waterR and dilute to 50 mL with the same
eliminate any excess pressure in the burette. Keeping the solvent.
burette vertical, fill the funnel with a freshly prepared mixture Reference solution Dissolve 10 mg of alanine CRS in waterR
of 21 mL of a 560 gIL solution of potassium hydroxide Rand and dilute to 50 mL with the same solvent.
130 mL of a 200 gIL solution of sodium dithionite R. Open Plate TLC silica gelplate R.
the upper tap slowly. The solution absorbs the oxygen and
Mobile phase glacial acetic acidR, waterR, butanol R
enters the burette. Allow to stand for 10 min without
(20:20:60 VIVIV).
shaking. Read the level of the liquid meniscus on the
graduated part of the burette. This figure represents the Application 5).lL.
percentage VIVof oxygen. The value read is 95.0 per cent to Development Over 2/3 of the plate.
105.0 per cent of the nominal value. Drying In air.
C. It complies with the limits of the assay. Detection Spray with ninhydrin solution R and heat at 105°C
for 15 min.
TESTS
Water vapour Results The principal spot in the chromatogram obtained
Maximum 67 ppm VIJ7, determined using a water vapour with the test solution is similar in position, colour and size to
detector tube (2.1. 6). the principal spot in the chromatogram obtained with the
reference solution.
STORAGE
D. Dissolve 0.5 g in a mixture of 0.25 mL of hydrochloric
As a compressed gas in suitable containers complying with
acidR1, 0.5 mL of a 100 gIL solution.of sodium nitrite Rand
the legal regulations or as a compressed gas supplied by a
1 ml, of waterR. Shake; gas is given off. Add 2 mL of dilute
pipe network, after mixing of the components.
sodium hydroxide solution R, followed by 0.25 mL of iodinated
LABELLING potassium iodide solution R. After about 30 min, a yellow
The label states the nominal content of O 2 in per cent VIV. precipitate is formed.
IMPURITIES TESTS
A. H 20: water. Solution S
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur Dissolve 2.5 g in distilled water R and dilute to 50 mL with
the same solvent.
Appearance of solution
The solution is clear (2.2.1) and not more intensely coloured
Alanine than reference solution BY6 (2.2.2, Method II).
Dilute 10 mL of solution S to 20 mL with water R.
(Ph. Bur. monograph 0752) Specific optical rotation (2.2.7)
+ 13.5 to + 15.5 (dried substance).
Dissolve 2.50 g in hydrochloric acidR1 and dilute to 25.0 mL
with the same acid.
Ninhydrin-positive substances
89.1 56-41-7 Amino acid analysis (2.2.56). For analysis, use Method 1.
The concentrations of the test solution and the reference
Action and use
solutions may be adapted according to the sensitivity of the
Amino acid.
equipment used. The concentrations of all solutions are
PhEur -,.... _ adjusted so that the system suitability requirements described
in general chapter 2.2.46 are fulfilled, keeping the ratios of
DEFINITION concentrations between all solutions as described.
(2S)-2-Aminopropanoic acid.
Solution A dilute hydrochloric acidR1 or a sample preparation
Content buffer suitable for the apparatus used.
98.5 per cent to 101.0 per cent (dried substance).

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 1-88 Albendazole 2020

Test solution Dissolve 30.0 mg of the substance to be Loss on drying (2.2.32)

examined in solution A and dilute to 50.0 mL with Maximum 0.5 per cent, determined on 1.000 g by drying in
solution A. an oven at 105°e.
Reference solution (a) Dilute 1.0 mL of the test solution to Sulfated ash (2.4.14)
100.0 mLwith solution A. Dilute 2.0 mL of this solution to Maximum 0.1 per cent, determined on 1.0 g.
10.0 mL with solution A.
ASSAY
Reference solution (b) Dissolve 30.0 mg of proline R in Dissolve 80.0 mg in 3 mL of anhydrous formic acid R.
solution A and dilute to 100.0 mL with solution A. Dilute Add 30 mL of anhydrous acetic acid R. Titrate with 0.1 M
1.0 mL of the solution to 250.0 mL with solution A. perchloric acid, determining the end-point potentiometrically
Reference solution (c) Dilute 6.0 mL of ammonium standard (2.2.20).
solution (l00 ppm NH4J R to 50.0 mL with solution A. Dilute 1 mL of 0.1 M perchloric acid is equivalent to 8.91 mg of
1.0 mL of this solution to 100.0 mL with solution A. C3H7N02 •
Reference solution (d) Dissolve 30 mg of isoleucine Rand
30 mg of leucine R in solution A and dilute to 50.0 mL with
STORAGE
solution A. Dilute 1.0 mL of the solution to 200.0 mL with Protected from light.
solutionA. IMPURITIES
Blank solution Solution A. Otherdetectable impurities (the following substances would, if
Inject suitable, equal amounts of the test, blank and reference present at a sufficient level, be detected by one or other of the tests
solutions into the amino acid analyser. Run a program in the monograph. They are limited by the general acceptance
suitable for the determination of physiological amino acids. criterion for other/unspecified impurities and/or by the general
monograph Substances for pharmaceutical use (2034). It is
System suitability Reference solution (d):
therefore not necessary to identify these impurities for
- resolution: minimum 1.5 between the peaks due to
demonstration of compliance. See also 5.10. Control of impurities
isoleucine and leucine.
in substances for pharmaceutical use) A, B'.
Calculation of percentage contents:
- for any ninhydrin-positive substance detected at 570 nm,
use the concentration of alanine in reference solution (a);
, - for any ninhydrin-positive substance detected at 440 nm,
use the concentration of proline in reference solution (b);
A. (2S)-2-aminobutanedioic acid (aspartic acid),
if a peak is above the reporting threshold at both
wavelengths, use the result obtained at 570 nm for
quantification.
Limits:
- any ninhydrin-positive substance: for each impurity,
maximum 0.10 per cent; B. (2S)-2-aminopentanedioic acid (glutamic acid).
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
- total: maximum 0.5 per cent;
- reporting threshold: 0.05 per cent.
Chlorides (2.4.4)
Maximum 200 ppm.
Dilute 5 mL of solution S to 15 mL with waterR. Albendazole
Sulfates (2.4.13) (Ph. Bur. monograph 1386)
, Maximum 300 ppm.
Dilute 10 mL of solution S to 15 mL with distilled water R.
' H
N
0
}-OCH3
Ammonium
Amino acid analysis (2.2.56) as descnbed in the test for
ninhydrin-positive substances with the following
HC
3 ~s DI s;
""'" N
}--NH

modifications.
265.3 54965-21-8
Injection Test solution, reference solution (c) and blank
solution. Action and use
Limit: Benzimidazole antihelminthic.
- ammonium at 570 nm: not more than the area of the Preparations
corresponding peak in the chromatogram obtained with Albendazole Oral Suspension
reference solution (c) (0.02 per cent), taking into account
Albendazole Oral Suspension with Minerals
the peak due. to ammonium in the chromatogram
obtained with the blank solution. PhEur _ _-'-- ~ _

Iron (2.4.9) DEFlNmON

Maximum 10 ppm. Methyl N- [5-(propylsulfanyl)-lH-benzimidazol-2-yl]
In a separating funnel, dissolve 1.0 gin 10 mL of dilute carbamate.
hydrochloric acid R. Shake with 3 quantities, each of 10 mL, Content
of methyl isobutyl ketoneR1, shaking for 3 min each time.
99.0 per cent to 101.0 per cent (dried substance).
To the combined organic layers add 10 mL of water Rand
shake for 3 min. Use the aqueous layer. CHARACTERS
Appearance
White or slightly yellowish powder.

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2020 Albendazole 1-89

Solubility Calculation of percentage contents:

Practically insoluble in water, freely soluble in anhydrous - correction factors: multiply the peak areas of the following
formic acid, very slightly soluble in methylene chloride, impurities by the corresponding correction factor:
practically insoluble in ethanol (96 per cent). impurity A = 1.7; impurities Band C = 1.4;
It shows polymorphism (5.9). impurity D = 1.9; impurity E = 1.4;
- for each impurity, use the concentration of albendazole in
IDENTIFICATION reference solution (a).
Infrared absorption spectrophotometry (2.2.24).
Limits:
Comparison albendazole CRS. - impurity H: maximum 0.6 per cent;
If the spectra obtained in the solid state show differences, - impurity F: maximum 0.5 per cent;
dissolve the substance to be examined and the reference - impurity A: maximum 0.4 per cent;
substance separately in methylene chloride R, evaporate to - sum of impurities Band C: maximum 0.4 per cent;
dryness and record new spectra using the residues. - impurity E: maximum 0.3 per cent;
TESTS - impurity D: maximum 0.2 per cent;
Appearance of solution - unspecified impurities: for each impurity, maximum
The solution is clear (2.2.1) and not more intensely coloured 0.10 per cent;
than reference solution BY6 (2.2.2) Method II). - total: maximum 1.3 per cent;
- reporting threshold: 0.05 per cent.
Dissolve O.IO.g in a mixtureof 10 volumes of anhydrous
formic-acid Rand 90 volumes of methylene chloride Rand Loss on drying (2.2.32)
diluteto 10 mL with the same mixture of solvents. Maximum 0.5 per cent, determined on 1.000 g by drying in
an oven at 105 DC for 4 h.
Related substances
Liquid chromatography (2.2.29). Prepare the solutions Sulfated ash (2.4.14)
immediately before use. Maximum 0.2 per cent, determined on 1.0 g.
Solvent mixture sulfuric acid R, methanolR (1:99 VIV)o ASSAY
Test solution Dissolve 25.0 mg of the substance to be In order to avoid overheating duringthe titration) mix thoroughly
examined in 5 mL of the solvent mixture and immediately throughout and stop the titration immediately after the end-point
dilute to 50.0 mL with the mobile phase. has been reached.
Reference solution (a) Dilute 1.0 mL of the test solution to Dissolve 0.250 g in 3 mL of anhydrous formic acid R and add
100.0 mL with the mobile phase. Dilute 1.0 mL of this 40 mL of anhydrous acetic acid R. Titrate with 0.1 M
solution to 10.0 mL with the mobile phase. perchloric acid, determining the end-point potentiometrically
(2.2.20).
Reference solution (b) Dissolve 5 mg of albendazole for system
suitability CRS (containing impurities B, C, E, F and H) in 1 mL of 0.1 M perchloric acid is equivalent to 26.53 mg
I mL of the solvent mixture and dilute to 10 mL with the of ClzH15N30ZS.
mobile phase. STORAGE
Reference solution (c) Dilute I mL of the solvent mixture to Protected from light.
10 mL with the mobile phase. Use I mL of this solution to
IMPURITIES
dissolve the contents of a vial of albendazole
Specifiedimpurities A) B) C) D) E) F) H.
impurity mixture CRS (containing impurities A and D).
Other detectable impurities (thefollowing substances would) if
Column:
present at a sufficientlevel) be detected by one or otherof the tests
- size: l = 0.25 m, (2) = 4.6 mm;
in the monograph. They arelimited by the general acceptance
- stationaryphase: end-capped octadecylsilyl silica gelfor
criterion for other/unspecified impurities and/or by the general
chromatographyR (5 urn).
monograph Substances for pharmaceutical use (2034). It is
Mobilephase 1.67 gIL solution of ammonium dihydrogen therefore not necessary to identify these impurities for
phosphate R, methanol R (30:70 V/V). demonstration of compliance. See also 5.10. Control. of impurities
Flow rate 0.7 mIJmin. in substances for pharmaceutical use) G) I) J) K) L.
Detection Spectrophotometer at 254 nm,
Injection 20 JlL.
Run time Twice the retention time of albendazole.
Identification of impurities Use the chromatogram supplied
with albendazole impurity mixture CRS and the chromatogram
obtained with reference solution (c) to identify the peaks due A. 5-(propylsulfanyl)-IH-benzimidazol-2-amine,
to impurities A and D; use the chromatogram supplied with
albendazole for system suitability CRS and the chromatogram
obtained with reference solution (b) to identify the peaks due
to impurities B + C, E, F and H. HC
3 ~s
*
...
DI
s;
~
H
N

N
)-NH
0
} - OCH3

Relative retention With reference to albendazole (retention II

o
time = about II min): impurity D = about 0.35;
impurities Band C = about 0.40; impurity E = about 0.45;
B. methyl N-[5-(propylsulfinyl)~lH-benzimidazol-2­
impurity A = about 0.48; impurity F =about 0.57;
yl]carbamate,
impurity H = about 0.66.
System suitability Reference solution (b):
- resolution: minimum 1.5 between the peaks due to
impurities B + C and E.

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1-90 Alcuronium Chloride 2020

H 0
N }-OCH3
HC
3 ~s

o 0
DI
,,~
s;
~ N
}-NH

L.methyl N-[5-[(propan-2-yl)sulfanyl] -lH-benzimidazol-2-

C. methyl N- [5-(propylsulfonyl)-1H-benzimidaz~l- 2- yl]carbamate.
yl]carbamate, _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur

AlcuroniumChloride
(Ph. Bur. monograph 1285)
D. (2-amino-1H-benzimidazol-5-yl)propyl-A6-sulfanedione,

2 cr
E. methyl N-(1H-benzimidazol-2-yl)carbamate,

738 15180-03-7

F. methyl N-[5-(methylsulfanyl)-IH-benzimidazol-2- Action and use

yl]carbamate, Non-depolarizing neuromuscular blocker.
PhEur _--'-
o ~_

CI
1):I ~'r
-::? .

~ N
NH
) - OCH3 DEFINITION
(lR,3aS,10S,11aS,12R,14aS,19aS,20bS,21S,22aS,23B,26E)-
23,26-bis(2-Hydroxyethylidene)-1,12-bis(prop-2-enyl)-
2,3,11,lla, 13,14,22,22a-oetahydro:"10H,21H-l,21:1O, 12-
G. methyl N-(5-chloro-lH-benzirnidazol-2-yl)carbamate, diethano-19aH,20bH-[l,5]diazocino[1,2,3-lm:5,6,7-l'm']
dipyrrolo [2' ,3'-d:2" ,3" :d']dicarbazolediium dichloride (4,4'-
didesmethyl-4,4'-bis(prop-2-enyl)toxiferin I dichloride).
Content
98.0 per cent to 102.0 per cent (anhydrous substance).
CHARACTERS
H. methyl N-[5-[ (2-methyl-4-oxopentan-2-yl)sulfanyl] -IH- Appearance
benzimidazol-2-yl]carbamate, White or slightly greyish-white, crystalline powder.

·I 'r
~
o
}-OCH,
Solubility
Freely soluble in water and in methanol, soluble in ethanol
HC
3 ~o.
.
D ~~ N
NH (96 percent), practically insoluble in cyclohexane.
Carry out the identification, tests and assay as rapidly as possible
avoiding exposure to actinic light.
1. methyl N-(5-propoxy-lH-benzimidazol-2-yl)carbamate, IDENTIFICATION
Firstidentification: A, C.
o
CI.. \X..I ~}-OCH3
~
'r N
NH
Secondidentification: B, C.
A. Infrared absorption spectrophotometry (2.2.24).
Comparison alcuronium chloride CRS.
CI B. Thin-layer chromatography (2.2.27).
Test solution Dissolve 10 mg of the substance to be
J. methyl N-(4,6-dicbloro-lH-benzimidazol-2-yl)carbamate, examined in methanolR and dilute to 10 mL with the same
solvent.
Reference solution Dissolve 10 mg of alcuronium chloride CRS
in methanolR and dilute to 10 mL with the same solvent.
Plate TLC silica gelplate R.
Mobile phase Mix 15 volumes ofa 58.4 gIL solution of
K. methyl N-[5-(butylsulfanyl)-lH-benzimidazol-2-yl] sodium chloride R, 35 volumes of dilute ammonia R2 and
carbamate, 50 volumes of methanolR.

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 2020 Alcuronium Chloride 1-91

Application 10 llL. - resolution: minimum 4.0 between the peaks due to

Development Over a path of 15 cm. N-allylstrychnine and alcuronium.
Drying In air for 10 min. Limits:
Detection Spray with 0.1 M ammonium and cerium nitrate. - impurities A, B: for each impurity, not more than the area
of the principal peak in the chromatogram obtained with
Results The principal spot in the chromatogram obtained reference solution (a) (0.5 per cent) and not more than
with the test solution is similar in position, colour and size to
one of the peaks has an area greater than the area of the
the principal spot in the chromatogram obtained with the
principal peak in the chromatogram obtained with
reference solution. reference solution (b) (0.2 per cent);
C. It gives reaction (a) of chlorides (2.3.1). - total: not more than twice the area of the principal peak in
TESTS the chromatogram obtained with reference solution (a)
Solution S (1 per cent);
Dissolve 0.250 g in carbon dioxide-free waterR and dilute to - disregard limit: the area of the principal peak in the
25.0 mL with the same solvent. chromatogram obtained with reference solution (c)
(0.05 per cent).
Appeuanceofsmution
Solution S is clear (2.2.1) and not more intensely coloured Water (2.5.12)
than reference solution Y6, BY6 or B6 (2.2.2, Method 1). Maximum 5.0 per cent, determined on 0.500 g.
Acidity or alkalinity Sulfated ash (2.4.14)
To 10 mLof solution S add 0.1 mL of methyl redsolution R Maximum 0.1 per cent, determined on 1.0 g.
and 0.2 mL of 0.01 M hydrochloric acid. The solution is red. ASSAY
Add 0.4 mL of 0.01 M sodium hydroxide. The solution is Dissolve 0.300 g by stirring in 70 mL of acetic anhydride R
yellow. for 1 min. Titrate with 0.1 M perchloric acid until the colour
Specific optical rotation (2.2.7) changes from violet-blue to greenish-blue, using 0.1 mL of
-430 to -451 (anhydrous substance), determined on crystal violet solution R as indicator.
solution S. 1 mL of 0.1 M perchloric acid is equivalent to 36.9 mg
Propan-2-o1 (2.4.24, System A) of C44HsoClzN40z.
MaximumLfl per cent. STORAGE
Related substances In an airtight container under nitrogen, protected from light,
Liquid chromatography (2.2.29). at a temperature of 2 °C to 8°C.
Solvent mixture Mix 100 mL of methanol R, 200 mL of IMPURITIES
acetonitrile R and 200 mL of a 6.82 gIL solution of potassium Specified impurities A., B.
dihydrogen phosphate R. Dissolve 1.09 g of sodium
laurylsulfonate for chromatography R in the mixture and adjust
the apparent pH to 8.0 with a 100 gIL solution of sodium
hydroxide R.
Test solution Dissolve 0.20 g of the substance to be
examined in the solvent mixture and dilute to 100.0 mL with 2Cf
. the solvent mixture.
Reference solution (a) Dilute 0.5 mL of the test solution to
100.0 mL with the solvent mixture.
Reference solution (b) Dilute 4.0 mL of reference solution (a)
to 10.0 mL with the solvent mixture. A. (lR,3aS, 9R, 9aR, lOR, 11as, 12R, 14aS, 19a5,20R,20aR,
Reference solution (c) Dilute 1.0 mL of reference solution (a) 20bS,21R,22aS)-1,12-bis(prop-2-enyl)-
to 10.0 mL with the solvent mixture. 2,3,9a, II,l1a,13, 14,19a,20a,21,22,22a-dodecahydro-
Reference solution (d) To 5.0 mL of the test solution add 1OH,20bH-l,23: 12,27-dimethano-9, 1O:20,21-bis
5.0 mg of allylstrychnine bromide CRS, dissolve in the solvent (epoxyprop [2]eno)-9H,20H-[1,5]diazocino [1,2,3-lm:5,6,7-
mixture and dilute to 100.0 mL with the solvent mixture. rm'] dipyrrolo [2' ,3'-d:2" ,3" :d']dicarbazolediium
Column: dichloride (4,4'-diallylcaracurin V dichloride),
=
- size: 1 0.25 m, 0 = 4 mrn;
- stationary phase: octylsilyl silica gelfor chromatography R
(5lJ.IIl).
Mobile phase Mix 200 mL of methanol R, 400 mL of
acetonitrile Rand 400 mL of a 6.82 gIL solution of potassium
dihydrogen phosphate R. Dissolve 2.18 g of sodium
laurylsulfonate for chromatography R in the mixture and adjust
the apparent pH to 5.4 with a 100 gIL solution of phosphoric
acidR. B. (4bS, 7R, 7 as,8aR, 13R, 13aR, 13bS)-13-hydroxy-7-(prop-2-
Flow rate 1.2 mlJrnin. enyl)-5,6,7a,8,8a,11,13,13a,13b,14-decahydro-7,9-
Detection Spectrophotometer at 254 nm. methano-7H-oxepino [3,4-a]pyrrolo [2, 3-d]carbazolium
Injection 10 llL.
«
chloride 4R, 17R)-4-allyl-17,18-epoxy-17-hydroxy-19,20-
didehydrocuranium chloride) .
. Run time Twice the retention time of alcuronium. _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
System suitability Reference solution (d):

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1-92 Alfacalcidol 2020

Column:
Alfacalcidol - size: 1 = 0.25 m, (2) = 4.6 mm;
(Ph. Eur. monograph 1286) - stationary phase: end-capped octadecylsilyl silica gelfor
chromatography R (5 urn).
Mobile phase ammonia R, waterR, acetonitrile R
(1:200:800 V/V/V).
Flow rate 2.6 mLImin.
Detection Spectrophotometer at 265 run.
Injection 100 JlL of the test solution and reference
solutions (b) and (c).
Run time Twice the retention time of alfacalcidol.
Identification of impurities Use the chromatogram supplied
with alfacalcidol for system suitability CRS and the
chromatogram obtained with reference solution (c) to identify
400.6 41294-56-8 the peaks due to impurities A and B.
Relative retention With reference to alfacalcidol (retention
Action and use =
time about 21 min): pre-alfacalcidol = about 0.88;
Vitamin D analogue. impurity A = about 0.93; impurity B = about 1.1.
PhEur _ System suitability Reference solution (c):
- resolution: minimum 15 between the peaks due to pre-
DEFINITION alfacalcidol and impurity A and minimum 1.5 between
(5Z,7£)-9,1 0-Secocholesta-5,7,10(l9)-triene-l cx:,3P-diol. the peaks due to impurity A and alfacalcidol.
Content Limits:
97.0 per cent to 102.0 per cent. - impurities A, B: for each impurity, maximum 0.5 per cent;
A reversible isomerisation to pre-alfacalcidol takes place in - unspecified impurities: for each impurity, maximum
solution, depending on temperature and time. The activity is 0.10 per cent;
due to both compounds (see Assay). - total: maximum 1.0 per cent;
- disregard limit: the area of the principal peak in the
CHARACTERS chromatogram obtained with reference solution (b)
Appearance (0.05 per cent); disregard the peak due to pre-alfacalcidol.
White or almost white crystals.
ASSAY
Solubility
Liquid chromatography (2.2.29) as described in the test for
Practically insoluble in water, freely soluble in ethanol
related substances with the following modifications.
(96 per cent), soluble in fatty oils.
Injection Test solution and reference solutions (a) and (c).
It is sensitive to air, heat and light.
System suitability Reference solution (c):
IDENTIFICATION - repeatability: maximum relative standard deviation of
A. Infrared absorption spectrophotometry (2.2.24). 1 per cent for the peak due to alfacalcidol after
Comparison Ph. Eur. reference spectrum of alfacalcidol. 6 injections.
B. Examine the chromatograms obtained in the test for Calculate the percentage content of CZ7H440Z taking into
related substances. account the assigned content of alfacalcidol CRS and, if
Results The principal peak in the chromatogram obtained necessary, the peak due to pre-alfacalcidol.
with the test solution is similar in retention time and size to STORAGE
the principal peak in the chromatogram obtained with Under nitrogen, in an airtight container, protected from light,
reference solution (a). at a temperature of 2 °C to 8°C.
TESTS The contents of an opened container are to be used
Related substances immediately.
Liquid chromatography (2.2.29): use the normalisation IMPURITIES
procedure. Carry out the testas rapidly as possible, avoiding
Specified impurities A, B.
exposure to light and air.
Other detectable impurities (thefollowing substances would, if
Testsolution Dissolve 1.0 mg of the substance to be
present at a sufficient level, be detected by oneor otherof the tests
examined without heating in 10.0 mL of the mobile phase.
in the monograph. They are limited by the general acceptance
Reference solution (a) Dissolve 1.0 mg of alfacalcidol CRS criterion for other/unspecified impurities and/or by the general
without heating in 10.0 mL of the mobile phase. monograph Substances for pharmaceutical use (2034). It is
Reference solution (b) Dilute 1.0 mL of reference solution (a) therefore not necessary to identify these impurities for
to 100.0 mL with the mobile phase. Dilute 1.0 mL of this demonstration of compliance. See also 5.10. Control of impurities
solution to 20.0 mL with the mobile phase. in substances for pharmaceutical use) C.
Reference solution (c) In order to prepare pre-alfacalcidol in
situ, dissolve the contents of a vial of alfacalcidoi for system
suitability CRS (containing impurities A and B) in 25 mL of
the mobile phase, heat in a water-bath at 80°C under a
reflux condenser for 2 h and cool.

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2020 Alfadex 1-93

PhEur ~

CH3 DEFINITION
Cyclohexakis-(l ~ 4)-(a.-D-glucopyranosyl)
(cyclomaltohexaose or a.-cydodextrin).
Content
97.0 per cent to 102.0 per cent (dried substance).
CHARACTERS
Appearance
White or almost white, amorphous or crystalline powder.
A. (5B,7E)-9,10-secocholesta-5,7,1 0(19)-triene-la.,3f3-diol Solubility
(trans-alfacalcidol), Freely soluble in water, slightly soluble in propylene glycol,
practically insoluble in anhydrous ethanol and in methylene
chloride.
CH3 IDENTIFICATION
A. Specific optical rotation (see Tests).
B. Examine the chromatograms obtained in the assay.
Results The principal peak in the chromatogram obtained
with test solution (b) is similar in retention time and size to
the principal peak in the chromatogram obtained with
reference solution (c).
C.Dissolve 0.2g in 2 mL of iodine solution R4 by warming
on a water-bath, and allow to stand at room temperature;
B. (SZ,7E)-9,10-secocholesta-5,7 ,10(19)-triene-l~,3f3-diol a yellowish-brown precipitate is formed.
(1 ~-calcidol),
TESTS
Solution S
Dissolve 1._000 g in carbon dioxide-free waterR and dilute to
100.0 mL with the same solvent.
Appearance of solution
Solution S is clear (2.2.1).
pH (2.2.3)
5.0 to 8.0.
HO·- Mix 1 mL of a 223.6 gIL solution of potassium chloride Rand
H 30 mL of solution S.
Specific optical rotation (2.2.7)
C. 6~-[(3S,SR)-3,5-dihydroxy-2-methylcyclohex-l-en-1-yl]-2­ + 147 to + 152 (dried substance), determined on solution S.
phenyl-2,S,10-triaza-4,19-dinor-9~-cholest-7-ene-l,3­
Reducing sugars
dione.
Maximum 0.2 per cent.
______________ ~ PhEur
Test solution To 1 mL of solution S add 1 mL of cupri-
tartaric solution R4. Heat on a water-bath for 10 min, cool to
_room temperature. Add 10 mL of ammonium molybdate
reagent R1 and allow to stand for 15 min.
Alfadex Reference solution Prepare a reference solution at the same
Alphacydodextrin time and in the same manner as the test solution, using
1 mL of a 0.02 gIL solution of glucose R.
(Ph. Bur. monograph 1487)
Measure the absorbance (2.2.25) of the test solution and the
reference solution at the absorption maximum at 740 nm
using water R as the compensation liquid. The absorbance of
the test solution is not greater than that of the reference
solution.
light-absorbing impurities
Examine solution S between 230 nm and 750 nm. Between
230 nm and 350 nm, the absorbance {2.2.25) is not greater
than 0.10. Between 350 nm and 750 nm, the absorbance
(2.2.25) is not greater than 0.05.
Related substances
Liquid chromatography (2.2.29).
Test solution (aJ Dissolve 0.25 g of the substance to be
973 10016-20-3 examined in water R with heating, cool and dilute to
25.0 mL with the same solvent.
Action and use
Cyclodextran; carrier molecule for drug delivery systems.

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 1-94 Alfentanil Hydrochloride 2020

Test solution (b) Dilute 5.0 mL of test solution (a) to

50.0 mL with water R. HO..-C\OH"O"9:H~OH
Reference solution (a) Dissolve 25.0 mg of betadex CRS
0' 0 0 '0
(impurity A), 25.0 mg of gammacyclodextrin CRS
(impurity B) and 50.0 mg of alfadex CRS in water R, then H0J:::r:OHHO HO »~,'OH
0 OH
dilute to 50.0 mL with the same solvent. HO", 0
Reference solution (b) Dilute 5.0 mL of reference solution (a) 6"r-f0H HO b
HO~~O" ~:~~--OH
to 50.0 mL with water R.
Reference solution (c) Dissolve 25.0 mg of alfadex CRS in
water R and dilute to 25.0 mL with the same solvent.
Column: HO ~ OH
- size: 1= 0.25 m, 0 = 4.6 rom;
- stationary phase: end-capped octadecylsilyl silica gelfor
HO OH
chromatography R (10 urn). A. cyc1oheptakis-(l ~4)-(ct-D-g1ucopyranosyl) (betadex or
Mobile phase methanol R, water R (10:90 VIV). cyclomaltoheptaose or ~-cyc1odextrin),
" Flow rate 1.5 mUmin.
Detection Differential refractometer.
Equilibration With the mobile phase for about 3 h.
HO H~H OH
Injection 50 IlL of test solution (a) and reference HO,,)--,"O-' 0-S'°-rr-,OH
HOV/)--O,~OH OH HO HOY»""'!~,OH
solutions (a) and (b).
Run time 3.5 times the retention time of alfadex.
Relative retention With reference to alfadex (retention 0-
time = about 10 min): impurity B = about 0.7; _ 0 HO
HO- HoJ)::~o~>H~OH OH
impurity A = about 2.2.
System suitability Reference solution (a):
- resolution: minimum 1.5 between the peaks due to
impurity Band alfadex; if necessary, adjust the
concentration of methanol in the mobile phase. HO ~ OH
Limits:
- impurities A, B: for each impurity, not more than
HO OH
0.5 times the area of the corresponding peak in the B. cyc1ooctakis-(1 ~4)-(ct-D-g1ucopyranosyl)
chromatogram obtained with reference solution (b) (cyc1omaltooctaose or y-cyc1odextrin).
(0.25 per cent);
- sum of impurities other than A and B: not more than - - - - - - - - - - - c - - - - - - - - - - - PhEur
0.5 times the area of the peak due to alfadex in the
chromatogram obtained with reference solution (b)
(0.5 per cent).
Loss on drying (2.2.32)
Alfentanil Hydrochloride Hydrate
Maximum 11 per cent, determined on 1.000 g by drying in Alfentanil Hydrochloride
an oven at 120 °C for 2 h.
(Ph. Bur. monograph 1062)
Sulfated ash (2.4.14)
Maximum 0.1 per cent, determined on 1.0 g.
ASSAY
Liquid chromatography (2.2.29) as described in the test for
related substances with the following modifications.
Injection Test solution (b) and reference solutions (a)
and (c).
System suitability:
- repeatability: maximum relative standard deviation of
2.0per cent for the peak due to alfadex after 5 injections
of reference solution (a).
Calculate the percentage content of [C6HlOOS16 from the
assigned content of alfadexCRS. Anhydrous alfentanil hydrochloride 69049-06-5
STORAGE Action and use
In an airtight container. Opioid receptor agonist; analgesic.
IMPURITIES PhEur _
Specified impurities A, B.
DEFINITION
N-[1-[2-(4-Ethyl-5-oxo-4,5-dihydro-lH-tetrazol-l-yl)ethyl]-4-
(methoxymethyl)piperidin-4-yl]-N-phenylpropanamide
hydrochloride hydrate.

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2020 Alfentanil Hydrochloride 1-95

Content Time Mobile phase A Mobile phase B

(min) (per cent V/JI) (per cent VIJ')
98.5 per cent to 101.5 per cent (anhydrous substance).
0-15 90 -> 40 10 -> 60
It contains a variable quantity of water.
15 - 20 40 60
CHARACTERS 20 - 25 40 -> 90 60 -> 10
Appearance
White or almost white powder. Flow rate 1.5 mIJmin.
Solubility Detection Spectrophotometer at 220 nm.
Freely soluble in water, in ethanol (96 per cent) and in
Injection 10 ut,
methanol.
Identification of impurities Use the chromatogram obtained
mp with reference solution (a) to identify the peak due to
About 140°C, with decomposition. impurity E; use the chromatogram obtained with reference
It shows polymorphism (5.9). solution (b) to identify the peak due to impurity D.
IDENTIFICATION Relative retention With reference to alfentanil (retention
A. Infrared absorption spectrophotometry (2.2.24). time = about 8 min): impurity D = about 0.8;
Comparison alfentanilhydrochloride hydrateCRS. impurity E = about 0.9.
If the spectra obtained in the solid state show differences, System suitability Reference solution (a):
dissolve the substance to be examined and the reference - resolution: minimum 4.0 between the peaks due to
substance separately in methanolR, evaporate to dryness and impurity E and alfentanil.
record new spectra using the residues. Calculation of percentage contents:
B. Dissolve 50 mg in a mixture of 0.4 mL of ammonia Rand - for each impurity, use the concentration of alfentanil
2 mL of water R. Mix, allow to stand for 5 min and filter. hydrochloride hydrate in reference solution (c).
Acidify the filtrate with dilute nitricacid R. It gives Limits:
reaction (a) of chlorides (2.3.1). - impurity D: maximum 0.2 per cent;
- unspecified impurities: for each impurity, maximum
TESTS
0.10 per cent;
Appearance of solution - total: maximum 0.4 per cent;
The solution is clear (2.2.1) and colourless (2.2.2,
- reporting threshold: 0.05 per cent.
Method If).
Water (2.5.12)
Dissolve 0.2 g in water R and dilute to 20 mL with the same
3.0 per cent to 4.0 per cent, determined on 0.500 g.
solvent.
Related substances ASSAY
Liquid chromatography (2.2.29). Carry out the testprotected Dissolve 0.350 g in 50 mL of a mixture of 1 volume of
from light. ethanol (96 per cent) Rand 4 volumes of waterR and add
5.0 mL of 0.01 M hydrochloric acid. Titrate with 0.1 M
Test solution Dissolve 0.100 g of the substance to be
sodium hydroxide, determining the end-point
examined in methanol R and dilute to 10.0 mL with the same
potentiometrically (2.2.20). Read the volume added between
solvent.
the 2 points of inflexion.
Reference solution (a) In order to prepare impurity E in situ,
1 mL of 0.1 M sodium hydroxide is equivalent to 45.30 mg of
dissolve 10 mg of the substance to be examined in 10.0 rnL
of dilute hydrochloric acid R. Heat on a water-bath under a CZIH33CIN603'
reflux condenser for 4 h. Neutralise with lO.OmL of dilute STORAGE
sodium hydroxide solution R and evaporate to dryness on a Protected from light.
water-bath. Cool and take up the residue in 10 mL of IMPURITIES
methanolR. Filter.· Specified impurities D.
Reference solution (b) Dissolve the contents of a vial of Other detectable impurities (the following substances would, if
alfentanilimpurity D CRS in 1 mL of methanol R. present at a sufficient level, be detected by one or otherof the tests
Reference solution (c) Dilute 1.0 mL of the test solution to in the monograph. They are limitedby the general acceptance
100.0 mL with methanolR. Dilute 1.0 mL of this solution to criterion for other/unspecified impurities and/orby the general
10.0 mL with methanolR. monograph Substances for pharmaceutical use (2034). It is
Blank solution methanolR. therefore not necessary to identify these impurities for
Column: demonstration of compliance. .See also 5.10. Control of impurities
- size: l = 0.1 m, 0 = 4.6 mm; in substances for pharmaceutical use) A, B, C, E, F, G, H.
- stationary phase: end-capped octadecylsilyl silica gelfor
chromatography R (3 urn).
Mobz1e phase:
- mobile phase A: 5 giL solution of ammonium carbonate R in
a mixture of 10 volumes of tetrahydrofuran Rand
90 volumes of waterfor chromatography R;
- mobile phase B: acetonitrile for chromatography R;

A. (ls,4s)-1-[2";(4-ethyl-5-oxo-4,5-dihydro-1H-te~azol-l-yl)
ethyl]-4-(methoxymethyl)-4-(N-phenylpropanamido)
piperidine t'-oxide,

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1-96 Alfuzosin Hydrochloride 2020

B. (lr,4r)-1-[2-( 4-ethyl-5-oxo-4,5-dihydro-1H-tetrazol-1-yl) H. N- [1-[2-(4-ethyl-5-oxo-4,5-dihydro-1H-tetrazol-1-yl)

ethyl]-4-(methoxymethyl)-4-(N-phenylpropanamido) ethyl]-4-(methoxymethyl)piperidin-4-yl]-N-
piperidine I-oxide, phenylbutanamide.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur

~. I ·
Q ,I~H Alfuzosin Hydrochloride
H3C'i(N ~
OCH 3 (Ph. Eur. monograph 1287)
CH
C. N-[4-(methoxymethyl)piperidin-4-yl]-N-
H3CO~N'V~~~ H./1
phenylpropanamide, I I '( "o.-J , HC)
H ~ AN 0
3CO

NH2 and enantiomer

425.9 81403-68-1

Action and use

Alpha }-adrenoceptor antagonist.
Preparations
D. N-[1-[2-( 4-ethyl-5-oxo-4,5-dihydro-1H-tetrazol-1-yl)
Alfuzosin Tablets
ethyl]-4-(methoxymethyl)piperidin-4-yl]-N-
phenylacetamide, Alfuzosin Prolonged-release Tablets
PhEur . _

DEFINITION
(2RS)-N-[3-[ (4-Amino-6, 7-dimethoxyquinazolin-2-yl)
methylamino]propyl] oxolan-2-carboxamide hydrochloride.
Content
99.0 per cent to 101.0 per cent (anhydrous substance).
CHARACTERS
E. 1-ethyl-4-[2-[4-(methoxymethyl)-4-(phenylamino) Appearance
piperidin-l-yl] ethyl]-1,4-dihydro-5H-tetrazol-5-one, White or almost white, crystalline powder, slightly
hygroscopic.
Solubility
Freely soluble in water, sparingly soluble in ethanol
(96 per cent), practically insoluble in methylene chloride -.
IDENTIFICATION
A. Infrared absorption spectrophotometry (2.2.24).
Comparison alfuzosin hydrochloride CRS.
F. N- [1-(2-hydroxyethyl)-4-(methoxymethyl)piperidin-4-yl]- B. It gives reaction (a) of chlorides (2.3.1).
N-phenylpropanamide,
TESTS
pH (2.2.3)
4.0 to 5.5.
Dissolve 0.500 g in carbon dioxide-free water R and dilute to
25.0 mL with the same solvent. Use a freshly prepared
solution.
Related substances
Liquid chromatography (2.2.29).
Test solution Dissolve 40 mg of the substance to be
examined in the mobile phase and dilute to 100.0 mL with
G. N-[1-[2-(4-ethyl-5-oxo-4,5-dihydro-1H-tetrazol-1-yl)
the mobile phase.
ethyl]-4- [(propanoyloxy)methyl]piperidin-4-yl]-N-
phenylpropanamide, Reference solution (a) . Dilute 1.0 mL of the test solution to
100.0 mL with the mobile phase. Dilute 1.0 mL ofthis
solution to 10.0 mL with the mobile phase.

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2020 Alfuzosin Hydrochloride 1-97

Reference solution (b) Dissolve 4 mg of aljuzosin for system perchloric acid, determining the end-point potentiometrically
suitability A CRS (containing impurities B, F and G) in the (2.2.20).
mobile phase and dilute to 10.0 mL with the mobile phase. 1 mL of O.I M perchloric acid is equivalent to 42.59 mg
Reference solution (c) Dissolve 4 mg of aljuzosin for peak of C19HzsCINS04'
identification CRS (containing impurity D) in the mobile
STORAGE
phase and dilute to 10.0 mL with the mobile phase.
In an airtight container, protected from light.
Column:
- size: 1= 0.15 m, (2) = 4.6 mm; IMPURITIES
- stationary phase: base-deactivated end-capped octadecylsilyl Specified impurities DJ F.
silica gelfor chromatography R (5 JlIIl); Other detectable impurities (the following substances toould, if
- temperature: 25°C; if necessary, increase the temperature present at a sufficient leoel, be detected by one or otherof the tests
slightly to achieve the required resolution between the in the monograph. They are limitedby thegeneral acceptance
peaks due to impurity G and alfuzosin. criterion for other/unspecified impurities and/or by the general
Mobile phase Mix 1 volume of tetrahydrofuran R, 20 volumes monograph Substances for pharmaceutical use (2034). It is
of acetonitrile Rand 80 volumes of a solution prepared as therefore not necessary to identify these impurities for
follows: dilute 5.0 mL of perchloric acid R in 900 mL of water demonstration of compliance. See also 5.10. Control of impurities
for chromatography R, adjust to pH 3.5 with dilute sodium in substances for pharmaceutical use) A J B J CJ EJ G.
hydroxide solution R and dilute to 1000 mL with waterfor
chromatography R.
Flow rate 1.5 mlJmin.
Detection Spectrophotometer at 254 nm.
Injection 10 ~L.
Run time Twice the retention time ofalfuzosin.
A. N- [3-{(4-amino-6,7-dimethoxyquinazolin-2-yl)
Identification of-impurities Use the chromatogram supplied methylamino]propyl] furan-2-carboxamide,
with aljuzosinfor system suitabilityA CRS and the
chromatogram obtained with reference solution (b) to
identify the peaks due to impurities B, F and G; use the
chromatogram supplied with alfuzosinfor peak
identification CRS and the chromatogram obtained with
reference solution (c) to identify the peak due to impurity D.
Relative retention With reference to alfuzosin (retention B. 2-chloro-6,7-dimethoxyquinazolin-4-amine,
time = about 9 min): impurity D = about 0.4;
impurity B = about 0.57; impurity F = about 0.63;
impurity G = about 0.9.
System suitabiluy Reference solution (b):
- resolution: minimum 1.5 between the peaks due to
impurities Band F; minimum 1.5 between the peaks due
to impurity G and alfuzosin. C. (2RS)-N-[3-[(4-amino-6,7-dimethoxyquinazolin-z-yl)
Limits: amino]propyl] ,;.N-methyloxolan-2-carboxamide,
- correction factor. for the calculation of content, multiply the
peak area of impurity F by 0.6;
- impurity D: not more than twice the area of the principal
peak in the chromatogram obtained with reference
solution (a) (0.2 per cent);
- impurity F: not more than 1.5 times the area of the
principal peak in the chromatogram obtained with
reference solution (a) (0.15 per cent); D. N Z-(3-aminopropyl)-6,7-dimethoxy-N'-methylquinazolin-
- unspecified impurities: for each impurity, not more than the 2,4-diamine,
area of the principal peak in the chromatogram obtained
with reference solution (a) (0.10 per cent);
- total: not more than 3 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.3 per cent);
- disregard limit: 0.5 times the area of the principal peak in
the chromatogram obtained with reference solution (a)
(0.05 per cent). E. N- [3-[ (4-amino-6,7-dimethoxyquinazolin-2-yl)
methylamino] propyl] formamide,
Water (2.5.12)
Maximum 0.5 per cent, determined on 1.00 g.
Sulfated ash (2.4.14)
Maximum 0.1 per cent, determined on 1.0 g.
ASSAY
Dissolve 0.300 g in a mixture of 40 mL of anhydrous acetic
acid Rand 40 mL of acetic anhydride R. Titrate with 0.1 M
F. 6,7-dimethoxy-N' ,N2-dimethylquinazoline-2,4-diamine,

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1-98 Alginic Acid 2020

Loss on drying (2.2.32) _

Maximum 15.0 per cent, determined on 0.1000 g by drying
in an oven at 105°C for 4 h.
Sulfated ash (2.4.14)
Maximum 8.0 per cent (dried substance), determined on
0.100 g.
G. N Z- [3- [(4-amino-6,7-dimethoxyquin azolin-2-yl)amino] Microbial contamination
propyl] -6,7-dimethoxy-N''-methylquinazoline-2,4-diamine. TAMC: acceptance criterion lO z CFU/g (2.6.12).
_ _ _ _ _ _ _ _ _ _ _ _-c- PhEur
Absence of Escherichia coli (2.6.13).
Absence of Salmonella (2.6.13).
ASSAY
Alginic Acid To 0.2500 g add 25 mL of waterR, 25.0 mL of 0.1 M
sodium hydroxide and 0.2 mL of phenolphthalein solution R.
(Ph. Eur. monograph 0591) Titrate with 0.1 M hydrochloric acid.
Action and use 1 mL of 0.1 M sodium hydroxide is equivalent to 4.502 mg of
Treatment of gastro-oesophageal reflux disease; excipient; carboxyl groups (-COzH).
thickening agent. FUNCTIONALITY-RELATED CHARACTERISTICS
PhEur _ This section provides information on characteristics that are
recognised as being relevant control parameters for one or more
DEFINITION functions of the substance when usedas an excipient (see chapter
Mixture of polyuronic acids [(C 6Hs0 6)n] composed of 5.15). Some of the characteristics described in the Functionality-
residues of n-mannuronic and L-guluronic acids, obtained , related characteristics section may also bepresent in the mandatory
mainly from algae belonging to the Phaeophyceae. A small part of the monograph since they also represent mandatory quality
proportion of the carboxyl groups may be neutralised. criteria. In such cases, a cross-reference to the tests described in the
Content mandatorypart is included in the Functionality-related
19.0 per cent toZfi.O per cent of carboxyl groups (-COzH) characteristics section. Control of the characteristics can contribute
(dried substance). to the quality of a medicinal product by improving the consistency
of the manufacturing process and the performance of the medicinal
CHARACTERS productduringuse. W'here control methods are cited, they are
Appearance recognised as being suitable for the purpose, but othermethods can
White or pale yellowish-brown, crystalline or amorphous also be used. W'herever results for a particular characteristic are
powder. reported, the control methodmust be indicated.
Solubility The following characteristics may be relevant for alginic acid used
Very slightly soluble or practically insoluble in ethanol as disintegrant and/orbinder.
(96 per cent), practically insoluble in organic solvents.
Particle-size distribution (2.9.31 or 2.9.38)
It swells in water but does not dissolve; it dissolves in
solutions of alkali hydroxides. Settling volume
Place 75 mL of waterR in a 100 mLgraduated cylinder and
IDENTIFICATION add 1.5 g of the substance to be examined in 0.5 g portions,
A. To 0.2 g add 20 mL of water Rand 0.5 mL of sodium shaking vigorously after each addition. Dilute to 100.0 mL
carbonate solution R. Shake and filter. To 5 mL of the filtrate with waterR and shake again until the substance is
add 1 mL of calcium chloride solution R. A voluminous homogeneously distributed. Allow to stand for. 4 hand
gelatinous mass is formed. determine the volume of the settled mass.
B. To 5 mL of the filtrate obtained in identification test A The following characteristic may be relevant for alginic acid used
add 0.5 mL of a 123 gIL solution of magnesium sulfateR. as gelling agentor viscosity-increasing agent.
No voluminous gelatinous mass is formed.
Apparent viscosity
C. To 5 mg add 5 mL of water R, 1 mL of a freshly Determine the' dynamic viscosity using a rotating viscometer
prepared 10 gIL solution of l;13-dihydroxynaphthalene R in (2.2.10).
ethanol (96 per cent) R and 5 mL of hydrochloric acid R. Boil
Prepare a 20 gIL suspension of alginic acid (dried substance)
gently for 3 min, cool, add 5 mL of waterR, and shake with
and add 0.1 M sodium hydroxide until a solution is obtained.
15 mL of di-isopropyl etherR. Carry out a blank test.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _-'-- PhEur
The upper layer obtained with the substance to be examined
exhibits a deeper bluish-red colour than that obtained with
the blank.
TESTS
Chlorides
Maximum 1.0 per cent.
To 2.50 g add 50 mL of dilute nitric acid R, shake for 1 h
and dilute to 100.0 mL with dilute nitric acid R. Filter.
To 50.0 mL of the filtrate add 10.0 mL of 0.1 M siloer nitrate
and 5 mL of toluene R. Titrate with 0.1 M ammonium
thiocyanate, using 2 mL of ferric ammonium sulfate solution R2
as indicator and shaking vigorously towards the end-point.
1 mL of 0.1 M siioer nitrate is equivalent to 3.545 mgof Cl.

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2020 Alimemazine Tartrate 1-99

the solvent mixture and dilute to 10.0 mL with the solvent

Alimemazine Tartrate mixture.
(Alimemazine Hemitartrate Ph. Eur. monograph Column:
2650) = =
- size: 1 0.15 m, (2') 4.6 mm;
- stationaryphase: base-deactivated end-capped octadecylsilyl
silica gelfor chromatography R (3 urn);
- temperature: 40°C.
Mobile phase acetonitrile R, methanol R, 3.854 gIL solution of
ammonium acetate R (10:40:50 VIVlv).
Flow rate 1.3 mIJmin.
Detection Spectrophotometer at Z53 TIm.
373.5 4330-99-8 Injection ZO .ilL.
Run time Twice the retention time of alimemazine.
Action and use Identification of impurities Use the chromatogram supplied
Histamine H b receptor antagonist; sedative. with alimemazinefor system suitability CRS and the
Preparations chromatogram obtained with reference solution (b) to
Paediatric Alimemazine Oral Solution identify the peaks due to impurities A, Band C.
Strong Paediatric Alimemazine Oral Solution Relative retention With reference to alimemazine (retention
Alimemazine'Tablets time = about Z7 min): impurity A = about 0.1;
impurity B = about 0.5; impurity C = about 1.4.
Ph Eur ~_-".: --,-- _
System suitabz7ity Reference solution (b):
DEFINITION - resolution: minimum 5.0 between the peaks due to
(2R5)-N.,N.,Z':'Trimethyl-3-(1 OH-phenothiazin-1 O-yl)propan- alimemazine and impurity C.
I-amine hemi[(ZR,3R)-Z,3-dihydroxybutanedioate]. Calculation of percentage contents:
Content - correction factors: multiply the peak areas of the following
99.0 per cent to 101.0 per cent (dried substance). impurities by the corresponding correction factor:
impurity A = 4.4; impurity C = 0.4;
CHARACTERS - for each impurity, use the concentration of alimemazine in
Appearance reference solution (a).
White or very slightly yellowish powder.
Limits:
Solubility " - impurity B: maximum 0.3 per cent;
Freely soluble in water, sparingly soluble in ethanol - impurities A., C: for each impurity, maximum
(96 per cent), practically insoluble in toluene. 0.15 per cent;
It deteriorates when exposed to air and light. - unspecified impurities: for each impurity, maximum
0.10 per cent;
IDENTIFICATION
- total: maximum 0.5 per cent;
Infrared absorption spectrophotometry (2.2.24).
- reporting threshold: 0.05 per cent.
Comparison alimemazine hemitartrate CRS.
Loss on drying (2.2.32)
TESTS Maximum 0.5 per cent, determined on 1.000 g by drying in
Appearance of solution an oven at 105°C for 3 h.
The solution is not more opalescent than reference Sulfated ash (2.4.14)
suspension II (2.2.1) and not more intensely coloured than Maximum 0.1 per cent, determined on 1.0 g.
reference solution BY s (2.2.2., Method II).
Dissolve 1.0 g in water R and dilute to 10 mL with the same
ASSAY
solvent. Dissolve 0.300 g in 50 mL of anhydrousacetic acid R. Titrate
with 0.1 M perchloric acid, determining the end-point
pH (2.2.3) potentiometrically (2.2.20).
5.0 to 6.5. Carry out the test protected from light and use a
1 mL of 0.1 M perchloric acid is equivalent to 37.35 mg of
freshly prepared solution.
CzoHzsNz03S.
Dissolve 1.0 g in carbon dioxide-free water R and dilute to
50 mL with the same solvent. STORAGE
In an airtight container, protected from light.
Related substances
Liquid chromatography (2.2.29). Carry out the testprotected IMPURITIES
from light and usefreshly prepared solutions. Specifiedimpurities A., B., C.
Solvent mixture acetonitrile R, water R (ZO:80 VIV).
Test solution Dissolve 35 mg of the substance to be
examined in the solvent mixture and dilute to 100.0 mL with
and enantiomer
the solvent mixture.
Reference solution (a) Dilute 1.0 mL of the test solution to
100.0 mL with the solvent mixture. Dilute 1.0 mL of this
solution to 10.0 mL with the solvent mixture.
Reference solution (b) Dissolve 3.5 mg of alimemazinefor A. (ZRS)-N,N,2-trlmethyl-3-(5-oxido-1 OH-phenothiazin-l 0-
system suitabilityCRS (containing impurities A, B and C) in yl)propan-l-amine,

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1-100 Allantoin 2020

~
~ TESTS
# r>;«> "CH s Solution S

8'0 CH, N H"l ~ and enantiomer

Dissolve 0.5 g in carbon dioxide-free water R, with heating if
necessary, and dilute to 100 mL with the same solvent.
Acidity or alkalinity
To 5 mL of solution S add 5 mL of carbon dioxide-free
B. (2RS)-N,2-dimethyl-3-(1 OH-phenothiazin-l 0-yl)propan-1- 'Water R, 0.1 rnL of methyl redsolution Rand 0.2 mL of
amine, 0.01 M sodium hydroxide. The solution is yellow. Add 0.4 mL
of O. 01 M hydrochloric acid. The solution is red.
Optical rotation (2.2.7)
The angle of optical rotation, determined on solution S, is
-0.10° to + 0.10°.
Reducing substances
Shake 1.0 g with 10 mL of water R for 2 min. Filter.
Add 1.5 mL of 0.02 M potassium permanganate. The solution
C. 10H-phenothiazine. must remain violet for at least 10 min.
_ _ _ _ _ _ _ _ _ _ _ _ _ _---' PhEur
Related substances
Examine by thin-layer chromatography (2.2.27), using a
suitable cellulose for chromatography R as the coating
substance.
Allantoin Test solution (a) Dissolve 0.10 g of the substance to be
examined in 5.0 rnL of 'Water R with heating. Allow to cool.
(Ph. Eur. monograph 1288) Dilute to 10 mL with methanol R. Use the solution immediately
afterpreparation.
Test solution (b) Dilute 1 mL of test solution (a) to 10 mL
and enantiomer
with a mixture of 1 volume of methanol Rand 1 volume of
'Water R.
Reference solution (a) Dissolve 10 mg of allantoin CRS in a
158.1 97-59-6 mixture of 1 volume of methanolRand 1 volume of waterR
and dilute to 10 mL with the same mixture of solvents.
Action and use
Reference solution (b) Dissolve 10 mg of urea R in 10 mL of
Astringent; keratolytic,
water R. Dilute 1 mL of this solution to 10 mL with
PhEur _ methanol R.
Reference solution (c) Mix 1 mL of reference solution (a)
DEFINITION
and 1 mL of reference solution (b).
Allantoin contains not less than 98.5 per cent and not more
than the equivalent of 101.0 per cent of (RS)-(2,5- Apply to the plate 10 ~ of test solution (a) and 5· JlL each
dioxoimidazolidin-4-yl)urea. of test solution (b), reference solution (a), reference
solution (b) and reference solution (c). Develop over a path
CHARACTERS of 10 em using a mixture of 15 volumes of glacial acetic
A white or almost white, crystalline powder, slightly soluble acid R, 25 volumes of water Rand 60 volumes of butanolR.
in water, very slightly soluble in alcohol. Allow the plate to dry in air. Spray the plate with a 5 gIL
It melts at about 225°C, with decomposition. solution of dimethylaminobenzaldehyde R in a mixture of
IDENTIFICATION 1 volume of hydrochloric acidR and 3 volumes of methanol R.
Dry the plate in a current of hot air. Examine in daylight
First identification: A.
after 30 min. Any spot in the chromatogram obtained with
Second identification: B, C, D. test solution (a), apart from the principal spot, is not more
A. Examine by infrared absorption spectrophotometry intense than the spot in the chromatogram obtained with
(2.2.24), comparing with the spectrum obtained with reference solution (b) (0.5 per cent). The test is not valid
allantoin CRS. unless the chromatogram obtained with reference solution (c)
B. Examine the chromatograms obtained in the test for shows two clearly separated principal spots.
related substances. The principal spot in the chromatogram Loss on drying (2.2.32)
obtained with test solution (b) is similar in position, colour Not more than 0.1 per cent, determined on 1.000 g by
and size to the principal spot in the chromatogram obtained drying in an oven at 105°C. .
with reference solution (a).
Sulfated ash (2.4.14)
C. Boil 20 mg with a mixture of 1 mL of dilute sodium Not more than 0.1 per cent, determined on 1.0 g.
hydroxide solution Rand 1 mL of water R. Allow to cool.
Add 1 mL of dilute hydrochloric acid R. To 0.1 mL of the ASSAY
solution add 0.1 mL of a 100 gIL solution of potassium Dissolve 120.0 mg in 40 mL of water R. Titrate with 0.1 M
bromide R, 0.1 mL of a 20 gIL solution of resorcinol Rand sodium hydroxide, determining the end-point
3 mL of sulfuric acid R. Heat for 5 min to 10 min on a water- potentiometrically (2.2.20).
bath. A dark blue colour develops, which becomes red after 1 mL of 0.1 M sodium hydroxide is equivalent to 15.81 mg of
cooling and pouring into about 10 mL of waterR. C4H6N403 •
D. Heat about 0.5 g. Ammonia vapour is evolved, which
turns red litmus paper R blue.

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2020 Allergen Products 1-101

IMPURITIES Hymenoptera venoms. Other source materials include certain

insects and foods.
HyCOzH The source materials are defined, where possible, by their
o origin, nature, method of collection-or' production and pre-
treatment. Control methods and acceptance criteria relating
A. glyoxylic acid, to identity and purity are established. The acceptance criteria
must ensure the consistency of the allergenic source material
o from a qualitative and quantitative point of view. The source
)l materials are stored under controlled conditions justified by
HzN NHz stability data.
The collection or production, as well as the handling of the
B. carbamide (urea). source materials, are such that consistent composition is
___________ ~ PhEur ensured from batch to batch.
When applicable, pesticides, heavy metals and residual
solvents are limited according to the principles defined in
general chapters 2.8.13. Pesticide residues, 2.4.27. Heavy metals
Allergen Products in herbal drugs and herbaldrugpreparations and 2.4.24.
Identification and control of residual solvents, respectively.
(Ph. Eur. monograph 1063) Microbial contamination of the source material may be
PhEur ~ _ unavoidable and should be monitored according to a justified
sampling plan; if a determination of microbial contamination
TIns monograph does not apply to: chemicals that are used solely is not applicable, this must be justified.
for diagnosis ofcontact dermatitis; chemically synthesised products;
allergens derived by-recombinant DNA technology. It does not The scientific name (species, variety, strain etc.) of the source
necessarily apply to allergen products for veterinary use. material is indicated and the part used is stated,if applicable.
Foods must be of a quality suitable for human consumption.
DEFINITION
The origin of the food stuff as well as its processing stage is
Allergen products are pharmaceutical preparations derived stated.
from extracts of naturally occurring source materials
containing allergens, which are substances that lead to and/or MANUFACTURING PROCESS
provoke allergic reactions. The allergenic components are Allergen products are generally obtained by extraction, and
most often of a proteinaceous nature. Allergen products are may be purified, from the source materials using appropriate
intended for in vivo diagnosis or treatment of allergic diseases methods shown to preserve the allergenic properties of the
attributed to these allergens. components. Allergens for which there are not enough
patients to determine the total allergenic activity in vivo or in
Allergen products areavailable as finished products, and as
vitro, the extraction ratio indicating the relative proportions
finished products used on a named-patient basis. Allergen
(mlV) of allergenic source materials and solvents is a
products are generally presented as parenteral preparations,
minimum requirement. Allergen products presented as
eye preparations, preparations for inhalation, preparations for
parenteral preparations, eye preparations, preparations for
oral use, sublingual preparations or preparations for skin
inhalation and preparations for skin testing are manufactured
tests.
under aseptic conditions.
For in vivo diagnostic use, allergen products are usually
In the manufacture, packaging, storage and distribution of
prepared as unmodified extracts in a 50 per cent VIV
allergen products intended for administration by other routes,
solution of glycerol for skin testing. For intradermal diagnosis
suitable measures are taken to ensure their microbial quality;
or for provocation tests by nasal, ocular or bronchial
recommendations on this aspect are provided in general
administration, suitable dilutions of allergen products may be
chapter 5.1.4. Microbiological quality of non-sterile
prepared by dilution of aqueous or glycerinated extracts, or
pharmaceutical preparations and substances for pharmaceutical
by reconstitution of unmodified freeze-dried extracts.
use.
For specific immunotherapy, allergen products may be either
All allergen preparations are manufactured under conditions
unmodified extracts or extracts modified chemically and/or
designed to minimise exogenous and endogenous enzymatic
by adsorption onto different carriers (for example, aluminium
degradation.
hydroxide, calcium phosphate or tyrosine).
Any purification procedure. is designed to minimise the
PRODUCTION content of any potential irritant low-molecular-mass
Where allergen products or source materials are components and non-allergenic components.
manufactured using materials of human or animal origin, the Allergen products may contain suitable antimicrobial
requirements of general chapter 5.1. 7. Viral safety apply. preservatives. The nature and concentration of the
SOURCE MATERIALS antimicrobial preservatives have to be justified and their
Source materials are obtained from qualified suppliers. efficacy complies with chapter 5.1.3. Efficacy of antimicrobial
The source materials comply with the requirements of the preservation.
appropriate individual monographs (where a relevant The manufacturing process comprises various stages:
monograph exists) and the statements in this section are - source material;
intended to be read in conjunction with the individual - active substance: it is generally a modified or an
monographs. unmodified allergen extract; where applicable it is stored
Source materials for the preparation of allergen products are under conditions ensuring its stability, for example freeze-
products of animal or vegetable origin, mostly pollens, dried;
moulds, mites, animal epithelia and outgrowths, and - finished product.

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 1-102 Allergen Products 2020

All other stages of the manufacturing process are considered TESTS

as intermediates. The tests are performed as late as possible in the
IN-HOUSE REFERENCE PREPARATION manufacturing process. In the case of products used on a
An appropriate representative preparation is selected as the named-patient basis, the control is performed on the active
in-house reference preparation (lliRP), characterised and substance and/or at the intermediate stage between the active
used to verify batch-to-batch consistency. The IHRP is substance and the finished product.
stored in suitably sized aliquots under conditions ensuring its Various biochemical and immunological tests have been
stability, for example freeze-dried. developed in order to characterise allergens qualitatively and
Characterisation of the in-house reference preparation quantitatively. In those cases where such methods cannot be
The extent of characterisation of the IHRP depends on the source applied, particularly for the determination of allergenic
material, knowledge of the allergenic components and availability activity and allergen and/or protein profile, justification must
of suitable reagents, as wellas the intendeduse. The characterised be provided.
IHRP is used as the reference in the batch control of active Water (2.5.12 or 2.5.32) or loss on drying (2.2.32)
substances and intermediates and, if possible, in the batchcontrol Maximum 5 per cent for freeze-dried products. In the case of
of finished products. orallyophilisates, the water content may be higher than
The IHRP is characterised by the protein content 5 per cent, where justified and authorised.
, determination and a protein profile using appropriate Sterility (2.6.1)
methods (such as isoelectrlc focusing, sodium dodecyl sulfate Allergen products presented as parenteral preparations, eye
polyacrylamide gel electrophoresis, immunoelectrophoresis, preparations, preparations for inhalation or preparations for
capillary electrophoresis, chromatographic techniques and skin testing comply with the test for sterility.
mass spectrometry). Microbial contamination
Allergenic components may be detected by appropriate For non-sterile allergen products, recommendations are
methods (for example, immunoblotting or crossed radio- provided in general chapter 5.1.4. Microbiological quality of
immunoelectrophoresis). Characterisation of the allergenic non-sterile pharmaceutical preparations and substances for
components may include identification of relevant allergens pharmaceutical use.
based on serological or other techniques using pooled or
Protein content (2.5.33)
individual sera from allergic patients, or allergen-specific
80 per cent to 120 per cent of the stated content, unless
polyclonal 01' monoclonal antibodies.
otherwise justified and authorised. If the biological potency
Determination of the content of relevant allergens is can be determined then the test for protein content is
performed wherever possible. This determination may be performed as a batch-to-batch consistency test and the
made using individual allergen-specific reference standards, protein content is within 50 per cent to 150 per cent of the
when available. The choice of the relevant allergen stated content. When the finished product contains
components subjected to the determination must be justified. proteinaceous excipients, the test for protein content is
Individual allergens are identified and named according to performed as late as possible during production before
internationally established nomenclature wherever possible. addition of the proteinaceous excipient.
The biological potency of the first IHRP is determined in Protein profile
patients by in vivo techniques such as skin testing, and The protein profile determined by suitable methods
expressed in units of biological activity, except when not corresponds to that of the IHRP. The presence of relevant
enough patients are available. In this case, the potency of the allergen components is verified, where possible. The choice
first IHRP is determined by an in vitro method. .of relevant allergen components to be tested for must be
Subsequently, the biological activity of future IHRPs is justified.
demonstrated by in vitro methods by comparison with the
Various additional tests, some with increasing selectioity,
results obtained with the first lliRP. The in vitro potency
depending on the allergen productconcerned can be applied, but in
may be measured by a suitable immunoassay (for example,
any case for allergen products intended for therapeutic use, a
an assay based on the inhibition of the binding capacity of
validatedtest measuring the potency (total allergenic aaioity,
specific immunoglobulin E antibodies).
determination of individual allergens or any otherjustified tests)
IDENfIFICATION must be applied.
The tests for identification are performed as late as possible Aluminium (2.5.13)
in the manufacturing process. In the case of products used 80 per cent to 120 per cent of the stated amount but in any
on a named-patient basis, the control is performed on the case not more than 1.25 mg per human dose unless
active substance and/or at the intermediate stage between the otherwise justified and authorised, when aluminium
active substance and the finished product. hydroxide or aluminium phosphate is used as adsorbent.
Identity is confirmed by comparison with the IHRP using
Calcium (2.5.14)
protein profiling by appropriate methods (for example,
80 per cent to 120 per cent of the stated amount when
isoelectrlc focusing, sodium dodecyl sulfate polyacrylamide
calcium phosphate is used as adsorbent.
gel electrophoresis, immunoelectrophoresis, immunoblotting,
liquid chromatography or mass spectrometry). Allergen profile
Relevant allergenic components are identified by means of
In exceptional cases, if no IHRP is available, a representative
suitable techniques using allergen-specific human or animal
batch may be used to confirm identity.
antibodies.
Identity may also be confirmed by comparison with
individual allergen-specific reference standards, when Total allergenic activity
available. 50 per cent to 150 percent of the stated amount as assayed
by inhibition of the binding capacity of specific

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2020 Allopurinol 1-103

immunoglobulin E antibodies or a suitable equivalent in vitro A. Ultraviolet and visible absorption speetrophotomet.ry
method. (2.2.25).
Individual allergens Test solution Dissolve 10 mg in 1 mL of a 4 gIL solution of
50 per cent to 200 per cent of the stated amount of each sodium hydroxide R and dilute to 100.0 mL with a 10.3 gIL
relevant allergen component, determined by a suitable solution of hydrochloric acid R. Dilute 10.0 mL of this
method. solution to 100.0 mL with a 10.3 gIL solution of hydrochloric
acid R.
STORAGE
Adsorbed allergen products are not to be frozen, unless Spectralrange 220-350 nm.
otherwise justified and authorised. Absorption maximum At 250 nm.
LABELLING Absorption minimum At 231 nm.
The label states: =
Absorbance ratio A2311A25o 0.52 to 0.62.
- the name of the allergen product; B. Infrared absorption spectrophotometry (2.2.24).
- the biological potency and/or the protein content and/or Comparison .allopurinol CRS.
the extraction concentration; C. Dissolve 0.3 gin 2.5 mLof dilute sodium hydroxide
- the route of administration and the intended use; solution R and add 50 mL of water R. Add slowly and with
- the storage conditions; shaking 5 mL of silvernitrate solution R1. A white precipitate
-,- where applicable, the name and amount of added is formed which does not dissolve on the addition of 5 mL of
antimicrobial preservative; ammonia R.
--where applicable, for freeze-dried preparations:
D. Thin-layer chromatography (2.2.27).
- the name, composition and volume of the
reconstituting liquid to be added; Test solution Dissolve 20 mg of the substance to be
- the period of time within which the preparation is to examined in concentrated ammonia R and dilute to 10 mL
be used after reconstitution; with the same solvent.
--where applicable, that the preparation is sterile; Reference solution Dissolve 20 mg of allopurinol CRS in
- where applicable, the name and amount of adsorbent. concentrated ammonia R and dilute to ·10 mL with the same
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur solvent.
Plate TLC silica gel F254 plate R.
Mobile phase anhydrous ethanol R, methylene chloride R
(40:60 VIV).
Allopurinol Application 10 ~.
Development Over 2/3 of the plate.
(Ph. Bur. monograph 0576)
Drying In air.
o Detection Examine in ultraviolet light at 254 nm.

N~NH
~~IJL ...,
Results The principal spot in the chromatogram obtained
with the test solution is similar in position and size to the
~ N principal spot in the chromatogram obtained with the
reference solution.
136.1 315-30-0 TESTS
Related substances
Action and use Liquid chromatography (2.2.29). Usefreshly prepared solutions.
Xanthine oxidase inhibitor; treatment of gout and Store and inject them at 8°C, using a cooled autosampler.
hyperuricaemia. Test solution (a) Dissolve 25.0 mg of the substance to be
Preparations examined in 2.5 mL of a 4 gIL solution of sodium hydroxide R
Allopurinol Oral Suspension and dilute immediately to 50.0 mL with the mobile phase.
Allopurinol Tablets Test solution (b) Dissolve 20.0 mg of the substance to be
examined in 5.0 mL of a 4 gIL solution of sodium hydroxide R
PhEur _
and dilute immediately to 250.0 mL with the mobile phase.
DEFINITION Reference solution (a) Dilute 2.0 mL of test solution (a) to
1,5-Dihydro-4H-pyrazolo [3,4-d]pyrimidin-4-one. 100.0 mL with the mobile phase. Dilute 5.0 mL of this
Content solutionto 100.0 mL with the mobile phase.
97.0 per cent to 102.0 per cent (dried substance). Reference solution (b) Dissolve 5 mg of allopurinol
impurity A CRS, 5 mg of allopurinol impurity B CRS and
CHARACTERS 5.0 mg of allopurinol impurity C CRS in 5.0 mL of a 4 gIL
Appearance solution of sodium hydroxide R and dilute immediately to
White or almost white powder. 100.0 mL with the mobile phase. Dilute 1.0 mL of this
Solubility solution to 100.0 mL with the mobile phase.
Very slightlysoluble in water and in ethanol (96 per cent). Reference solution (c) Dissolve 20.0 mg of allopurinol CRS in
It dissolves in dilute solutions of alkali hydroxides. 5.0 mL of a 4 gIL solution of sodium hydroxide R and dilute
IDENTIFICATION immediately to 250.0 mL with the mobile phase.
First identification: B. Column:
Second. identification: A~ C, D. - size: 1= 0.2? m, 0= 4.6 mm;

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1-104 Allopurinol 2020

- stationary phase: octadecylsilyl silica gelfor chromatography R - resolution: minimum 2.0 between the peaks due to
(5 !Jl11). impurities D and E.
Mobile phase 1.25 g/L solution of potassium dihydrogen Limits:
phosphate R. - impurity D: not more than the area of the corresponding
Flow rate 1.4 mUmin. peak in the chromatogram obtained with the reference
solution (0.1 per cent);
Detection Spectrophotometer at 230 TIm.
- impurity E: not more than the area of the corresponding
Injection 20 JlL of test solution (a) and reference peak in the chromatogram obtained with the reference
solutions (a) and (b). solution (0.1 per cent).
Run time Twice the retention time of allopurinol.
ImpurityF
Elution order Impurity A, impurity B, impurity C, Liquid chromatography (2.2.29).
allopurinol.
Under the following conditions, any hydrazine in the sample
=
Retention time Allopurinol about 10 min. reacts with benzaldehyde to give benzaldehyde azine.
System suitabzlity Reference solution (b): Solvent mixture Mix equal volumes of dilute sodium hydroxide
- resolution: minimum 1.1 between the peaks due to solution R and methanolR.
impurities Band C.
SolutionA Dissolve 2.0 g of benzaldehyde R in the solvent
Limits: mixture and dilute to 50.0 mL with the solvent mixture.
- impurityA: not more than twice the area of the principal Prepare immediately before use.
peak in the chromatogram obtained with reference
Test solution Dissolve 250.0 mg of the substance to be
solution (a) (0.2 per cent);
examined in 5 mL of the solvent mixture. Add 4 mL of
- impurity B: not more than the area of the principal peak in
solution A, mix and allow to stand for 2.5 h at room
the chromatogram obtained with reference solution (a)
temperature. Add 5.0 mL of hexane R and shake for 1 min.
(0.1 per cent);
Allow the layers to separate and use the upper layer.
- impurity C: not more than the area of the corresponding
peak in the chromatogram obtained with reference Reference solution Dissolve 10.0 mg of hydrazine sulfate R in
solution (b) (O.lper cent); the solvent mixture by sonicating for about 2 min and dilute
- unspecified impurities: for each impurity, not more than the to 50.0 mL with the solvent mixture. Dilute 1.0 mL to
area of the principal peak in the chromatogram obtained 20.0 mL with the solvent mixture. Dilute 1.0 mL of this
with reference solution (a) (0.10 per cent); solution to 20.0 mL with the solvent mixture. To 5.0 mL of
- sum of impurities otherthan A, Band C: not more than the solution obtained, add 4 mL of solution A, mix and
3 times the area of the principal peak in the allow to stand for 2.5 h at room temperature. Add 5.0 mL of
chromatogram obtained with reference solution (a) hexane R and shake for 1 min. Allow the layers to separate
(0.3 per cent); and use the upper layer.
- disregard limit: 0.5 times the area of the principal peak in Blank solution To 5 mL of the solvent mixture add 4 mL of
the chromatogram obtained with reference solution (a) solution A, mix and allow to stand for 2.5 h at room
(0.05 per cent). temperature. Add 5.0 mL of hexane R and shake for 1 min.
Impurities D and E Allow the layers to separate and use the upper layer.
Liquid chromatography (2.2.29). Use freshly prepared solutions. Column:
Store and inject them at 8°C, using a cooled autosampler. - size: I = 0.25 m, 0 = 4.0 mID;
Solution A 1.25 gIL solution of potassium dihydrogen - stationary phase: cyanosilyl silica gelfor chromatography R
(5 urn) with a pore size of 10 TIm;
phosphate R.
- temperature: 30°C.
Testsolution Dissolve 50.0 mg of the substance to be
examined in 5.0 mL of a 4 gIL solution of sodium hydroxide R Mobilephase 2-propanol R, hexane R (5:95 VIV).
and dilute immediately to 100.0 mL with solution A. Flow rate 1.5 mUmin.
Reference solution Dissolve 5.0 mg of allopurinol Detection Spectrophotometer at 310 nm.
impurity D CRS and 5.0 mg of allopurinol impurity E CRS in Injection 20 J.!L.
5.0 mL of a 4 gIL solution of sodium hydroxide R and dilute Relative retention With reference to benzaldehyde (retention
immediately to 100.0 mL with solution A. Dilute 1.0 mL of time = about 2.8 min): benzaldehyde azine = about 0.8.
this solution to 100.0 mL with solution A. System suitability Reference solution:
Column: - resolution: minimum 2 between the peaks due to
- size: I = 0.05 m, 0 = 4.6 mm; benzaldehyde azine and benzaldehyde;
- stationary phase: base-deactivated octadecylsilyl silica gelfor - signal-to-noise ratio: minimum 20 for the peak due to
chromatography R (3 JlID). benzaldehyde azine.
Mobzle phase methanolR, 1.25 gIL solution of potassium Limit:
dihydrogen phosphate R (10:90 VIV). - impurity F: the area of the peak due to benzaldehyde azine
Flow rate 2 mUmin. in the chromatogram obtained with the test solution is not
Detection Spectrophotometer at 230 TIm. more than the area of the corresponding peak in the
chromatogram obtained with the reference solution
Injection 20 J.!L.
(10 ppm of hydrazine sulfate equivalent to 2.5 ppm of
Run time 1.5 times the retention time of impurity E. hydrazine).
=
Retention times Impurity D about 3.6 min;
Loss on drying (2.2.32)
impurity E = about 4.5 min.
Maximum 0.5 per cent, determined on 1.000 g by drying in
System suitability Reference solution: an oven at 105°C.

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2020 Almagate 1-105

Sulfated ash (2.4.14)

Maximum 0.1 per cent, determined on 1.0 g.
Almagate
ASSAY (Ph. Eur. monograph 2010)
Liquid chromatography (2.2.29) as described in the test for AlzMg6C202oH14,4H20 630 66827-12-1
related substances with the following modification.
Injection Test solution (b) and reference solution (c). Action and use
Calculate the percentage content of C5H~40 from the Antacid.
declared content of allopurinol CRS. Ph Eur ---'- --'-- _
IMPURITIES DEFINITION
Specified impurities A, B, C, D, E, F. Hydrated aluminium magnesium hydroxycarbonate.
Content
- aluminium: 15.0 per cent to 17.0 per cent (calculated
as Alz0 3) ,
- magnesium: 36.0 per cent to 40.0 per cent (calculated
as MgO),
- carbonic acid: 12.5 per cent to 14.5 per cent (calculated
A. 5-amino-1H-pyrazole-4-carboxamide, as CO 2) ,
CHARACTERS
Appearance
White or almost white, fine, crystalline powder.
Solubility
Practically insoluble in water, in ethanol (96 per cent) and in
methylene chloride. It dissolves with effervescence and
B. 5-(fonnylamino)-IH-pyrazole-4-carboxamide, heating in dilute mineral acids.
IDENTIFICATION
A. Infrared absorption spectrophotometry (2.2.24).
Comparison Ph. Eur. reference spectrum of almagate.
B. Dissolve 0.15 g in dilute hydrochloric acid R and dilute to
20 mL with the same acid. 2 ml, of the solution gives the
reaction of aluminium (2.3.1).
C. 2 mL of the solution prepared under identification test B
C. 5-(4H-l ,2,4-triazol-4-yl)-IH-pyrazole-4-carboxamide, gives the reaction of magnesium (2.3.1).
TESTS
pH (2.2.3)
9.1 to 9.7.
Disperse 4.0 g in 100 mL of carbon dioiide-free waterR, stir
for 2 min and filter.
Neutralising capacity
D. ethyl 5-amino-1H-pyrazole-4-carboxylate,
Carry out the test at 37 DC Disperse 0.5 g in 100 mL of
water R, heat, add 100.0 mL of 0.1 M hydrochloric acid,
previously heated and stir continuously; the pH (2.2.3) of the
solution between 5 min and 20 min is not less than 3.0 and
not greater than 4.5. Add 10.0 mL of 0.5 M hydrochloric acid,
previously heated, stir continuously for 1 h and titrate with
0.1 M sodium hydroxide to pH 3.5; not more than 20.0 mL of
E. ethyl 5-(formylamino)-lH-pyrazole-4-carboxylate, 0.1 M sodium hydroxide is required.
Chlorides (2.4.4)
Maximum 0.1 per cent.
Dissolve 0.33 gin 5 mL of dilute nitricacid R and dilute to
F. diazane (hydrazine). 100 mL with water R. Prepare simultaneously the standard
_ _ _ _ _ _ _ _ _...:..- ---'- PhEur by diluting 0.7 mL of dilute nitric acid R to 5 mL with
water R and adding 10 mL of chloride standardsolution (5 ppm
Cl) R.
Sulfates (2.4.13)
Maximum 0.4 per cent.
Dissolve 0.25 gin 5 mL of dilute hydrochloric acid R, and
dilute to 100 mL with distilled water R. Prepare
simultaneously the standard by adding 0.8 mL of dilute
hydrochloric acidR to 15 mL of sulfate standardsolution
(10 ppm SO,J R.

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1-106 Almond Oil 2020

Sodium Run time 16 min.

Maximum 150 ppm. System suitability:
Atomic absorption spectrometry (2.2.23~ Method 1). - average percentage of carbon in 5 reference samples must
Test solution Dissolve 0.25 g in 50 mL of a 103 gIL solution be within ± 0.2 per cent of the value assigned to
of hydrochloric acid R. the CRS; the difference between the upper and the lower
Reference solutions Prepare the reference solutions using values of the percentage of carbon in these samples must
sodium standard solution (200 ppm N a) R, diluted as necessary be below 0.2 per cent.
with a 103 gIL solution of hydrochloric acid R. Calculate the percentage content of carbonic acid in the test
sample according to the following formula:
Loss on ignition
43.0 per cent to 49.0 per cent, determined on 1.000 g by A
ignition at 900 ± 50°C. CxKx-.:..
m
Microbial contamination
C percentage content of carbonic acid in the reference sample;
T AMC: acceptance criterion 103 CFU/g (2.6.12). K mean value for the 5 reference samples of the ratio of the mass
TYMC: acceptance criterion 102 CFU/g (2.6.12). in milligrams to the area of the peak due to carbonic acid;
A area of the peak due to carbonic acid in the chromatogram
Absence of Escherichia coli (2.6.13). obtained with the test sample;
, Absence of Pseudomonas aeruginosa (2.6.13). m sample mass, in milligrams.

ASSAY
STORAGE
Aluminium
Dissolve 1.000 ginS mL of hydrochloric acid R, heating if In an airtight container.
_ _ _ _ _ _ _ _ _ _ _-:- PhEur
necessary. Allow to cool to room temperature and dilute to
100.0 mLwith water R (solution A). Introduce 10.0 mL of
solution A into a 250 mL conical flask, add 25.0 mL of
0.05 M sodium edetate, 20 mL of buffersolution pH 3.5 R,
40 mL of ethanolR and 2 mL of a freshly prepared 0.25 gIL Virgin Almond Oil
solution of dithizone R in ethanolR. Titrate the excess of
sodium edetate with 0.05 M zinc sulfate until the colour Almond Oil
changes from greenish-violet to pink. (Ph. Eur. monograph 0261)
1 mL of 0.05 M sodium edetate is equivalent to 2.549 mg Preparation
of Al2 0 3 . Almond Oil Ear Drops
Magnesium PhEur ~ _
Introduce 10.0 mL of solution A prepared in the assay of
aluminium into a 500 mL conical flask, add 200 mL of DEFINITION
water R, 20 mL of triethanolamine R with shaking, 10 mL of Fatty oil obtained by cold expression from the ripe seeds of
ammonium chloride buffersolution pH 10.0 Rand 50 mg of Prunus dulcis (Mill.) D.A.Webb var. dulcis or Prunus dulcis
mordant black 11 triturate R. Titrate with 0.05 M sodium (Mill.) D.A.Webb var. amara (DC.) Buchheim or a mixture
edetate until the colour changes from violet to pure blue. of both varieties.
1 mL of 0.05 M sodium edetate is equivalent to 2.015 mg CHARACTERS
of MgO. Appearance
Carbonic acid Yellow, clear liquid.
12.5 per cent to 14.5 per cent. Solubility
Test sample Place 7.00 mg of the substance to be examined Slightly soluble in ethanol (96 per cent), miscible with light
in a tin capsule. Seal the capsule. petroleum.
Reference sample Place 7.00 mg of almagate CRS in a tin Relative density
capsule. Seal the capsule. About 0.916.
Introduce separately the test sample and the reference sample It solidifies at about -18°C.
into a combustion chamber of a CRN analyser purged with IDENTIFICATION
helium for chromatography R and maintained at a temperature First identification: A~ C.
of 1020 DC. Simultaneously, introduce oxygen R at a pressure
Secondidentification: A~ B.
of 40 kPa and a flow rate of 20 mIJmin and allow complete
combustion of the sample. Sweep the combustion gases A. Absorbance (see Tests).
through a reduction reactor and separate the gases formed by B. Identification of fatty oils by thin-layer chromatography
gas chromatography (2.228). (2.3.2).
Column: Results The chromatogram obtained is similar to the
- size: I = 2 m, 0 = 4 1DIn; corresponding chromatogram shown in Figure 2.3.2.-1.
- stationary phase: ethyl'lJinylbenzene-divinylbenzene C. Composition of fatty acids (see Tests).
copolymer Rl.
TESTS
Carrier gas heliumfor chromatography R. Specific absorbance (2.2.25)
Flow rate 100 mUmin. Maximum 0.2, determined at the absorption maximum at
Temperature: 270 nm.The ratio of the absorbance measured at 232 nm to
- column: 65 DC; that measured at 270 nm is greater than 7.
- detector: 190 DC. To 0.100 g add cyclohexane R and dilute to 10.0 rnL with the
Detection Thermal conductivity. same solvent. Adapt the concentration of the solution so that

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 2020 Almond Oil 1-107

the absorbance lies between 0.5 and 1.5, measured in a 1 em IDENTIFICATION

cell. A. Identification of fatty oils by thin-layer chromatography
Acid value (2.5.1) (2.3.2).
Maximum 2.0, determined on 5.0 g. Results The chromatogram obtained is similar to the
Peroxide value (2.5.5, Method A) corresponding chromatogram shown in Figure 2.3.2.-1.
Maximum 15.0. B. Composition of fatty acids (see Tests).
Unsaponifiable matter (2.5.7) TESTS
Maximum 0.9 per cent, determined on 5.0 g. Specific absorbance (2.2.25)
Composition offatty acids (2.4.22, Method A) 0.2 to 6.0, determined at the absorption maximum at
Use the mixture of calibrating substances in Table 2.4.22.-3. 270 nm.
Composition of thefatty-acidfraction of the oil: To 0.100 g add cyclohexane R and dilute to 10.0 mL with the
- saturatedfatty acids of chain length less than C16 : maximum same solvent. Adapt the concentration of the solution so that
0.1 per cent, the absorbance lies between 0.5 and 1.5, measured in a 1 ern
- palmitic acid: 4.0 per cent to 9.0 per cent, cell.
- palmitoleic acid: maximum 0.8 per cent, Acid value (2.5.1)
- margaric acid: maximum 0.2 per cent, Maximum 0.5, determined on 5.0 g.
- stearic acid: maximum 3.0 per cent, Peroxide value (2.5.5, Method A)
- oleic acid: 62.0 per cent to 86.0 per cent, Maximum 5.0.
-, linoleic acid: 20.0 per cent to 30.0 per cent,
- linolenic acid: maximum 0.4 per cent, Unsaponifiable matter (2.5.7)
-.:.:::. "arachidic acid: maximum 0.2 per cent, Maximum 0.9 per cent, determined on 5.0 g.
~ eicosenoic acid: maximum 0.3 per cent, Composition of fatty acids (2.4.22, Method A)
- behenic acid: maximum 0.2 per cent, Use the mixture of calibrating substances in Table 2.4.22.-3.
- erucic acid: maximum 0.1 per cent. Composition of the fatty-acidfraction of the oil:
Sterols (2.4.23) - saturated fatty acids of chain length less than C16 : maximum
Composition of sterol fraction of the oil: 0.1 per cent;
- cholesterol: maximum 0.7 per cent, - palmitic add: 4.0 per cent to 9.0 per cent;
- campesterol: maximum 4.0 per cent, - palmitoleic acid: maximum 0.8 per cent;
- stigmasterol: maximum 3.0 ·per .cent, - margaric add: maximum 0.2 per cent;
- p-sitosterol: 73.0 per cent to 87.0 per cent, - stearic acid: maximum 3.0 per cent;
- 115-avenasterol: minimum 10.0 per cent, - oleic acid: 62.0 per cent to 86.0 per cent;
- 117-stigmastenol: maximum 3.0 percent, - linoleic acid: 20.0 per cent to 30.0 per cent;
- 117-avenasterol: maximum 3.0 per cent, - linolenic acid: maximum 0.4 per cent;
- brassicasterol: maximum 0.3 per cent. - arachidic acid: maximum 0.2 per cent;
Water (2.5.32) - eicosenoic acid: maximum 0.3 per cent;
Maximum 0.1 per cent, determined on 1.00 g. - behenic acid: maximum 0.2 per cent;
- erucic acid: maximum 0.1 per cent.
STORAGE
Sterols (2.4.23)
In a well-filled container, protected from light. Composition of the sterol fraction of the oil:
______- - - - - - - - - - - - - - - - - PhEur - cholesterol: maximum 0.7 per cent;
- campesterol: maximum 5.0 per cent;
- stigmasterol: maximum 4.0 per cent;
- f3-sitosterol: 73.0 per cent to 87.0 per cent;
Refined Almond Oil - L15-avenasierol: minimum 5.0 per cent;
- L1 7-stigmastenol: maximum 3.0 per cent;
- (ph. Bur. monograph 1064) - L1 7-avenasterol: maximum 3.0 per cent;
PhEur '-- _ - brassicasterol: maximum 0.3 per cent.
Water (2.5.32)
DEFINITION
Maximum 0.1 per cent, determined on 1.00 g.
Fatty oil obtained from the ripe seeds of Prunus dulcis
(Mill.) D.A. Webb var. dulcis or Prunusdulcis (Mill.) D.A. STORAGE
Webb var. amara (DC.) Buchheim or a mixture of both In a well-filled container, protected from light.
varieties by cold expression. It is then refined. A suitable _ _ _ _ _ _ _-'- PhEur
antioxidant may be added.
CHARACTERS
Appearance
Pale yellow, clear liquid.
Solubility
Slightly soluble in ethanol (96 per cent), miscible with light
petroleum.
Relative density
About 0.916.
,It solidifies at about -18°C.

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1-108 Alprazolam 2020

Alprazolam Application 5 J1L.

Development Over a path of 12 em.
(ph. Bur. monograph 1065) Drying In air.
Detection Examine in ultraviolet light at 254 nm.
System suitability Reference solution (b):
- the chromatogram shows 2 clearly separately spots.
Results The principal spot in the chromatogram obtained
with the test solution is similar in position and size to the
principal spot in the chromatogram obtained with reference
solution (a).
TESTS
Related substances
308.8 28981-97-7 Liquid chromatography (2.2.29).
Buffer solution Dissolve 7.7 g of ammonium acetate R in
Action and use 1000 mL of water R and adjust to pH 4.2 with glacial acetic
.Benzodiazepine, acid R."
PhEur _-:-- _ Test solution Dissolve 0.100 g of the substance to be
examined in dimethylformamide R and dilute to 10.0 mL with
DEFINITION the same solvent.
8-Chloro-l-methyl-6-phenyl-4H-[1,2,4]triazolo[4,3~a]
Reference solution (a) Dissolve 2 mg of alprazolam CRS and
[1,4]benzodiazepine.
2 mg of triazolam CRS in dimethylformamide R and dilute to
Content 100.0 mL with the same solvent.
99.0 per cent to 101.0 per cent (dried substance).
Reference solution (b) Dilute 5.0 mL of the test solution to
CHARACTERS 100.0 mL with dimethylformamide R. Dilute 0.5 mL of this
Appearance solution to 10.0 mL with dimethylformamide R.
White or almost white, crystalline powder. Column:
Solubility =
- size: 1 0.25 m, 0 = 4.6 mm;
Practically insoluble in water, freely soluble in methylene - stationary phase: phenylsilyl silica gel for chromatography R1
chloride, sparingly soluble in acetone and in ethanol (5 um),
(96 per cent). Mobile phase:
It shows polymorphism (5.9). - mobile phase A: buffer solution, methanol R (44:56 VIII);
- mobile phase B: buffer solution, methanol R (5:95 VIII);
IDENflFICATION
- temperature: 40°C;
First identification: B.
Second identification: A.1 C. Time Mobile phase A Mobile phase B
A. Dissolve the substance to be examined in the smallest (min) (per cent VIJ') (per cent VIJ')
necessary quantity of ethyl acetate R and evaporate to dryness 0-15 98 2
on a water-bath. Thoroughly mix 5.0 mg of the substance to 15 - 35 98 ~ 1 2 ~ 99
be examined with 5.0 mg of alprazolam CRS. The melting 35 - 40 1 99
point (2.2.14) of the mixture does not differ by more than
2 °C from the melting point of the substance to be Flow rate 2 mL/min.
examined.
Detection Spectrophotometer at 254 nm.
B. Infrared absorption spectrophotometry (2.2.24). Injection 10 J1L; inject dimethylformamide R as a blank.
Preparation Discs.
Retention time Triazolam = about 9 min;
Comparison alprazolam CRS. alprazolam = about 10 min.
If the spectra obtained in the solid state show differences, System suitability Reference solution (a):
dissolve the substance to be examined and the reference - resolution: minimum 1.5 between the peaks due to
substance separately in the minimum volume of ethyl triazolam and alprazolam.
acetate R, evaporate to dryness on a water-bath and record
Limits:
new spectra using the residues.
- total: not more than the area of the principal peak in the
C. Thin-layer chromatography (2.2.27). chromatogram obtained with reference solution (b)
Test solution Dissolve 10 mg of the' substance to be (0.25 per cent);
examined in methanol R and dilute to 10 mL with the same - disregard limit: 0.2 times the area of the principal peak in
solvent. the chromatogram obtained with reference solution (b)
Reference solution (a) Dissolve 10 mg of alprazolam CRS in (0.05 per cent).
methanol R and dilute to 10 mL with the same solvent. Loss on drying (2.2.32)
Reference solution (b) Dissolve 10 mg of alprazolam CRS and Maximum 0.5 per cent, determined on 1.000 g by drying in
10 mg of midazolam CRS in methanol R and dilute to 1'0 mL an ovenat 105°C.
with the same solvent. Sulfated ash (2.4.14)
Plate TLC silica gelGF254 plate R. Maximum 0.1 per cent, determined on 1.0 g.
Mobile phase glacial acetic acid R, water R, methanol R, ethyl
acetate R (2:15:20:80 VIVIVIII). '

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2020 Alprazolam 1-109
-----------------------------------

H3C
ASSAY )=N,
Dissolve 0.140 gin 50 mL of a mixture of Z volumes of N

~
acetic anhydride Rand 3 volumes of anhydrous acetic add R.
Titrate with 0.1 M perchloric add, determining the end-point
potentiometrically (2.2.20). Titrate to the 2n d point of CI
~..I Nlu el

inflexion.
I~
1 mL of 0.1 M perchloric add is equivalent to 15.44 mg #
of C 17H13CIN4 •
STORAGE F. [5-chloro.,.Z-[3-(chloromethyl)-5-methyl-4H-l,Z,4-triazol-
Protected from light. 4-yl]phenyl] phenylmethanone,
IMPURITIES
H3C

og
~I
r~'N
and enantiomer
CI '" "'" NH,

I~
#
A. (4RS)-3-amino-6-chloro-2-methyl-4-phenyl-3,4-
dihydroquinazolin-d-ol, G.7-chloro-l-methyl-5-phenyl[I,2,4]triazolo[4,3-a]quinolin-
4-amine,

B. ·[5-chloro-2-[3-(hydroxymethyl)-5-methyl-4H-1,Z,4-triazol- H. bis [[4-(2-benzoyl-4-chlorophenyl)-5-methyl-4H-1,2,4-

4-yl]phenyl]phenylmethanone, triazol-3-yl]methyl]amine,

H3C
yN
r ,

N_!(
CI
: I N
) N-N
'I ~

~ . , h
C. [5-chloro-2-[3-methyl-4H-1,2,4-triazol-4-yl]phenyl] CI
phenylmethanone,
1. [5-chloro-2-[3- [[(6RS)-8-chloro-6-hydroxy-l-methyl-6-
phenyl-4H-[1,Z,4]triazolo [4,3-a] [1,4]benzodiazepin-5
(6H)-yl] methyl]-5-methyl-4H-l ,2,4-triazol-4-yl]phenyl]
phenylmethanone,

D.8-chloro-1-ethenyl-6-phenyl-41l-[1,2,4]triazolo[4,3-a]
[1,4]benzodiazepine,

~'#
.~1fY
J. Z,17-dichloro-6,13-dimethyl-18b,19a-dipheny~

CI
. .. I NH2.0 8b,19adihydro-l0H,18bH-[1,Z,4]triazolo
[4'" ,3"':1 ",Z"]quinolo[3",4":4',5']oxazolo[3',Z'-d]-
~ 1,2,4.,.triazolo[4,3-a][1,4]benzodiazepine.
_ _ _ _ _ _ _ _ _-t--t-r- PhEur

E. (2-amino-5-chlorophenyl)phenylmethanone,

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 1-110 Alprenolol Hydrochloride 2020

Test solution (a) Dissolve 0.50 g of the substance to be

Alprenolol Hydrochloride examined in methanol R and dilute to 10 mL with the same
(Ph. Eur. monograph 0876) solvent.
Test solution (b) Dilute 1 mL of test solution (a) to 50 mL
H pH H with methanol R.

oc:
~N
~ I yCH3. HCI and enantiomer
Reference solution (a) Dissolve 10 mg of alprenolol
hydrochloride CRS in methanolR and dilute to 10 mL with the
~ :::::,... CH3
CH2 same solvent.
Reference solution (b) Dissolve 10 mg of alprenolol
285.8 13707-88-5 hydrochloride CRS and 10 mg of oxprenolol hydrochloride CRS
in methanol R and dilute to 10 mL with the same solvent.
Action and use
Reference solution (c) Dilute 5 mL of test solution (b) to
Beta-adrenoceptor antagonist.
50 mL with methanol R.
PhEur _ Plate TLC silica gel G plate R.
DEFINITION Mobile phase Place 2 beakers each containing 30 mL of
, (2RS)-1-[(I-Methylethyl)amino]-3-[2-(prop-2-enyl) ammonia R at the bottom of the tank containing a mixture of
phenoxy] propan- 2-01 hydrochloride. 5 volumes of methanol Rand 95 volumes of ethyl acetate R.
Content Application 5 flL.
99.0 per cent to 101.0 per cent (dried substance). Development Over a path of 15 em in a tank saturated for at
least 1 h.
CHARACTERS
Drying At 100°C for 15 min.
Appearance
White or almost white, crystalline powder or colourless Detection Expose to iodine vapour for up to 6 h.
crystals. System suitabz1t"ty Reference solution (b):
Solubility - the chromatogram shows 2 clearly separated spots.
Very soluble in water, freely soluble in ethanol (96 per cent) Limits Test solution (a):
and in methylene chloride. - impurityD: any spot with an R p value greater than that of
the principal spot is not more intense than the principal
IDENTIFICATION
spot in the chromatogram obtained with reference
First identification: B~ D. solution (c) (0.2 per cent).
Second identification: A~ C~ D.
Related substances
A. Melting point (2.2.14): 108°C to 112°C. Liquid chromatography (2.2.29).
B. Infrared absorption spectrophotometry (2.2.24). Test solution Dissolve 20.0 mg of the substance to be
Comparison alprenolol hydrochloride CRS. examined in the mobile phase and dilute to 10.0 mL with
C. Examine the chromatograms obtained in the test for the mobile phase.
impurity D. Reference solution (a) Dissolve 4.0 mg of alprenolol
Detection Examine in daylight, after exposure to iodine hydrochloride CRS and 0.8 mg of 4-isopropylphenol R in the
vapour for 30 min. mobile phase and dilute to 100.0 mL with the mobile phase.
Results The principal spot in the chromatogram obtained Reference solution (b) Dilute 4.0 mL of the test solution to
with test solution (b) is similar in position, colour and size to 100.0 mL with the mobile phase. Dilute 1.0 mL of this
the principal spot in the chromatogram obtained with solution to 10.0 mL with the mobile phase.
reference solution (a). Column:
D. It gives reaction (a) of chlorides (2.3.1). - size: 1 = 0.15 m, 0 = 4 mm;
- stationary phase: octylsilyl silica gelfor chromatography R
TESTS
(5 urn).
Solution S
Dissolve 1.0 g in carbon dioxide-free waterR and dilute to Mobile phase Mix 0.656 g of sodium octanesulfonate R with
150 mL of acetonitrile R and dilute to 500 mL with
50 mL with the same solvent.
phosphate buffer pH 2.8 prepared as follows: mix 1.78 g of
Appearance of solution phosphoric acid Rand 15.6 g of sodium dihydrogen phosphate R
Solution S is clear (2.2.1) and not more intensely coloured and dilute to 2000 mL with waterR.
than reference solution B 9 (2.2. 2~ Method II).
Flow rate 1 mUmin.
Acidity or alkalinity Detection Spectrophotometer at 280 nm.
To 10 mL of solution S add 0.2 mL of methylred solution R
and 0.2 mL of 0.01 M hydrochloric acid; the solution is red. Equilibration With the mobile phase for about 1 h.
Add 0.4 mL of 0.01 M sodium hydroxide; the solution is Injection 20 flL.
yellow. Run time Twice the retention time of alprenolol.
Impurity C Retention time Alprenolol = about 11 min;
Maximum 0.1 per cent. 4-isopropylphenol = about 18 min.
Dissolve 0.25 g in ethanol (96 per cent) R and dilute to System suitability Reference solution (a):
25 mL with the same solvent. The absorbance (2.2.25) ---.,... resolution: minimum 5 between the peaks due to alprenolol
measured at 297 nm is not greater than 0.20. and 4-isopropylphenol; if necessary, adjust the
ImpurityD concentration of sodium oetanesulfonate and/or
Thin-layer chromatography (2.2.27). acetonitrile in the mobile phase (increase the

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2020 Alprostadil 1-111

concentration of sodium oetanesulfonate to increase the

retention time of alprenolol and increase the
concentration of acetonitrile to decrease the
retention times of both compounds).
Limits:
- unspecified impurities: for each impurity, not more than D. 1,l'-[(1-methylethyl)imino]bis[3-[2-(prop-2-enyl)phenoxy]
0.25 times the area of the principal peak in the propan-Z-ol] .
chromatogram obtained with reference solution (b) _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
(0.10 per cent);
- total: not more than the area of the principal peak in the
chromatogram obtained with reference solution (b)
(0.4 per cent);
- disregard limit: 0.1 times the area of the principal peak in Alprostadil
the chromatogram obtained with reference solution (b)
(0.04 per cent). (Ph. Bur. monograph 1488)
Loss on drying (2.2.32)
Maximum 0.5 per cent, determined on 1.000 g by drying
over diphosphorus pentoxide R at a pressure not exceeding
2.7 kPa.
Sulfated ash (2.4.14)
Maximum 0.1 per cent; determined on 1.0 g.
ASSAY 354.5 745-65-3
Dissolve 0.400 g in 25 mL of a mixture of equal volumes of
anhydrousethanolR and waterR. Add 10 mL of 0.01 M Action and use
hydrochloric acid. Carry out a potentiometric titration (2.2.20), Prostaglandin E 1 (pGE}).
using 0.1 M sodium hydroxide. Read the volume added
PhEur _
between the 2 points of inflexion.
1 mL of 0.1 M sodiumhydroxide is equivalent to 28.58 mg DEFINITION
of ClsH24CINOZ. 7-[(lR,2R,3R)-3-Hydroxy-2-[(IE,3S)-3-hydroxyoct-l-enyl]-
STORAGE 5-oxocyclopentyl]heptanoic acid.
Protected from light. Content
95.0 per cent to 102.5 per cent (anhydrous substance).
IMPURITIES
Specified impurities C, D. CHARACTERS
Other detectable impurities (the following substances would, if Appearance
present at a sufficient level, be detected by one or other of the tests White or slightly yellowish, crystalline powder.
in the monograph. They arelimitedby the general acceptance Solubility
criterion for other/unspecified impurities and/orby the general Practically insoluble in water, freely solubleinethanol
monograph Substances for pharmaceutical use (2034). It is (96 per cent), soluble in acetone, slightlysoluble in ethyl
therefore not necessary to identify these impurities for acetate.
demonstration of compliance. See also 5.10. Control of impurities IDENfIFICATION
in substances for pharmaceutical use) A, B. A. Specific optical rotation (2.2.7): -70 to -60 (anhydrous
substance) .
H pH
O~OH and enantiomer
~v~
Immediately before use, dissolve 50 mg in ethanol
(96 per cent) R and dilute to 10.0 mL with the same solvent.
~CH2 B. Infrared absorption spectrophotometry (2.2.24).
Comparison alprostadil CRS.
A. (2RS)- 3-[2-(prop-2-enyl)phenoxy]propan-l,2-diol, C. Examine the chromatograms obtained in the assay.
Results The principal peak inthe chromatogram obtained
with the test solution is similar in retention time and size to
the principal peak in the chromatogram obtained with the
reference solution.
TESTS
B. 2-(prop-2-enyl)phenol,
Related substances
Liquid chromatography (2.2.29). Prepare the solutions protected
H pH H
from light.

OC
O~ N y. CH3 and enantiomer Test solution Dissolve 10.0 mg of the substance to be
~
I CH3 examined in a mixture of equal volumes of acetonitrile Rl and
~CH3
water R and dilute to 10.0 mL with the same mixture of
solvents.
C. (2RS)-I-[(l-methylethyl)amino]-3-[2-(prop-l-enyl) Reference solution (a) Dilute 100 ilL of the test solution to
phenoxy]propan-2-o1, 20.0 mL with a mixture of equal volumes of acetonitrile Rl
and water R.

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1-112 Alprostadil 2020

Reference solution (b) Dissolve 1.0 mg of dinoprostone Time Mobile phase A Mobile phase B
(min) (per cent VIJl) (per cent VIJI)
impurity C CRS (alprostadil impurity H) and 1.0 mg of the
substance to be examined in a mixture of equal volumes of 0-50 100 o
acetonitrile Rl and water R and dilute to 20.0 mL with the 50 - 51 100 ..... 0 0-> 100
same mixture of solvents. 51 - 61 o 100
61 - 62 0-> 100 100 ..... 0
Reference solution (c) In order to prepare impurities A and B
in situ, dissolve 1 mg of the substance to be examined in
62 -72 100 o
100 ul, of 1 M sodium hydroxide (the solution becomes
brownish-red), wait for 3 min and add 100 JlL of a 112 gIL Relative retention With reference to alprostadil (retention
solution of phosphoric acid R (yellowish-white opalescent time = about 7 min): impurity A = about 2.4;
solution); dilute to 5.0 mL with a mixture of equal volumes impurity B = about 2.6.
of acetonitrile Rl and waterR. System suitability:
System A - resolution: minimum 1.5 between the peaks due to
Column: impurity A and impurity B in the chromatogram obtained
=
- size: I 0.25 m, 0 = 4.0 mm; with reference solution (c).
- stationary phase: base-deactivatedoctylsilyl silica gelfor Carry out the test according to system A and B.
chromatography R· (4 urn) with a pore size of 6 nID; Limits:
- temperature: 35°C. - correction factors: for the calculation of content, multiply
Mobz7e phase: the peak areas of the impurities listed in Table 1488.-1 by
- mobile phase A: dissolve 3.9 g of sodium dihydrogen the corresponding correction factor;
phosphate R in water R and dilute to 1.0 L with the same
solvent; adjust to pH 2.5 with a 2.9 gIL solution of Table 1488.-1.
phosphoric acid R (approximately 600 mL is required); Impurity Relative retention Relative retention Correction factor
to 740 mL of the buffer solution add 260 mL of (system A) (system B)
acetonitrile Rl; impurity G 0.80 0.7
- mobile phase B: dissolve 3.9 g of sodium dihydrogen impurity F 0.88 0.8
phosphate R in water R and dilute to 1.0 L with the same impurity D 0.90 1.0
solvent; adjust to pH 2.5 with a 2.9 gIL solution of impurity H 0.96 0.7
phosphoric acid R (approximately 600 mL is required); impurity E 1.10 0.7
to 200 mL of the buffer solution add 800 mL of impurity C 1.36 1.9
acetonitrile Rl; impurity K 1.85 0.06
impurity A 2.32 0.7
Time Mobile phase A Mobile phase B impurity B 2.45 1.5
(min) (per cent VIJl) (per cent VIJl) impurity I 4.00 1.0
0-75 100 a impurity J 5.89 1.0
75 - 76 100 -> 0 0 ..... 100
76 - 86 a 100 - impurityA: not more than 3 times the area of the
86 - 87 0-> 100 100 ..... a principal peak in the chromatogram obtained with
87 - 102 100 a reference solution (a) (1.5 per cent);
- impurity B: not more than the area of the principal peak in
Flow rate 1 mIlmin. the chromatogram obtained with reference solution (a)
(0.5 per cent);
Detection Spectrophotometer at 200 nID'
- any other impurity: not more than 1.8 times the area of the
Injection 20 j.lL. principal peak in the chromatogram obtained with
Retention time Alprostadil = about 63 min. reference solution (a) (0.9 per cent), and not more than 1
System suitability: such peak has an area greater than the area of the
- resolution: minimum 1.5 between the peaks due to principal peak in the chromatogram obtained with
impurity Hand alprostadil in the chromatogram obtained reference solution (a) (0.5 per cent). Evaluate impurities
with reference solution (b). appearing at relative retentions less than 1.2 by system A
System B and impurities appearing at relative retentions greater than
Use the same conditions as for system A with the following 1.2 by system B;
mobile phase and elution programme: - total: not more than 3 times the area of the principal peak
- mobile phaseA: dissolve 3.9 g of sodium dihydrogen in the chromatogram obtained with reference solution (a)
phosphate R in water R and dilute to 1.0 L with the same (1.5 per cent);
solvent; adjust to pH 2.5 with a 2.9 gIL solution of - disregard limit: 0.1 times the area of the principal peak in
phosphoric acid R (approximately 600 mL is required); the chromatogram obtained with reference solution (a)
to 600 mL of the buffer solution add 400 mL of (0.05 per cent).
acetonitrile Rl; Water (2.5.32)
- mobile phase B: use mobile phase B as described under Maximum 0.5 per cent, determined on 50 mg.
system A; ASSAY
Liquid chromatography (2.2.29) as described in the test for
related substances, system A. Prepare the'solutions protected
from light.
Test solution Dissolve 10.0 mg of the substance to be
examined in a mixture of equal volumes of acetonitrile Rl and

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2020 Alprostadil 1-113

~C~H
water R and dilute to 25.0 mL with the same mixture of
solvents. Dilute 3.0 mL of the solution to 20.0 mL with a
mixture of equal volumes of acetonitrile RJ and water R.
Reference solution Dissolve 5.0 mg of alprostadil CRS in a HO~~CH3
mixture of equal volumes of acetonitrile Rl and water Rand H H OH
dilute to 25.0 mL with the same mixture of solvents. Dilute
6.0 mL of the solution to 20.0 mL with a mixture of F. 7-[(IS,2R,3R)-3-hydroxy-2-[(IE,3S)-3-hydroxyoct-l-
equal volumes of acetonitrile Rl. and waterR. enyl]-5-oxocyclopentyl]heptanoic acid
Injection 20 J.tL. (8-epiprostaglandin E1) ,
Calculate the percentage content of CZOH340S taking into

).J--~C~H
account the assigned content of alprostadil CRS.

HO~CH,
STORAGE
At a temperature of 2 °C to 8°C.
IMPURITIES H H OH

G. (5Z)-7-[(IR,2R,3R)-3-hydroxy-2-[(IE,3S)-3-hydroxyoct-
l-enyl]~5-oxocyclopentyl]hept-5-enoic acid '
(dinoprostone),

A. 7';;[(IR,2S)-2-[(IE,3S)-3-hydroxyoet-l-enyl]-5-
_·-oxocyclopent-3-enyl]heptanoic acid (prostaglandin AI),

o
H. (5E)-7-[(IR,2R,3R)-3-hydroxy-2-[(IE,3S)-3-hydroxyoct-
l-enyl]-5-oxocyclopentyl]hept-5-enoic acid ((5E)-
prostaglandin E 2 ) ,

B. 7-[2-[(IE,3S)-3-hydroxyoct-l-enyl]-5-oxocyclopent-l-
enyl]heptanoic acid (prostaglandin B 1) ,

1. ethyl 7- [(1R,2R,3R)-3-hydroxy-2- [(1E,3S)- 3-hydroxyoct-

l-enyl] -5-oxocyclopentyl]heptanoate . (prostaglandin E 1,
ethyl ester),
C.7-[(IR,2R,3R)-3-hydroxy-2-[(IE)-3-oxooct-l-enyl]-5-
oxocyclopentyl]heptanoic acid (15-oxoprostaglandin E 1) ,

J. l-methylethyl 7- [(1R,2R,3R)-3-hydroxy-2- [(1E,3S)-3-

hydroxyoct-1-enyl]-5-oxocyclopentyl] heptanoate
D.7-[(IR,2R,3R)-3-hydroxy-2-[(IE,3R)-3-hydroxyoet-l- (prostaglandin E 1, isopropyl ester),
enyl]-5-oxocyclopentyl]heptanoic acid (15-
epiprostaglandin E 1) , o

O-.6~-O·
~~I
j
~

K. triphenylphosphine oxide.
_ _ _ _ _ _ _ _ _ _- - - - - - PhEur
E. 7- [(1R,2R,3S)- 3-hydroxy-2- [(IE,3S)- 3-hydroxyoct-1-
enyl]-5-oxocyclopentyl]heptanoic acid (11-
epiprostaglandin E 1) ,

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1-114 Alteplase 2020

absorbance value (A2 80 - A 320 ) is divided by the specific

Alteplase for Injection absorption coefficient for alteplase of 1.9.
(ph. Bur. monograph 1170) Potency
The potency of alteplase is determined in an in vitro clot-lysis
I I
SYQVICRDEK TQMIYQQHQS WLRPVLRSNR VEYCWCNSGR
I
assay as described under Assay. The specific activity of bulk
I i I
alteplase is approximately 580 000 IV per milligram of
AQCHSVPVKS CSEPRCFNGG TCQQALYFSD FVCQCPEGFA
I I
I
alteplase.
GKCCEIDTRA TCYEDQGISY RGTWSTAESG AECTNWNSSA
......,
LAQKPYSGRR PDAIRLGLGN HNYCRNPDRD SKPWCYVFKA N-terminal sequence
I
I N-tenninal sequencing is applied to determine the correct
GKYSSEFCST PACSEGNSDC YFGNGSAYRG THSLTESGAS
N-tenninal sequence and to determine semiquantitatively
CLPWNSMILI
......, GKVYT~QNP~QALGLGKHN YCRNPDGDAK
I additional cleavage sites in the alteplase molecule, for
PWCHVLKNRR LTWEYCDVPS CSTCGLRQYS QPQFR example at position AA 275-276 or at position AA 27-28.
IKGGL The N-tenninal sequence must conform with the sequence of
FADIASHPWQ AAIFAKHRRS PGERFLCGGI LISSCWILSA human tissue plasminogen activator.
I
AHCFQERFPP HHLTVILGRT YRV~PG~EEQ I KFEV~KYIVH Isoelectric focusing
KEFDDDTYDN DIALLQLKSD SSRCAQESSV VRTVCLPPAD
The consistency in the microheterogeneity of glycosylation of
the alteplase molecule can be demonstrated by isoelectric
LQLPDWTECE LSGYGKHEAL SPFYSERLKE AHVRLYPSSR
focusing (IEF). A complex banding pattern with 10 major
CTS~HLLNRTJVTDNMLCAGD TRSGGPQANL HDA<;:QGDSGG and several minor bands in the pH range 6.5-8.5 is observed.
I
PLVCLNDGRM TLVGIISWGL GCGQKDVPGV YTKVTNYLDW Denaturing conditions are applied to achieve a good
IRDNMRP separation of differently charged variants of alteplase.
The broad charge distribution observed is due to a
Action and use population of molecules, which differ in the fine structure of
Tissue-type plasminogen activator; fibrinolytic. biantenary and triantenary complex-type carbohydrate
residues, with different degrees of substitution with sialic
PhEur _
acids. The banding pattern of alteplase test samples must be
DEFINITION consistent with the pattern of alteplase reference standard.
Alteplase foriniection is a sterile, freeze-dried preparation of Single-chain alteplase content
alteplase, a tissue plasminogen activator produced by The alteplase produced by CHO (Chinese hamster ovary)
recombinant DNA technology. It has a potency of not less cells in serum-free medium is predominantly single-chain
than 500 000 ill per milligram of protein. alteplase. The single-chain form can be separated from the
Tissue plasminogen activator binds to fibrin clots and two-chain form by gel-permeation liquid chromatography
activates plasminogen, leading to the generation of plasmin under reducing conditions as described under Single-chain
and to the degradation of fibrin clots or blood coagulates. content (see Tests). The single-chain alteplase content in
bulk samples must be higher than 60 per cent.
Alteplase consists of 527 amino acids with a calculated
relative molecular mass of 59 050 without consideration of Tryptic-peptide mapping
the carbohydrate moieties attached at positions Asn 117, The primary structure of the alteplase molecule is verified by
Asn 184 and Asn 448. The total relative molecular mass is tryptic-peptide mapping as described under Identification B.
approximately 65 000. Alteplase is cleaved by plasmin The reduced and carboxymethylated molecule is cleaved by
between amino-acids 275 and 276 into a two-chain form (A trypsin into about 50 peptides, which are separated by
chain and B chain) that are connected by a disulfide bridge reverse-phase liquid chromatography. A characteristic
between Cys 264 and Cys 395. The single-chain form and chromatogram (fingerprint) is obtained. The identity of the
the two-chain form show comparable fibrinolytic activity in tryptic-peptide map of a given alteplase sample with the
vitro. profile of a well-characterised reference standard is an
indirect confirmation of the amino-acid sequence, because
PRODUCTION
even single amino-acid exchanges in individual peptides can
Alteplase is produced by recombinant DNA synthesis in cell be detected by this sensitive technique. In addition, complex
culture; the fermentation takes place in serum-free medium. peaks of the glycopeptides can be isolated from the tryptic-
The purification process is designed to remove efficiently peptide map and separated in a second dimension, either by
potential impurities, such as antibiotics, DNA and protein reverse-phase liquid chromatography under modified
contaminants derived both from the host cell and from the conditions or by capillary electrophoresis. By this two-
production medium, and potential viral contaminants. dimensional separation of glycopeptide variants, lot-to-lot
If alteplase is stored in bulk form, stability (maintenance of consistency of the microheterogeneity of glycosylation can be
potency) in the intended storage conditions must be demonstrated.
demonstrated. The tryptic-peptide map of alteplase samples must be
The production, purification and product consistency are consistent with the. tryptic-peptide map of alteplase reference
checked by a number of analytical methods described below, standard.
carried out routinely as in-process controls. Monomer content
Protein content The monomer content of alteplase is measured by gel-
The protein concentration of alteplase solutions is permeation liquid chromatography under non-reduced
determined by measuring the absorbance (2.2.25) of the conditions as described under Monomer content (see Tests).
protein solution at 280 nm and at 320 nm, using formulation The monomer content of alteplase bulk samples must be
. buffer as the compensation liquid. If dilution of alteplase higher than 95 per cent.
samples is necessary, the samples are diluted in formulation
buffer. For the calculation of the alteplase concentration, the

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2020 Alteplase 1-115

Type IIType II alteplase content IDENTIFICATION

eHG cells produce 2 glycosylation variants of alteplase. A. The assay serves also to identify the preparation.
Type I alteplase contains 1 polymannose-type glycosylation at B. Tryptic-peptide mapping. Examine by liquid
position Asn 117 and 2 complex-type glycosylation sites at chromatography (2.2.29).
positions Asn 184 and Asn 448. Type IT alteplase is only
Test solution Dilute the preparation to be examined with
glycosylated at positions Asn 117 and Asn 448.
waterR to obtain a solution containing about 1 mg of
The ratio of Type IlType IT alteplase is constant in the range alteplase per millilitre. Dialyse about 2.5 mL of the solution
of 45 to 65 per cent of Type I and 35 to 55 per cent of for at least 12 h into a solution containing 480 gIL of urea R,
Type IT. The content of alteplase Type I and Type IT can be 44 gIL of tris(hydroxymethyl)aminomethane Rand 1.5 gIL of
determined by a densitometric scan of SDS-PAGE (sodium sodium edetate R and adjusted to pH 8.6, using a membrane
dodecyl sulfate polyacrylamide gel electrophoresis) gel. with a cut-off point corresponding to a relative molecular
Plasmin-treated samples of alteplase, which are reduced and mass of 10 000 for globular proteins. Measure the volume of
carboxymethylated before loading on the gel, are separated the solution, transfer it to a clean test-tube and add per
into 3 bands: Type I alteplase A-chain (AA 1-275), Type IT millilitre 10·J.lL of a 156 gIL solution of dithiothreitol R. Allow
alteplase A-chain eM 1-275) and alteplase B-chain to stand for 4 h, cool in iced water and add per millilitre of
(AA 276-527). The ratio of Type IIType IT alteplase is solution 25 J.lL of a freshly prepared 190 g/L solution of
determined from a calibration curve, which is obtained by a iodoacetic acid R. Allow to stand in the dark for 30 min.
densitometric scan of defined mixtures of purified Type I Add per millilitre 50 ul, of dithiothreitol solution to stop the
alteplase and Type IT alteplase standards. reaction. Dialyse for 24 h against an 8 gIL solution of
SDS..PAGE ammonium hydrogen carbonate R. Add 1 part of trypsin for
SDS-PAGE (silver staining) is used to demonstrate purity of peptide mapping R to 100 parts of the protein and allow to
the alteplase bulk material and the integrity of the alteplase stand for 6 h to 8 h. Repeat the addition of trypsin and allow
molecule. For alteplase bulk samples, no additional protein to stand for a total of 24 h.
bands compared to reference standard or degradation Reference solution Prepare as for the test solution using a
products must occur in SDS-PAGE gels at a loading amount suitable reference standard instead of the preparation to be
of 2.5 ug alteplase protein per lane and a limit of detection of examined.
5 ng per proteiri (BSA) band. The chromatographic procedure may be carried out using:
Bacterialendotoxins (2.6,14) - a column 0.1 ttl long and 4.6 mID in internal diameter
Less than 1 IV per milligram of alteplase. packed with octadecylsilyl silica gelfor chromatography R
Sialic acids (5 urn to 10 urn);
Proceed using a suitable validated method developed Mobile phase A 8 gIL solution of sodium dihydrogen
according to general chapter 2.2. 59. Gly~an analysis of phosphate R, adjusted to pH 2.85 with phosphoric acid R,
glycoproteins. The sialic acids content for the test samples filtered and degassed;
must be in the range of 70 to 130 per cent compared to Mobile phase B 75 per cent V/V solution of
alteplase reference standard, which contains about 3 moles of acetonitrile R in mobile phase A;
sialic acids per mole of alteplase. - as detector a spectrophotometer set at 210 nm.
Neutral sugars Equilibrate the system with mobile phase A at a flow rate of
Dilute alteplase samples and the reference standard in the I mlzmin. After injection of the solution, increase the
assay buffer, containing 34.8 gIL of arginine R, 0.1 gIL of proportion of mobile phase B at a rate of 0.44 per cent per
polysorbate 80 R and adjusted to pH 7.4 with phosphoric minute until the ratio of mobile phase A to mobile phase B is
acid R, to a protein concentration of 50 ug/ml., Prepare the 60:40, then increase the proportion of mobile phase B at a
following concentrations of mannose in the same assay buffer rate of 1.33 per cent per minute until the ratio of mobile
for a calibration curve: 20, 30, 40, 50 and 60 ug/ml., Pipette phase A to mobile phase B is 20:80 and then continue
2 mL of alteplase samples and reference standard, as well as elution with this mixture for a further 10 min. Record the
2 mL of each mannose concentration in duplicate in reagent chromatogram for the reference solution: the test is not valid
tubes. Add 50 ul, of phenolR, followed by 5 mL of sulfuric unless the resolution of peaks 6 (peptides 268-275) and 7
acid R, in each reagent tube. Incubate the mixture for 30 min (peptides 1-7) is at least 1.5; Whl and Wh2 are not more than
at room temperature. Measure the absorbance at 492 run for 0.4 min. Inject about 100 J.lL of the test solution and record
each tube. Read the content of neutral sugars from the the chromatogram. Verify the identity of the peaks by
mannose calibration curve. The neutral sugar content is comparison with the chromatograms of the reference
expressed in moles of neutral sugar per mole of alteplase, solution. There should not be any additional significant peaks
taking into account the. dilution factor for alteplase samples or shoulders, a significant peak or shoulder being defined as
and reference standard and using a relative molecular mass of one with an area response equal to or greater than 5 per cent
180.2 for mannose and a relative molecular mass of 59 050 of peak 19 (peptides 278-296); no. significant peak is missing.
for the alteplase protein moiety. The neutral sugar content of A type chromatogram for identification of the peaks cited is
the alteplase samples must be in the range of 70 to shown in Figure 1170.-1.
130 per cent compared to alteplase reference standard, which
TESTS
contains about 12 moles of neutral sugar per mole of
Appearance of solution
alteplase.
The reconstituted preparation is clear (2.2.1) and not more
CHARACTERS intensely coloured than reference solution Y7 (2.2.2,
White or slightly yellow powder or solid friable mass. Method II).
Reconstitute the preparation as stated on the labelimmediately pH (2.2.3)
before carryingout the Identification, Tests (except those for 7.1 to 7.5.
solubzlity and water) and Assay.

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1-116 Alteplase 2020

100

80

60

40 co co

....
20

0-r---r-----:r:---r---,---.,.----r--......----~----r---,--~-r------....--
o 20 40 80 100 120

Figure 1170.-1. - Chromatogram for tryptic-peptide mapping of alteplase

Solubility Calculate the relative amount of single-chain alteplase from

Add the volume of the liquid stated on the label. the peak area values.
The preparation dissolves completely within 2 min at 20 DC The test is not valid unless: the number of theoretical plates
to 25 DC. calculated on the basis of the single-chain alteplase peak is at
Protein content least 1000. The content of single-chain alteplase is not less
Prepare a solution of the substance to be examined with an than 60 per cent of the total amount of alteplase-related
accurately known concentration of about 1 gIL. Using a substances found.
34.8 gIL solution of arginine R adjusted to pH 7.3 with Monomer content
phosphoric add R, dilute an accurately measured volume of Examine by liquid chromatography (2.2.29).
the solution of the substance to be examined so that the
Test solution Reconstitute the preparation to be examined to
absorbance measured at the maximum at about 280 nm is
obtain a solution containing about 1 mg per millilitre.
0.5 to 1.0 (test solution). Measure the absorbance (2.2.25) of
The chromatographic procedure may be carried out using:
the solution at the maximum at about 280 nm and at
320 nm using the arginine solution as the compensation - a column 0.6 m long and 7.5 mm in internal diameter
packed with silica-based rigid, hydrophilic gel with
liquid. Calculate the protein content in the portion of
alteplase taken from the following expression: spherical particles 10 J.lIIl to 13 J.lIIl in diameter, suitable
for size-exclusion chromatography;
V(A 280 - A 320 ) - as mobile phase at a flow rate of 0.5 mUmin a solution
containing 30 gIL of sodium dihydrogen phosphate Rand
1.9
1 gIL of sodium dodecyl sulfate R, adjusted to pH 6.8 with
in which V is the volume of the test solution, A 280 is the dilute sodium hydroxide solution R;
absorbance at the maximum at about 280 nm and A 32 0 is the - as detector a spectrophotometer set at 214 nm.
absorbance at 320 nm. Inject the test solution and record the chromatogram.
Single-chain content The test is not valid unless the number of theoretical plates
Examine by liquid chromatography (2.2.29). calculated for the alteplase monomer peak is at least 1000.
Measure the response for all peaks, i.e. peaks corresponding
Test solution Dissolve the preparation to be examined in
to alteplase species of different molecular masses. Calculate
water R to obtain a solution containing about 1 mg of
the relative content of monomer from the area values of these
alteplase per millilitre. Place about 1 mL of the solution in a
peaks. The monomer content for alteplase must be at least
tube, add 3 mL of a 3 gIL solution of dithiothreitol R in the
95 per cent.
mobile phase, place a cap on the tube and heat at about
80 DC for 3 min to 5 min. Water (2.5.12)
Not more than 4.0 per cent, determined by the semi-micro
The chromatographic procedure may be carried out using:
determination of water.
- a column 0.6 m long and 7.5 mm in internal diameter
packed with silica-based, rigid, hydrophilic gel with Bacterial endotoxins (2.6.14)
spherical particles 10 J.lIIl to 13 urn in diameter, suitable Less than 1 ill per milligram of protein.
for size-exclusion chromatography; Sterility (2.6.1)
- as mobile phase at a flow rate of 0.5 mlJrnin a solution It complies with the test for sterility.
containing 30 gIL of sodium dihydrogen phosphate Rand
ASSAY
1 gIL of sodium dodecyl sulfate R, adjusted to pH 6.8 with
dilute sodium hydroxidesolution R; The potency of alteplase is determined by comparing its
- as detector a spectrophotometer set at 214 nm. ability to activate plasminogen to form plasmin with the same
capacity of a reference preparation calibrated in International
Inject about 50 ul, of the test solution and record the
Units. The formation of plasmin is measured by the
chromatogram. The chromatogram shows 2 major peaks
determination of the lysis time of a fibrin clot in given
corresponding to single-chain and two-chain alteplase.
conditions.

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2020 Altizide 1-117

The International Unit is the activity of a stated quantity of Calculate the specific activity in the portion of the substance
the International Standard of alteplase. The equivalence in to be examined from the following expression:
International Units of the International Standard is stated by
the World Health Organization.
Solvent buffer A solution containing 1.38 gIL of sodium
dihydrogen phosphate monohydrate R, 7.10 gIL of anhydrous in which P is the concentration of protein obtained in the test
disodium hydrogen phosphate R, 0.20 gIL of sodium azide Rand for protein content.
0.10 gIL of polysorbate 80 R.
The estimated potency is not less than 90 per cent and not
Human thrombin solution A solution of human thrombin R more than 110 per cent of the stated potency.
containing 33 IV/rnL in solvent buffer.
STORAGE
Human fibrinogen solution ' A 2 gIL solution of fibrinogen R in
Store in a colourless, glass container, under vacuum or under
solvent buffer.
an inert gas, protected from light, at a temperature of 2 DC to
Human plasminogen solution A 1 g/L solution of human 30 -c.
plasminogen R in solvent buffer.
Test solutions Using a solution of the substance to be LABELLING
examined containing 1 gIL, prepare serial dilutions using The labelstates:
solvent buffer, for example 1:5000, 1:10000, 1:20 000. - the number of International Units per container;
- the amount of protein per container;
Reference solutions Using a solution of a suitable reference
- the name and volume of the liquid to be used for
standard having an accurately known concentration of about
reconstitution.
1 gIL (580000 IU of alteplase per millilitre) , prepare 5 serial
_______ ~ PhEur
dilutions using water R to obtain reference solutions having
known concentrations in the range 9.0 IU/mL to 145 IU/mL.
To each .0La set of labelled glass test-tubes, add 0.5 mL of
human thrombin solution. Allocate each test and reference
solution to a separate tube and add to each tube 0.5 mL of Altizide
the solution allocated to it. To each of a second set of
(Ph. Bur. monograph 2185)
labelled glass tubes, add 20 ilL of human plasminogen
solution, and 1 mL of human fibrinogen solution, mix and
store on ice. Beginning with the reference/thrombin mixture
containing the lowest number of International Units per and enantiomer
millilitre, record the time and separately add 200 ilL of each
of the thrombin mixtures to the test tubes containing the
plasminogen-fibrinogen mixture. Using a vortex mixer,
intermittently mix the contents of each tube for a total of 383.9 5588-16-9
15 s and carefully place in a rack in a circulating water-bath
at 37 "C. A visibly turbid clot forms within 30 s and bubbles Action and use
subsequently form within the clot, Record the clot-lysis time Thiazide diuretic.
as the time between the first addition of alteplase solution PhEur --'- ~ _
and the moment when the last bubble rises to the surface.
Using a least-squares fit, determine the equation of the line DEFINITION
using the logarithms of the concentrations of the reference (3R5)-6-Chloro-3- [(prop-2-enylsulfanyl)methyl]-3,4-dihydro-
preparation in International Units per millilitre versus the 2H-l ,2,4-benzothiadiazine-7-sulfonamide 1, I-dioxide.
logarithms of the values of their clot-lysis times in seconds, Content
according to the following equation: 97.5 per cent to 102.0 per cent (anhydrous substance).
CHARACTERS
Appearance
in which t is the clot-lysis time, Us the activity in White or almost white powder.
International Units per millilitre of the reference preparation, Solubility
b is the slope and a the y-intercept of the line. The test is not Practically insoluble in water, soluble in methanol, practically
valid unless the correlation coefficient is -0.9900 to -1.0000. insoluble in methylene chloride.
From the line equation and the clot-lysis time for the test It shows polymorphism (5.9).
solution, calculate the logarithm of the activity UA from the
following equation: IDENTIFICATION
Infrared absorption spectrophotometry (2.2.24).
[(loglOt) - a] Comparison altizide CRS.
I oglO UA =. b
If the spectra obtained show differences, dissolve 50 mg of
the substance to be examined and 50 mg of the reference
Calculate the alteplase activity in International Units per substance separately in 2 mL of acetone R and evaporate the
millilitre from the following expression: solvent. Precipitate by adding 1 mL of methylene chloride R.
Evaporate to dryness and record new spectra using the
residues.
in which D is the dilution factor for the test solution. TESTS
Impurity B
Thin-layer chromatography (2.2.27).

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1-118 Alum 2020

Test solution Dissolve 0.200 g of the substance to be - unspecified impurities: for each impurity, not more than the
examined in acetone R and dilute to 2.0 mL with the same area of the principal peak in the chromatogram obtained
solvent. with reference solution (a) (0.10 per cent);
Reference solution (a) Dissolve 10.0 mg of altizide - total: not more than 5 times the area of the principal peak
impurity B CRS in acetone R and dilute to 25.0 mL with the in the chromatogram obtained with reference solution (a)
same solvent. (0.5 per cent);
Reference solution (b) To 1.0 mL of reference solution (a) - disregard limit: 0.5 times the area of the principal peak in
add 1.0 mL of the test solution. the chromatogram obtained with reference solution (a)
Reference solution (c) Dilute 5.0 mL of reference solution (a) (0.05 per cent).
to 10.0 mL with acetone R. Water (2.5.32)
Plate TLC silica gelF 254 plate R. Maximum 0.5 per cent, determined on 50.0 mg.
Mobilephase acetone R, methylene chloride R (25:75 VIV). Sulfated ash (2.4.14)
Application 10 ~L of the test solution and reference Maximum 0.1 per cent, determined on 1.0 g.
solutions (b) and (c). ASSAY
Development Over 2/3 of the plate. Liquid chromatography (2.2.29) as described in the test for
Drying In air. related substances, with the following modifications.
Detection Spray with a mixture of equal volumes of a 10 gIL Test solution Dissolve 25.0 mg of the substance to be
solution of potassium permanganate R and a 50 gIL solution of examined in 2 mL of acetonitrile R and dilute to 25.0 mL
sodium carbonate R, prepared immediately before use. Allow with the mobile phase.
to stand for 30 min and examine in daylight. Reference solution Dissolve 25.0 mg of altizide CRS in 2 mL
System suitability Reference solution (b): of acetonitrile R and dilute to 25.0 mL with the mobile phase.
- the chromatogram shows 2 clearly separated spots. Calculate the percentage content of CllH14ClN304S3 from
Limit Any spot due to impurity B is not more intense than the declared content of altizide CRS.
the principal spot in the chromatogram obtained with IMPURITIES
reference solution (c) (0.2 per cent). Specified impurities A, B.
Related substances
Liquid chromatography (2.2.29). Prepare the solutions
immediately before use, except reference solution (b).
Test solution Dissolve 50 mg of the substance to be
examined in 5 mL of acetonitrile R and dilute.to 25 rnl, with
the mobile phase.
Reference solution (a) Dilute 1.0 mL of the test solution to A. 4-amino-6-chlorobenzene-I,3-disulfonamide,
100.0 mL with the mobile phase. Dilute 1.0 mL of this
OCH3
solution to 10.0 mL with the mobile phase.
Reference solution (b) In order to produce impurity A in situ, H3CO~S~CH2
dissolve 50 mg of the substance to be examined in 5 mL of
acetonitrile R and dilute to 25 mL with waterR. Allow to B. 3-[(2,2-dimethoxyethyl)sulfanyl]prop-1-ene.
stand for 30 min. _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
Reference solution (c) Dissolve 4 mg of furosemide CRS in
2 mL of acetonitrile R, add 2 mL of the test solution and
dilute to 100 mL with the mobile phase.
Column: Alum
=
- size: 1 0.15 m, 0 = 3.9 mrn;
- stationary phase: end-capped octadecylsilyl silica gelfor Potash Alum
chromatography R (5 urn); Aluminium Potassium Sulphate
- temperature: 30 "C.
Aluminium Potassium Sulfate
Mobilephase acetonitrile R, water R previously adjusted to
(Ph. Bur. monograph 0006)
pH 2.0 with perchloric acid R (25:75 VIV).
Flow rate 0.7 mUmin. AlK(S04)2,12H20 474.4 7784-24-9
Detection Spectrophotometer at 270 nm. Action and use
Injection 5~. Astringent.
Run time Twice the retention time of altizide. PhEur _
Relative retention With reference to altizide (retention
time = about 25 min): impurity A = about 0.15; DEFINITION
furosemide = about 1.05. Content
System suitability Reference solution (c): 99.0 per cent to 100.5 per cent of AlK(SOJ2,12H20.
- resolution: minimum 1.0 between the peaks due to altizide CHARACTERS
and furosemide; Appearance
Limits: Granular powder or colourless, transparent, crystalline
- impurity A: not more than 3 times the area of the masses.
principal peak in the chromatogram obtained with
reference solution (a) (03 per cent);

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2020 Aluminium Glycinate 1-119

Solubility B. Dilute 0.3 mL of solution 52 to 2 mL with water R.

Freely soluble in water, very soluble in boiling water, soluble The solution gives the reaction of aluminium (2.3.1).
in glycerol, practically insoluble in ethanol (96 per cent). TESTS
IDENTIFICATION Solution S1
A. Solution S (see Tests) gives the reactions of sulfates Dissolve 10.0 g in distilled water R and dilute to 100 mL with
(2.3.1). the same solvent.
B. Solution S gives the reaction of aluminium (2.3.1). Solution S2
C. Shake 10 mL of solution 5 with 0.5 g of sodium hydrogen Dilute 50 mL of solution 51 to 100 mL with water R.
carbonate R and filter. The filtrate gives reaction (a) of Appearance of solution
potassium (2.3.1). Solution 82 is clear (2.2.1) and not more intensely coloured
TESTS than reference solution B7 (2.2.2~ Method II).
Solution S Sulfates (2.4.13)
Dissolve 2.5 g in water R and dilute to 50 mL with the same Maximum 100 ppm, determined on solution 81.
solvent. Iron (2.4.9)
Appearance of solution Maximum 10 ppm, determined on solution S1.
Solution S is clear (2.2.1) and colourless (2.2.2~ Method II). Alkali and alkaline-earth metals
pH (2.2.3) Maximum 0.5 per cent.
3.0 to 3.5. To 20 mL of solution S2 add 100 mL of water R and heat to
Dissolve LOg in carbon dioxide-free water R and dilute to boiling. To the hot solution add 0.2 mL of methyl red
10 mL with 'the .same solvent. solution R. Add dilute ammonia Rl until the colour of the
AmmOniU111 (2.4.1) indicator changes to yellow and dilute to 150 mL with
Maximum 02 per cent. water R. Heat to boiling and filter. Evaporate 75 mL of the
filtrate to dryness on a water-bath and ignite to constant
To 1 mL of solution 5 add 4 mL of water R. Dilute 0.5 mL
mass. The residue weighs a maximum of 2.5 mg.
of this solution to 14 mL with water R.
Water (2.5.12)
Iron (2.4.9)
42.0 per cent to 48.0 per cent, determined on 50.0 mg.
Maximum 100 ppm.
Dilute 2 mL of solution S to 10 mL with water R. Use in this ASSAY
test 0.3 mL of thioglycollic add R. Dissolve 0.500 g in 25.0 mL of water R. Carry out the
complexometric titration of aluminium (2.5.11). Titrate with
ASSAY 0.1 M zinc sulfate until the colour of the indicator changes
Dissolve 0.900 g in 20 mL of water R and carry out the from greyish-green to pink. Carry out a blank titration.
complexometric titration of aluminium (2.5.11).
1 mL of 0.1 M sodium edetate is equivalent to 24.14 mg
1 mL of 0.1 M sodium edetate is equivalent to 47.44 mg of AlCI3,6HzO.
of AlK(S04)z,12HzO.
_ _----' PhEur STORAGE
In an airtight container.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur

Aluminium Chloride Hexahydrate

(ph. Bur. monograph 0971) Aluminium Glycinate
AlCI3,6H zO 241.4 7784-13-6
NH2
Action and use
Astringent.
Preparation
Aluminium Chloride Solution
o £ 0
/
'" /OH
AI
'OH
,xHzO

PhEur _
135.1 41354-48-7

DEFINITION Action and use

Content Antacid.
95.0 per cent to 101.0 per cent.
DEFINITION
CHARACTERS Aluminium Glycinate is a basic aluminium monoglycinate,
Appearance partly hydrated. It contains not less than 34.5% and not
White or slightly yellow, crystalline powder or colourless more than 38.5% of Alz0 3 and not less than 9.9% and not
crystals, deliquescent. more than 10.8% of N, both calculated with reference to the
Solubility dried substance.
Very soluble in water, freely soluble in ethanol (96 per cent), CHARACTERISTICS
soluble in glycerol. A white or almost white powder.
IDENTIFICATION Practically insoluble in water and in organic solvents.
A. Dilute 0.1 mL of solution 52 (see Tests) to 2 mL with It dissolves in dilute mineral acids and in aqueous solutions
water R. The solution gives reaction (a) of chlorides (2.3.1); of the alkali hydroxides.

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1-120 Aluminium Hydroxide 2020

IDENTIFICATION Hydrated Aluminium Hydroxide for

A. Add 0.1 g to 10 mL of a solution prepared by dissolving
0.84 g of citric acid in 8 mL of 1M sodium hydroxide and Adsorption
diluting to 20 mL with water. Add 0.5 mL of a 0.1 % w/v (Ph. Eur. monograph 1664)
solution of ninhydrin in methanol and warm. A purple colour
is produced. [AlO(OH)],xHzO
PhEur _
B. Suspend 1 gin 25 mL of 0.5M hydrochloric acid and heat
gently until a clear solution is produced. Reserve half of the DEFINITION
solution. To 2 mL of the solution add 0.15 mL of liquefied Content
phenol, shake and add carefully without shaking 5 mL of 90.0 per cent to 110.0 per cent of the content of aluminium
dilute sodium hypochlorite solution. A blue colour is produced, stated on the label.
C. The solution reserved in test B yields the reaction NOTE: shake the gel vigorously for at least30 s immediately
characteristic of aluminium salts, Appendix VI. before examining.
TESTS CHARACTERS
Acidity or alkalinity Appearance
pH of a suspension of 1 g in 25 mL of carbon dioxide-free White or almost white, translucent, viscous, colloidal gel.
water, 6.5 to 7.5, Appendix V L. A supernatant may be formed upon standing.
Neutralising capacity Solubility
Shake 0.2 g vigorously with 25 mL of O.lM hydrochloric acid .A clear or almost clear solution is obtained with alkali
for 5 minutes and allow to stand for 5 minutes. The pH of hydroxide solutions and mineral acids.
the mixture is greater than 3.0, Appendix V L.
IDENTIFICATION
Arsenic
Solution S (see Tests) gives the reaction of aluminium.
Dissolve 2.0 g in 18 mL of brominated hydrochloric acid and
32 mL of water. 25 mL of the resulting solution complies To 10 mL of solution S add about 0.5 mL of dilute
with the limit testfor arsenic, Appendix vn (1 ppm). hydrochloric acid R and about 0.5 mL of thioacetamide
reagent R. No precipitate is formed. Add dropwise 5 mL of
Mercuric salts dilute sodium hydroxide solution R. Allow to stand for 1 h.
Dissolve 2.0 gin 10 mL of 1M sulfuric acid, transfer to a
A gelatinous white precipitate is formed which dissolves upon
separating funnel with the aid of water, dilute to about
addition of 5 mL of dilute sodium hydroxide solution R.
50 mL with water and add 50 mL of 0.5M sulfuric acid.
Gradually add 5 mL of ammonium chloride solution Rand
Add 100 mL of water, 2 g of hydroxylamine hydrochloride,
allow to stand for 30 min. The gelatinous white precipitate is
1 mL of 0.05M disodium edetate and 1 mL of glacial acetic
re-formed.
acid. Add 5 mL of chloroform, shake, allow to separate and
discard the chloroform layer. Titrate the aqueous layer with a TESTS
solution of dithizone in chloroform containing 8 ug per mL Solution S
until the chloroform layer remains green. After each addition, Add 1 g to 4 mL of hydrochloric acid R. Heat at 60°C for
shake vigorously, allow the layers to separate and discard the 1 h, cool, dilute to 50 mL with distilled waterR and filter if
chloroform layer. Repeat the operation using a solution necessary.
prepared by diluting 1 mL of mercurystandardsolution pH (2.2.3)
(5 ppm Hg) to 100 mL with 0.5M sulfuric acid and beginning 5.5 to 8.5.
at the words 'Add 100 mL of water. . . '. The volume of the
Adsorption power
dithizone solution required by the substance being examined
Dilute the substance to be examined with distilled waterR to
does not exceed that required by the mercury standard
obtain an aluminium concentration of 5 mg/mL. Prepare
solution.
bovine albumin R solutions with the following concentrations
Chloride of bovine albumin: 0.5 mg/mL, 1 mg/mL, 2 mg/mL,
Dissolve 1.0 g in 10 mL of 2M nitric acid and dilute to 3 mg/mL, 5 mg/mL and 10 mg/mL. If necessary, adjust the
100 mL with water. 15 mL of the resulting solution complies gel and the bovine albumin R solutions to pH 6.0 with dilute
with the limit testfor chlorides, Appendix vn (330 ppm). hydrochloric acid R or dilute sodium hydroxide solution R.
Loss on drying For adsorption, mix 1 part of the diluted gel with 4 parts of
When dried to constant weight at 1300, loses not more than each of the solutions of bovine albumin R and allow to stand
12.0% of its weight. Use 1 g. at room temperature for 1 h. During this time shake the
ASSAY mixture vigorously at least 5 times. Centrifuge or filter
For A1203 through a non-protein-retaining filter. Immediately determine
Dissolve 0.25 g in a mixture of 3 mL of 1M hydrochloric acid the protein content (2.5.33, Method 2) of either the
and 50 mL of water, add 50 mL of 0.05M disodium edetate VS supernatant or the filtrate.
and neutralise with 1M sodium hydroxide using methyl red It complies with the test if no bovine albumin is detectable in
solution as indicator. Heat the solution to boiling, allow to the supernatant or filtrate of the 2 mg/mL bovine albumin R
stand for 10 minutes on a water bath, cool rapidly, add solution (maximum level of adsorption) and in the
about 50 mg of xylenol orange triturate and 5 g of hexamine supernatant or filtrate of bovine albumin R solutions of lower
and titrate the excess of disodium edetate with 0.05M lead concentrations. Solutions containing 3 mg/mL, 5 mg/mL and
nitrate VS until the solution becomes red. Each mL of 0.05M 10 mg/mL bovine albumin R may show bovine albumin in the
disodium edetate VS is equivalent to 2.549 mg of Alz0 3 . supernatant or filtrate, proportional to the amount of bovine
albumin in the solutions.

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2020 Aluminium Hydroxide 1-121

Sedimentation LABELLING
If necessary, adjust the substance to be examined to pH 6.0 The label states the declared content of aluminium.
using dilute hydrochloric acid R or dilute sodium hydroxide -------- PhEur
solution R. Dilute with distilled water R to obtain an
aluminium concentration of approximately 5 mg/mL. If the
aluminium content of the substance to be examined is lower
than 5 mglmL, adjust to pH 6.0 and dilute with a 9 gIL
solution of sodium chloride R to obtain an aluminium Dried Aluminium Hydroxide
concentration of about 1 mg/mL. After shaking for at least
(Hydrated Aluminium Oxide, Ph. Bur. monograph
30 s, place 25 mL of the preparation in a 25 mL graduated
0311)
cylinder and allow to stand for 24 h.
It complies with the test if the volume of the clear Action and use
supernatant is less than 5 mL for the gel with an aluminium Antacid.
content of about 5 mg/mL. Preparations
It complies with the test if the volume of the clear Aluminium Hydroxide Chewable Tablets
supernatant is less than 20 mL for the gel with an aluminium Aluminium Hydroxide Oral Suspension
content of about 1 mg/mL. Compound Magnesium Trisilicate Chewable Tablets
Chlorides (2.4.4) Co-magaldrox Oral Suspension
Maximum033'per cent. Co-magaldrox Tablets
Dissolve05g inJOmL of dilute nitric acid R and dilute to
PhEur _
SOO mL with water R.
Nitrates DEFINITION
Maximum 100 ppm. Content
Place 5 g in a test-tube immersed in ice-water, add 0.4 mL 47.0 per cent to 60.0 per cent of Al2 0 3 (Mr 102.0).
of a 100 gIL solution of potassium chloride R, 0.1 mL of CHARACTERS
diphenylamine solution Rand, dropwise with shaking, 5 mL of Appearance
sulfuric acidR. Transfer the tube to a water-bath at 50°C. White or almost white, amorphous powder.
After .15 min, any blue colour in the solution is not· more
Solubility
intense than that in a standard prepared at the same time
Practically insoluble in water. It dissolves in dilute mineral
and in the same manner using 5 mL of nitrate standard
acids and in solutions of alkali hydroxides.
solution (100 ppm NOJJ R.
Sulfates (2.4.13) IDENTIFICATION
Maximum 0.5 per cent. Solution S (see Tests) gives the reaction of aluminium
(2.3.1).
Dilute 2 mL of solution S to 20 mL with water R.
Ammonium (2.4.1, Method B) TESTS
Maximum 50 ppm, determined on 1.0 g. Solution S
Dissolve 2.5 g in 15 mL of hydrochloric acid R, heating on a
Prepare the standard using 0.5 mL of ammonium standard
water-bath. Dilute to 100 mL with distilled water R.
solution (100 ppmNH4J R.
Appearance of solution
Arsenic (2.4.2, Method A)
Solution S is not more opalescent than reference
Maximum 1 ppm, determined on 1·g.
suspension II (2.2.1) and not more intensely coloured than
Iron (2.4.9) reference solution GY6 (2.2.2, Method II).
Maximum 15 ppm, determined on 0.67 g.
Alkaline impurities
Bacterial endotoxins (2.6.14) Shake 1.0 g with 20 mL of carbon dioxide-free water R for
Less than 5 IV of endotoxin per milligram of aluminium, if 1 min and filter. To 10 mL of the filtrate add 0.1 mL of
intended for use in the manufacture of an adsorbed product phenolphthalein solution R. Any pink colour disappears on the
without a further appropriate procedure for the removal of addition of 0.3 mL of 0.1 M hydrochloric acid.
bacterial endotoxins.
Neutralising capacity
ASSAY Carry out the test at 37°C Disperse 0.5 gin 100 mL of
Dissolve 2.50 g in 10 mL of hydrochloric acid R, heating for water R, heat, add 100.0 mL of 0.1 M hydrochloric acid,
30 min at 100°C on a water-bath. Cool and dilute to 20 mL previously heated, and stir continuously; the pH (2.2.3) of
with water R. To 10 mL of the solution, add concentrated the solution after 10 min, 15 min and 20 min is not less than
ammonia R until a precipitate is obtained. Add the smallest 1.8, 2.3 and 3.0 respectively and is at no time greater than
quantity of hydrochloric'acid R needed to dissolve the 4.5. Add 10.0 mL of 0.5 Mhydrochloric acid, previously
precipitate and dilute to 20 mL with water R. Carry out the heated, stir continuously for 1 h and titrate with 0.1 M
complexometric titration of aluminium (2.5.11). Carry out a sodium hydroxide to pH 3.5; not more than 35.0 mL of 0.1 M
blank titration. sodium hydroxide is required.
STORAGE Chlorides (2.4.4)
At a temperature not exceeding 30°C. Do not allow to Maximum 1 per cent.
freeze. If the substance is sterile, store in a sterile, airtight, Dissolve 0.1 g with heating in 10 mL of dilute nitric acid R
tamper-proof container. and dilute to 1OOmL with water R. Dilute 5 mL of the
solution to 15 mL with water R.

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1-122 Aluminium Magnesium Silicate 2020

Sulfates (2.4.13) Viscosity (mf'a-s) AI content I Mg co"fitent

Maximum 1 per cent. Type
min. max. min. max.
Dilute 4 mL of solution S to 100 mL with distilled water R.
IA 225 600 0.5 1.2
Arsenic (2.4.2, Method A)
Maximum 4 ppm, determined on 10 mL of solution S. IE 150 450 05 1.2
Microbial contamination Ie 800 2200 0.5 1.2
TAMC: acceptance criterion 103 CFU/g (2.6.12).
IIA 100 300 1.4 2.8
TYMC: acceptance criterion 102 CFU/g (2.6.12).
Absence of bile-tolerant gram-negative bacteria (2.6.13).
Absence of Escherichia coli (2.6.13). CHARACTERS
Appearance
ASSAY Almost white, coarse powder, granules or plates (types IA,
Dissolve 0.800 gin 10 mL of hydrochloric acid Rl, heating on IC and IIA); almost white, fine powder (type IB).
a water-bath. Cool and dilute to 50.0 mL with water R.
To 10.0 mL of the solution add dilute ammonia Rl until a Solubility
Practically insoluble in water and in organic solvents.
.precipitate begins to appear. Add the smallest quantity of
dilute hydrochloric acid R needed to dissolve the precipitate It swells in water to produce a colloidal dispersion.
and dilute to 20 mL with waterR. Carry out the IDENTIFICATION
complexometric titration of aluminium (2.5.11). Carry out either tests A, B, C, F or tests D, E.
1 mL of 0.1 M sodium edetate is equivalent to 5.098 mg A. In a platinum crucible mix 1.0 g with 5.0 g of anhydrous
of AI~03' lithium metaborate R. Heat slowly at first and ignite at
STORAGE 1000-1200 °C for 15 min. Allow to cool and crush the
In an airtight container, at a temperature not residue. 0.25 g of the residue gives the reaction of silicates
exceeding 30°C. (2.3.1).
FUNCTIONALITY-RELATED CHARACTERISTICS B. Dissolve 1.0 g of the residue obtained in identification
test A in a mixture of 5 mL of dilute hydrochloric acid Rand
This section provides information on characteristics that are
10 mL of waterR. Filter to obtain a clear solution and add
recognised as beingrelevantcontrol parameters for one or more
ammonium chloride buffersolution pH 10.0 R. A white,
junctions of the substance when usedas an excipient (see chapter
gelatinous precipitate is formed. Centrifuge and keep the
5.15). Some of the characteristics described in the Functionality-
supernatant for identification test C. Dissolve the precipitate
related characteristics section may also be present-in the mandatory
in dilute hydrochloric acid R. Add dropwise dilute sodium
part of the monograph since they also represent mandatory quality
hydroxide solution R. A white gelatinous precipitate is formed.
criteria. In such cases, a cross-reference to the tests described in the
Filter and add a few drops of phenolphthalein solution R to the
mandatorypart is includedin the Functionality-related
residue. The residue turns pink. Wash the residue with
characteristics section. Control of the characteristics can contribute
waterR until the pink colour is completely discharged and
to the quality of a medicinalproduct by improving the consistency
the residue remains white upon addition of a drop of
of the manufacturing process and theperformance of the medicinal
phenolphthalein solution R. Sprinkle a few crystals of sodium
productduring use. Where control methods are cited, they are
fluoride R on the residue. The residue, in contact with the
recognised as being suitable for the purpose, but othermethods can
crystals, tums pink again in a short time.
also be used. Wherever results for a particular characteristic are
reported, the control method must be indicated. C. To 2 mL of the supernatant obtained after centrifugation
in identification test B, add 1 mL of dilute ammonia R1 and
The following characteristics may be relevantfor hydrated
1 mL of ammonium chloride solution R. Upon the addition of
aluminium oxideused as adsorbent.
dilute ammonia R1 a white precipitate may form, which
Particle-size distribution (2.9.31) dissolves after addition of the ammonium chloride solution R.
Specific surface area (2.9.26) Add 1 mL of disodium hydrogen phosphate solution R. A white
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur precipitate is formed.
D. X-ray diffraction (2.9.33), oriented sample.
Add 2 g in small portions to 100 mL of waterR, with
vigorous shaking. Allow to stand for at least 12 h to ensure
Aluminium Magnesium Silicate complete hydration. Place 2 mL of the resulting mixture on a
suitable glass slide and allow to dry in air at room
(Ph. Eur. monograph 1388) temperature to produce an oriented film. Place the slide in a
Action and use vacuum desiccator over ethylene glycol R. Evacuate the
Excipient. desiccator and close the stopcock so that the ethylene glycol
saturates the chamber. Allow to stand for at least 12 h.
PhEur --
Record the X-ray diffraction pattern and calculate the
DEFINITION d values: the largest peak corresponds to a d value between
Mixture of colloidal-size particles of montmorillonite and 1.5 nm and 1.72 nm.
saponite, practically free from grit and non-swellable ore. E. X-ray diffraction (2.9.33), random sample.
The requirements for viscosity and ratio of aluminium Prepare a random powder sample, record the X-ray
content to magnesium content differ for the several types of diffraction pattern and determine the. d values in the region
aluminium magnesium silicate, as shown in the table below. between 0.148 nm and 0.154 nm. Peaks are found between
0.1492 nm and 0.1504 nm and between 0.1510 nm and
0.1540 nm.

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2020 Aluminium Magnesium Silicate 1-123

F. It complies with the limits of the assay. Lead

Maximum 15 ppm.
TESTS
pH (2.2.3) Atomic absorption spectrometry (2.2.23~ Method 1).
9.0 to 10.0. Test solution Transfer 10.0 g to a 250 mL beaker containing
Disperse 5.0 gin 100 mL of carbon dioxide-free waterR. 100 mL of dilute hydrochloric acidR. Mix, cover with a watch
glass and boil for 15 min. Allow to cool to room temperature
Viscosity (2.2.10)
and allow the insoluble matter to settle. Decant the
Weigh a quantity of the substance to be examined equivalent
supernatant through a rapid-flow filter paper into a 400 mL
to 25.0 gof the dried substance and immediately transfer to
beaker. To the insoluble matter in the 250 mL beaker add
a suitable 1 L blender jar containing a quantity of water R, at
25 mL of hot waterR. Stir, allow the insoluble matter to
25 ± 2 DC, that is sufficient to produce a mixture weighing
settle and decant the supernatant through the filter into the
500 g. Blend for exactly 3 min, at 14 000-15 000 r/min.
400 mL beaker. Repeat the extraction with 2 additional
The heat generated during blending causes the temperature
quantities, each of 25 mL, of waterR, decanting each time
to rise to above 30 DC. Transfer the contents of the blender
the supernatant through the filter into the 400 mL beaker.
to a 600 mL beaker and allow to stand for 5 min.
Wash the filter with 25 mL of hot water R, collecting this
The sample temperature should be 33 ± 3 DC.
filtrate in the 400 mL beaker. Concentrate the combined
Using a suitable rotating viscometer equipped with a spindle filtrates to about 20 mL by gently boiling. If a precipitate
as specified below, operate the viscometer at 60 r/min for appears, add about 0.1 mL of nitric acidR, heat to boiling
exactly 6 min and record the scale reading. and allow to cool to room temperature. Filter the
For type IA, use a spindle with a cylinder 1.87 em in concentrated extracts through a rapid-flow filter paper into a
diameter and p.69cm high attached to a shaft 0.32 ern in 50 mL volumetric flask. Transfer the remaining contents of
diameter, the distance from the top of the cylinder to the the 400 mL beaker through the filter paper and into the flask
lower tip of the shaft being 2.54 em, and an immersion with waterR. Dilute this solution to 50.0 mL with water R.
depth of 5.00 em (No.2 spindle); if the scale reading is Reference solutions Prepare the reference solutions using lead
greater than 90 per cent of the full scale, repeat the standardsolution (10 ppm Pb) R, diluted as necessary with
measurement using a spindle similar to the No.2 spindle but waterR.
with a cylinder 1.27 em in diameter and 0.16 cm high (No.3
Source Lead hollow-cathode lamp.
spindle).
Wavelength 217 nm.
For type IC, use a No.3 spindle; if the scale reading is
greater than 90 per cent of the full scale, repeat the Atomisation device Oxidising air-acetylene flame.
measurement using a spindle with a cylindrical shaft 0.32 em Loss on drying (2.2.32)
in diameter and an immersion depth of 4:05 em (No.4 Maximum 8.0 per cent, determined on 1.000 g by drying in
spindle). an oven at 110 DC.
For types IB and IIA, use a No.2 spindle. Microbial contamination
TAMC: acceptance criterion 103 CFU/g (2.6.12).
Viscosity (mPaos) TYMC: acceptance criterion 102 CFU/g (2.6.12).
Type
min. max. Absence of Escherichia coli (2.6.13).
ASSAY
IA 225 600
Aluminium
IB 150 450 Atomic absorption spectrometry (2.2.23~ Method 1).
Ie 800 2200 Test solution In a platinum crucible mix 0.200 g with 1.0 g
of anhydrous lithium metaborate R. Heat slowly at first and
IIA 100 300
ignite at 1000-1200 DC for 15 min. Allow to cool, place the
crucible in a 100 mL beaker containing 25 mL of a 50 gIL
Arsenic (2.4.2~ Method A) solution of nitric acid R and add 50 mL of a 50 gIL solution
Maximum 3 ppm. of nitric acid R, :filling and submerging the crucible. Place a
Transfer 16.6 g to a 250 mL beaker containing 100 mL of polytetrafiuoroethylene-coated magnetic stirring bar in the
dilute hydrochloric acid R. Mix, cover with a watch glass and crucible and stir gently with a magnetic stirrer until
boil gently, with occasional stirring, for 15 min. Allow the dissolution is complete. Transfer the solution to a 200 mL
insoluble matter to settle and decant the supernatant through volumetric flask, wash the beaker, crucible and magnetic
a rapid-flow filter paper into a 250 mL volumetric flask, stirrer bar with waterR, collecting the washings in the
retaining as much sediment as possible in the beaker. To the volumetric flask, and dilute to 200.0 mL with waterR
residue in the beaker add 25 mL of hot dilute hydrochloric (solution A). To 20.0 mL of solution A add 20 mL of a
acid R, stir, heat to boiling, allow the insoluble matter to 10 gIL solution of sodium chloride R and dilute to 100.0 mL
settle and decant the supernatant through the filter into the with waterR.
volumetric flask. Repeat the extraction with 4 additional Reference solutions Dissolve, with gentle heating, 1.000 g of
quantities, each of 25 mL, of hot dilute hydrochloric acid R, aluminium R in a mixture of 10 mL of hydrochloric acid Rand
decanting each supernatant through the filter into the 10 mLof water R. Allow to cool, then dilute to 1000.0 mL
volumetric flask. At the last extraction, transfer as much of with waterR (l mg of aluminium per millilitre). Into 4
the insoluble matter as possible onto the filter. Allow the identical volumetric flasks, each containing 0.20 g of sodium
combined filtrates to cool to room temperature and dilute to chloride R, introduce 1.0 mL, 2.0 mL, 3.0 mL and 4.0 mL of
250.0 mL with dilute hydrochloric acid R. Dilute 5.0 mL of this solution respectively, and dilute to 100.0 mL with
this solution to 25.0 mL with dilute hydrochloric acid R. waterR.

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1-124 Aluminium Phosphate 2020

Blank solution Dissolve 0.20 g of sodium chloride R in

water R and dilute to 100.0 mL with the same solvent.
Dried Aluminium Phosphate
Source Aluminium hollow-cathode lamp. (Aluminium Phosphate, Hydrated, Ph. Bur.
Wavelength 309 nm. monograph 1598)
Atomisation device Acetylene-nitrous oxide flame. AlP04,xHzO 122.0
Magnesium (anhydrous substance)
Atomic absorption spectrometry (2.2.23~ Method I).
Action and use
Test solution Dilute 25.0 mL of solution A, prepared in the
Antacid.
assay for aluminium, to 50.0 mL with water R. To 5.0 mL of
this solution add 20.0 mL of lanthanum chloride solution R PhEur _
and dilute to 100.0 mL with water R.
DEFINITION
Reference solutions Place 1.000 g. of magnesium R in a
Content
250 mL beaker containing 20 mL of waterR and carefully
94.0 per cent to 102.0 per cent of AlP0 4 (M r 122.0) (ignited
add 20 mL of hydrochloric acid R, warming if necessary to
substance) .
dissolve. Transfer the solution to a volumetric flask and
dilute to 1000.0 mL with water R (l mg of magnesium per It contains a variable quantity of water.
milli1itre). Dilute 5.0 mL of this solution to 500.0 mL with CHARACTERS
water R. Into 4 identical volumetric flasks, introduce 5.0 mL, Appearance
10.0 mL, 15.0 mL and 20.0 mL of the solution respectively. White or almost white powder.
To each flask add 20.0 mL of lanthanum chloride solution R
Solubility
and dilute to 100.0 mL with water R.
Very slightly soluble in water, practically insoluble in ethanol
Blank solution Dilute 20 mL of lanthanum chloride solution R (96 per cent). It dissolves in dilute solutions of mineral acids
to 100.0 mL with water R. and alkali hydroxides.
Source Magnesium hollow-cathode lamp.
IDENTIFICATION
Wavelength 285 nino A. Solution S (see Tests) gives reaction (b) of phosphates
Aiomisation device Air-acetylene flame. (2.3.1).
LABELLING B. Solution S gives the reaction of aluminium (2.3.1).
The label states the ratio of aluminium content to TESTS
magnesium content, the viscosity and the corresponding type Solution S
(see Definition). Dissolve 2.00 g in dilute hydrochloric acid R and dilute to
FUNCTIONAUTY-RELATED CHARACTERISTICS 100 mL with the same acid.
This section provides information on characteristics that are Appearance of solution
recognised as being relevant control parameters for one or more Solution S is clear (2.2.1) and colourless (2.2.2, Method II).
functions of the substance when used as an excipient (see chapter pH (2.2.3)
5.15). Some of the characteristics described in the Functionality- 5.5 to 7.2
related characteristics section may also be present in the mandatory
part of the monograph since they also represent mandatory quality Shake 4.0 g with carbon dioxide-free water Rand dilute to
100 mL with the same solvent.
criteria. In such cases, a cross-reference to the tests described in the
mandatory part is induded in the Functionality-related Chlorides (2.4.4)
characteristics section. Control of the characteristics can contribute Maximum 1.3 per cent.
to the qual£tyof a medicinal product by improving the consistency Dissolve 50.0 mg in 10 mL of dilute nitric acid R and dilute
of the manufacturing process and the performance of the medicinal to 200 mL with water R.
product during.use. "Where control methods are cued, they are Soluble phosphates
recognised as being suitablefor the purpose, but other methods can Maximum 1.0 per cent, calculated as pol';'.
also be used. Wherever results for a particular characteristic are
Test solution Stir 5.0 g with 150 mL of tuater.R for 2 h.
reported, the controlmethod must be indicated.
Filter and wash the filter with 50 mL of water R. Combine
The following characteristics may be relevantfor aluminium the filtrate and the washings and dilute to 250.0 mL with
magnesium silicate used as viscosity-increasing agent and water R. Dilute 10.0 mL of this solution to 100.0 mL with
stabiliser. water R.
Acid demand Reference solution (a) Dissolve 2.86 g of potassium dihydrogen
Weigh a quantity of the substance to be examined equivalent phosphate R in water R and dilute to 100 mL with the same
to 5.00 g of the dried substance and disperse in 500 mL of solvent.
water R using a suitable blender fitted with alL jar. With
Reference solution (b) Dilute 1 mL of reference solution (a)
constant mixing, add 3.0 mL portions of 0.1 M hydrochloric
to 5 mL with water R.
acid at 5 s, 65 s, 125 s, 185 S, 245 s, 305 s, 365 s, 425 s,
485 s, 545 s, 605 s, 665 s and 725 s and add a 1.0 mL Reference solution (c) Dilute 3 mL of reference solution (a)
portion at 785 s. Determine the pH potentiometrically at to 5 mL with water R.
840 S. The pH is not greater than 4.0. Treat each solution as follows. To 5.0 mL add 4 mL of dilute
Viscosity sulfuric acid R, 1 mL of ammonium molybdate solution R, 5 mL
(see Tests). of water Rand 2 mL of a solution containing 0.10 g of
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
4-methylaminophenol sulfate R, 0.5 g of anhydrous sodium
sulfite Rand 20.0 g of sodium metabisulfue R in 100 mL of
water R. Shake and allow to stand for 15 min. Dilute to

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2020 Aluminium Phosphate Gel 1-125

25.0 mL with water R and allow to stand for a further TESTS

15 min. Measure the absorbance (2.2.25) at 730 nm, Solution S
Calculate the content of soluble phosphates from a Dissolve 2.00 g in dilute hydrochloric acid R and dilute to
calibration curve prepared using reference solutions (a), 100 mL with the same acid.
(b) and (c) after treatment. pH (2.2.3)
Sulfates (2.4.13) 6.0 to 8.0.
Maximum 0.6 per cent. Peroxides
Dilute 8 mL of solution S to 100 mL with distilled water R. Maximum 150 ppm, expressed as hydrogen peroxide.
Arsenic (2.4.2) Test solution Dissolve with heating 1.0 g of the substance to
Maximum 1 ppm. be examined in 5 mL of dilute hydrochloric acid R, then add
1.0 g complies with limit test A. 5 mL of water Rand 2 mL of divanadium pentoxide solution in
sulfuric acid R.
Loss on ignition
Reference solution Dilute 1.0 mL of dilute hydrogen peroxide
10.0 per cent to 20.0 per cent, determined on 1.000 gat
solution R to 200.0 mL with water R. To 1 mL of this
800 ± 50 -c.
solution add 9 mL of water Rand 2 mL of divanadium
Neutralising capacity pentoxide solution in sulfuric acid R.
Add 0.50 g to 30 mL of 0.1 M hydrochloric acid previously The test solution is not more intensely coloured than the
heated to 37 "C and maintain at this temperature for 15 min reference solution.
while stirring. The pH (2.2.3) of the mixture after 15 min at
, Chlorides (2.4.4)
37 -c is 2.0 to 2.5.
Maximum 500 ppm.
ASSAY Dissolve 1.3 g in 5 mL of dilute nitric acid R and dilute to
Dissolve 0.400 gin 10 mL of dilutehydrochloric acid Rand 200 mL with water R.
dilute to 100.0 ml, with water R. To 10.0 mL of the
Soluble phosphates
solution, add 10.0 mL of 0.1 M sodium edetate and 30 mL of
Maximum 0.5 per cent, expressed as P04 •
a mixture of equal volumes of ammonium acetate solution R
and dilute acetic acid R. Boil for 3 min, then cool. Add 25 mL Test solution Centrifuge 10.0 g until a clear supernatant is
of ethanol (96 per cent) Rand 1 mL of a freshly prepared obtained. To 2.00 mL of the supernatant add 20.0 mL of a
0.25 gIL solution of dithizone R in ethanol (96 per cent) R. 10.3 gIL solution of hydrochloric acidR and dilute to
Titrate the excess of sodium edetate with 0.1 M zinc sulfate 100.0 mL with waterR. To 10.0 mL of this solution add
until the colour changes to pink. 10.0 mL of nitro-molybdovanadic reagent R and dilute to
50.0 mL with waterR. Allow to stand protected from light
1 mL of 0.1 M sodium edetate is equivalent to 12.20 mg of
for 15 min.
AlP04 •
Reference solution Add 10.0 mL of nitro-molybdovanadic
STORAGE reagent R to 10.0mL of a 143 mg/L solution of potassium
In an airtight container. dihydrogen phosphate R and dilute to 50.0 mL with water R.
_ _ _ _ _--'-- ~ PhEur Allow to stand protected from light for 15 min.
Measure the absorbances (2.2.25) of the 2 solutions at
400 nm. The absorbance of the test solution is not greater
than that of the reference solution.
Aluminium Phosphate Gel Sulfates (2.4.13)
Maximum 0.2 per cent.
(Ph. Bur. monograph 2166) Dilute 25 mL of solution S to 100 mL with distilled water R.
Action and use Soluble aluminium
Antacid; vaccine adjuvant. Maximum 50 ppm.
PhEur _ To 16.0 g add 50 mL of water R. Heat to boiling for 5 min.
Cool and centrifuge. Separate the supernatant. Wash the
DEFINITION residue with 20 mL of waterR and centrifuge. Separate the
Hydrated AlP0 4 in gel form. supernatant and add to the first supernatant. To the
Content combined supernatants add 5 mL of hydrochloric acid Rand
19.0 per cent to 21.0 per cent of AlP0 4 . 20 mL of waterR. Introduce all of this solution into a
500 mL conical flask and carry out the complexometric
CHARACTERS titration of aluminium (2.5.11) using 0.01 M sodium edetate.
Appearance
Arsenic (2.4.2, MethodA)
Gel.
Maximum 1 ppm, determined on 1.0 g.
Solubility
Acid neutralising capacity
Practically insoluble in water, in ethanol (96 per cent) and in
Add 2.0 g to 30 mL of 0.1 M hydrochloric acid heated to
methylene chloride. It dissolves in dilute solutions of mineral
acids.. 37°C and maintain at 37 °C while shaking. Determine the
pH after 15 min. The pH (2.2.3) of the mixture is 2.0 to 2.5.
IDENTIFICATION Residue on ignition
A. Solution S (see Tests) gives reaction (b) of phosphates 19.0 per cent to 23.0 per cent.
(2.3.1).
Heat 0.500 g at 50°C for 5 hours, then ignite at
B. Solution S gives the reaction of aluminium (2.3.1). 500 ± 50°C until constant mass.
C. It complies with the assay. Microbial contamination
TAMC: acceptance criterion 10 3 CFU/g (2.6.12).

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1-126 Aluminium Powder 2020

TYMC: acceptance criterion 102 CFU/g (2.6.12). Lead

Absence of bile-tolerant gram-negative bacteria (2.6.13). Use two solutions prepared in the following manner.
For solution (1) boil 0.40 g with 20 mL of 2M hydrochloric
Absence of Escherichia coli (2.6.13).
acid and 10 mL of water until effervescence ceases, add
ASSAY 0.5 mL of nitric acid, boil for 30 seconds and cool; add 2 g of
Dissolve with heating 0.300 gin 5 mL of dilute hydrochloric ammonium chloride. and 2 g of ammonium thiocyanate, extract
acid R. Add 45 mL of water R, 10.0 mL of 0.1 M sodium with three 10 mL quantities of a mixture of equal volumes of
edetate and 30 mL of a mixture of equal volumes of amyl alcohol and ether, discard the extracts and add 2 g of
ammonium acetate solution R and dilute acetic acid R. Heat to citric acid. For solution (2) dissolve 2 g of citric acid in 10 mL
boiling and maintain boiling for 3 min. Cool, then add of 2M hydrochloric acid and add 4 mL of lead standard solution
25 mL of ethanol (96 per cent) R. Titrate with 0.1 M zinc (10 ppm Pb). Make solutions (1) and (2) alkaline with
sulfate, determining the end-point potentiometrically (2.2.20). 5M ammonia and to each add 1 mL of potassium cyanide
1 mL of 0.1 M zinc sulfate is equivalent to 12;2 mg of AlP0 4 • solution PbT. The solutions should be not more than faintly
opalescent. If the colours of the solutions differ, equalise by
STORAGE
the addition of about 0.2 mL of a highly diluted solution of
In an airtight container.
burnt sugar or other non-reactive substance. Dilute each
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
solution to 50 mL with water, add 0.1 mL of a 10% w/v
solution of sodium sulfide to each and mix thoroughly.
The colour produced in solution (1) is not more intense than
that produced in solution (2), when viewed against a white
Aluminium Powder background (100 ppm).
AI 26.98 7429-90-5 Other metals
Dissolve 2 g in 40 mL of 2M hydrochloric acid. Dilute 20 mL
Action and use of the solution to 100 mL with water, make alkaline to litmus
Topical protective. paper by the addition of 5M ammonia, boil and filter.
Preparation Evaporate the filtrate to dryness, add 0.05 mL of sulfuric acid
Compound AIuminium Paste and ignite. The residue weighs not more than 2 mg.
Lubricant
DEFINITION To 2 g add 100 mL of hot water, cover and add, drop wise,
Aluminium Powder consists mainly of metallic aluminium in sufficient of a mixture of equal volumes of hydrochloric acid
the form of very small flakes, usually with an appreciable and water to dissolve the metal almost completely. Heat to
proportion of aluminium oxide; it is lubricated with stearic complete dissolution, cool, filter through a hardened filter
acid to prevent oxidation. It contains not less than 86.0% of paper and wash the vessel and filter paper thoroughly with
AI, calculated with reference to the substance freed from water; dry both the vessel and paper at room temperature.
lubricant and volatile matter. . Extract the paper with three 100-mL quantities of boiling,
CHARACTERISTICS freshly distilled acetone, using the original vessel to contain
A silvery grey powder. the solvent and then wash the paper with five 10-mL
quantities of freshly distilled acetone. Evaporate the combined
Practically insoluble in water and in ethanol (96%).
filtrate and washings to dryness using a rotary evaporator.
It dissolves in dilute acids and in aqueous solutions of alkali
The residue, after drying at 105° for 30 minutes and allowing
hydroxides, with the evolution of hydrogen.
to cool, weighs 10 to 60 mg.
IDENTIFICATION When the basin containing the residue is floated in a beaker
A solution in 2M hydrochloric acid yields the reaction of water suitably stirred and heated, the residue melts
characteristic of aluminium salts, Appendix VI. between 40° and 60°. The residue is almost completely
TESTS soluble, with effervescence, in hot dilute sodium carbonate
Surface-covering power solution.
Not less than 4000 cm 2 per g when determined by the Volatile matter
following method. Fill with water a shallow trough measuring When heated to constant weight at 105°, loses not more than
approximately 60 em x 12 em x 1.5 em, fitted with a 0.5% of its weight. Use 1 g.
movable partition so constructed that it is a sliding fit and ASSAY
can be used to divide the trough into two rectangular areas.
Transfer 0.2 g, previously freed from lubricant by successive
Place the movable partition near one end and sprinkle 50 mg
washing with acetone and drying, to a three-necked 500 mL
of the substance being examined on the surface of the liquid
flask fitted with alSO mL dropping funnel, an inlet tube
confined in the smaller area. Using a glass rod, spread the
connected to a cylinder of carbon dioxide and an outlet tube
powder evenly over the liquid surface until an unbroken film
dipping into a water trap; Add 60 mL of water and disperse
covers the entire surface. Move the partition so as to increase
the substance being examined; replace the air by carbon
the area confined and again spread the powder to cover the
dioxide andadd 100 mLof a solution containing 56 g of
increased surface. Continue this process and determine the
ammonium iron(IlI) sulfate and 7.5 mL ofsuljuric add in water.
maximum unbroken surface area obtained. The surface-
While maintaining an atmosphere of carbon dioxide in the
covering power is the area covered per g of the powder at the
flask, heat to boiling, boil for 5 minutes after the sample has
breaking point of the film.
dissolved, cool rapidly to 20°· and dilute to 250 mL with
Iron water. To 50 mL add 15 mL of otthophosphoric acid and
Dissolve 10 mg in 20· mL of 2M hydrochloric acid and dilute titrate with 0.02M potassiumpermanganate VS. Each mL of
to 100 mL with water. 10 mL of the resulting solution 0.02M potassium permanganate VS is equivalent to 0.8994 mg
complies with the limit testfor iron, Appendix VII (1.0%). of AI.

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2020 Aluminium Sodium Silicate 1-127

acid R, heat to boiling and allow to cool to room

Aluminium Sodium Silicate temperature. Filter the concentrated extracts through a rapid-
(Ph. Eur. monograph 1676)
flow filter paper into a 25 mL volumetric flask. Transfer the
remaining contents of the beaker through the filter paper and
PhEur _
into the volumetric flask with water R and dilute to 25.0 mL
DEFINITION with the same solvent.
Silicic acid aluminium sodium salt of synthetic origin. Reference solutions Into 4 separate 100 mL volumetric flasks,
introduce respectively 3.0 mL, 5.0 mL, 10.0 mL and
Content
- aluminium (Al; M r 26.98): 2.7 per cent to 7.9 per cent
15.0 mL of lead standard solution (10 ppm Pb) R, add
0.20 mL of heavy metal-free nitric acid R and dilute to
(dried substance);
- sodium (Na; M r 22.99): 3.7 per cent to 6.3 per cent (dried
100.0 mL with water R.
substance). Source Lead hollow-cathode lamp.
Wavelength 217.0 nm.
CHARACTERS
Appearance Atomisation device Air-acetylene flame.
White or almost white, fine, light, amorphous powder. Loss on drying (2.2.32)
Solubility Maximum 8.0 per cent, determined on 1.000 g by drying in
Practically insoluble in water and in organic solvents. an oven at 105 DC for 4 h. .
Loss on ignition
IDENTIFICATION
5.0 per cent to 11.0 per cent (dried substance), determined
A. Transfer 1.0 g to a 100 mL beaker and add 10 mL of
on 1.000 g by ignition in a platinum crucible to constant
dilute hydrochloric acid R. Mix, cover with a watch glass and
mass at 1000 ± 25°C.
boil for 15 min. Allow to cool to room temperature, mix and
centrifuge the solution. 2 mL of the supernatant gives the Microbial contamination
reaction of aluminium (2.3.1). TAMC: acceptance criterion 103 CFU/g (2.6.12).
B. 2 mL of the supernatant obtained in identification test A TYMC: acceptance criterion 102 CFU/g (2.6.12).
gives reaction (a) of sodium (2.3.1). Absence of Escherichia coli (2.6.13).
C. 0.2 g gives the reaction of silicates (2.3.1). ASSAY
TESTS Aluminium
pH (2.2.3) Atomic absorption spectrometry (2.2.23, Method 1).
9.5 to 11.5. Acid mixture Add 50 mL of nitric acid R to 500 mL of
Disperse 5.0 gin 100 mL of carbon dioxide-free water R. water R. Dissolve in this solution 17 g of tartaric acid Rand
dilute to 1000 mL with water R.
Arsenic (2.4. 2~ Method A)
Maximum 3 ppm. Blank solution Dissolve 1.4 g of anhydrous lithium
metaborate R in 60 mL of the acid mixture and dilute to
Transfer 8.3 g to a 250 mL beaker containing 50 mL of
200 mL with water R.
dilute hydrochloric acid R. Mix, cover with a watch glass and
boil gently, with occasional stirring, for 15 min. Centrifuge, Test solution In a platinum crucible mix 0.200 g with 1.4 g
and decant the supernatant through a rapid-flow filter paper of anhydrous lithium metaborate R. Heat slowly at first and
into a 250 mL volumetric flask. To the residue in the beaker, ignite at 1100 ± 25°C for 15 min. Cool, then place the
add 25 mL of hot dilute hydrochloric acid R, stir, centrifuge, crucible in a 100 mL beaker containing 60 mL of the add
and decant the supernatant through the same filter into the mixture. Place a polytetrafluoroethylene..coated magnetic
volumetric flask. Repeat the extraction with 3 additional stirring bar in the crucible and stir gently with a magnetic
quantities, each of 25 mL, of hot dilute hydrochloric acid R, stirrer for 16 h. Transfer the contents of the crucible into a
filtering each supernatant through this filter into the 200 mL volumetric flask. Wash the crucible, the magnetic
volumetric flask. Allow the combined filtrates to cool to room stirring bar and the beaker with water R and dilute to
temperature and dilute to 250.0 mL with dilute hydrochloric 200.0 mL with the same solvent (solution A). To 10.0 mL of
acid R. Dilute 10.0 mL of the solution to 25.0 mL with this solution, add 1.0 mL of lanthanum chloride solution Rand
water R. dilute to 50.0 mL with water R.
Reference solutions Into 5 separate 50 mL volumetric flasks,
Lead
Maximum 5 ppm. introduce respectively 1.0 mL, 2.5 mL, 5.0 mL, 7.5 mL and
10.0 mL of aluminium standard solution (100 ppm AD R, add
Atomic absorption spectrometry (2.2.23, Method l). 1 mL of lanthanum chloride solution R and 10 mL of the blank
Test solution Transfer 5.0 g to a 250 mL beaker containing solution, and dilute to 50.0 mL with water R.
50 mLof dilute hydrochloric acid R. Mix, cover with a watch Source .Aluminium hollow-cathode lamp.
glass and boil for 15 min. Allow to cool to room
temperature. Centrifuge, and decant the supernatant through Wavelength 309.3 nm.
a rapid-flow filter paper into a 250 mL beaker. To the Atomisation device Acetylene-nitrous oxide flame.
insoluble matter add 25 mL of hot water R. Stir vigorously, Sodium
centrifuge, and decant the supernatant through the same Atomic emission spectrometry (2.2.22, Method l).
filter into the beaker. Repeat the extraction with 2 additional Test solution To 2.0 mL of solution A, prepared in the assay
quantities, each of 25 mL, of hot water R, decanting each of aluminium, add 1 mL of a 12.5 gIL solution of caesium
supernatant through the filter into the beaker. Wash the filter chloride R and dilute to 20.0 mL with water R.
with 25 rnl..of hot water R, collecting the filtrate in the
Reference solutions Into 5 separate 200 mL volumetric flasks,
beaker. Concentrate the combined filtrates by gently boiling
each containing 10 mL of a 12.5 gIL solution of caesium
to about 15 mL. Add about 0.05 mL of heavy metal-free nitric
chloride R, introduce respectively 1.0 mL, 2.0 mL, 4.0 mL,

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 1-128 Aluminium Stearate 2020

6.0 mL and 10.0 mL of sodium standard solution To 10 mL of the filtrate add 0.05 mL of bromothymol blue
(200 ppm Na) R and dilute to 200.0 mL with water R. solution R4. Not more than 0.05 mL of 0.1 M hydrochloric
Wavelength 589.0 run. acid or 0.1 M sodium hydroxide is required to change the
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur colour of the indicator.
Chlorides (2.4.4)
Maximum 0.1 per cent.
Dilute 0.5 mL of solution S to 15 mL with water R.
Aluminium Stearate Sulfates (2.4.13)
Maximum 0.5 per cent.
(Ph. Eur. monograph 1663) Dilute 0.3 mL of solution S to 15 mL with distilled water R.
PhEur _
Cadmium
DEFINITION Maximum 3 ppm.
Aluminium salts of a mixture of solid organic acids consisting Atomic absorption spectrometry (2.2.23, Method II).
mainly of variable proportions of aluminium stearate and For the preparation of all aqueous solutions and for the rinsingof
aluminium palmitate. The organic acids are obtained from glassware before use, employ water that has been passedthrough a
, sources of vegetable or animal origin. strong-acid, strong-base, mixed-bedion-exchange resin before use.
Content Select all reagents to have as Iowa content of cadmium, lead and
- aluminium (Al; A r 26.98): 3.0 per cent to 9.0 per cent nickelas practicable and store all reagent solutions in containers of
(dried substance); borosilicate glass. Clean glassware before use by soakingin warm
- stearic acid in the fatty acidfraction: minimum 8 M nitric acidfor 30 min and by rinsing with deionised water.
40.0 per cent; , Blank solution Dilute 25 mL of cadmium- and lead-free nitric
- sum of stearic acid and palmitic acid in the fatty acidfraction: acid R to 100.0 mL with water R.
minimum 90.0 per cent. Modifiersolution Dissolve 20 g of ammonium dihydrogen
CHARACTERS phosphate R and 1 g of magnesium nitrate R in water Rand
Appearance dilute to 100 mL with the same solvent. Alternatively, use an
White or almost white, very fine, light powder. appropriate matrix modifier as recommended by the graphite
furnace atomic absorption (GFAA) spectrometer
Solubility
manufacturer.
Practically insoluble in water and in anhydrous ethanol.
Test solution Place 0.100 g of the substance to be examined
IDENTIFICATION in a polytetrafluoroethylene digestion bomb and add 2.5 mL
First identification: C~ D. of cadmium- and lead-free nitric acid R. Close and seal the
Secondidentification: A~ B~ D. bomb according to the manufacturer's operating instructions.
A. Freezing point (2.2.18): minimum 53°C, determined on W'hen using a digestion bomb, be thoroughly familiar with the
the residue obtained in the preparation of solution S safety and operating instructions. Carefully follow the bomb
(see Tests). manufacturer's instructions regarding care and maintenance of
B. Acid value (2.5.1): 195 to 210. these digestion bombs. Do not use metal-jacketed bombs or liners
that have been used with hydrochloric aciddue to contamination
Dissolve 0.200 g of the residue obtained in the preparation of
from corrosion of the metaljacket by hydrochloric acid. Heat the
solution S in 25 mL of the prescribed mixture of solvents.
bomb in an oven at 170°C for 3 h. Cool the bomb slowly in
C. Examine the chromatograms obtained in the assay of air to room temperature according to the bomb
stearic acid and palmitic acid. manufacturer's instructions. Place the bomb in a fume
Results The 2 principal peaks in the chromatogram obtained cupboard and open carefully as corrosive gases may be
with the test solution are similar in retention time to the expelled. Dissolve the residue in waterR and dilute to
2 principal peaks in the chromatogram obtained with the 10.0 mL with the same solvent.
reference solution. Reference solution Prepare a solution containing
D. 1 mL of solution S gives the reaction of aluminium 0.00165 ug/ml, of cadmium nitrate tetrahydrate R in the blank
(2.3.1). The addition of 0.5 mL of dilute hydrochloric acid R solution (equivalent to 0.006 ug/ml, of Cd).
described in the general method is omitted. Dilute 1.0 mL of the test solution to 10.0 mL with the blank
TESTS solution. Prepare mixtures of this solution, the reference
Solution S solution and the blank solution in the following proportions:
To 5.0 g add 50 mL of peroxide-free etherR, 20 mLof dilute (1.0:0:1.0 VIVIV), (1.0:0.25:0.75 VIVIV),
nitric acid Rand 20 mL of distilled water R and heat gently (1.0:0.5:0.5 VIVIV), (1.0:0.75:0.25 VIVIV). To each mixture
a
under reflux condenser until dissolution is complete. Allow add 50 JlL of the modifier solution and mix..These solutions
contain respectively 0 ug, 0.0015 ug, 0.0030 ug and
to cool. Ina separating funnel, separate the aqueous layer
and shake the ether layer with 2 quantities, each of 4 mL, of 0.0045 ug of cadmium per millilitre from the reference
distilled water R. Combine the aqueous layers, wash with solution. Keep the remaining test solution for use in the test
15 mL of peroxide-free etherR and dilute to 50.0 mL with for lead and nickel.
distilled water R (solution S). Evaporate the ether layer to Source Cadmium hollow-cathode lamp.
dryness and dry the residue at 100-105 °C.Keep the residue Wavelength 228.8 nm.
for identification tests A and B. Atomisation device Furnace.
Acidity or alkalinity Platform Pyrolytically coated with integrated tube.
To 1~O g add 20 mL of carbon dioxide-free water R and boil
Operating conditions Use the temperature programme
for 1 min with continuous shaking. Cool and filter.
recommended for cadmium by the GFAA manufacturer.

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2020 Aluminium Stearate 1-129

An example of temperature parameters for GFAA analysis of solvent. Alternatively, use an appropriate matrix modifier as
cadmium is shown below. recommended by the GFAA spectrometer manufacturer.
Test solution Use the solution described in the test for
Stage Final temperature Ramp time Hold time cadmium.
CC) (s) (s)
Reference solution Prepare a solution of 0.050 ug/ml, ofNi
Drying 110 10 20
by suitable dilutions of a 0.2477 ug/ml., solution of nickel
Ashing 600 10 30
nitratehexahydrate R with the blank solution.
Atomisation 1800 0 5
Prepare mixtures of the test solution, the reference solution
and the blank solution in the following proportions:
Lead (1.0:0:1.0 VIVIV), (1.0:0.5:0.5 VIVIV), (1.0:1.0:0 VIVIV).
Maximum 10 ppm. To each mixture add 50 J.l.L of the modifier solution and mix.
Atomic absorption speettometry (2.2.23~ Method II). These solutions contain respectively 0 ug, 0.0125 ug and
For the preparation of all aqueous solutions and for the rinsing of 0.025 J.l.g of nickel per millilitre from the reference solution.
glassware before use, employ water that has beenpassedthrough a Source .Nickel hollow-cathode lamp.
strong-acid, strong-base, mixed-bedion-exchange resin before use. Wavelength 232.0 nm.
Select all reagents to have as low a content of cadmium, lead and
Atomisation device Furnace.
nickel as practicable and store all reagent solutions in containers of
borosilicate glass. Clean glassware before use by soakingin warm Platform . Pyrolytically coated with integrated tube.
8 M nitric acid fof30 min and by rinsing with deionised water. Operating conditions Use the temperature programme
Blank solution Use the solution described in the test for recommended for nickel by the GFAA manufacturer.
cadmium. An example of temperature parameters for GFAA analysis of
nickel is shown below.
Modifier solution "Use the solution described in the test for
cadmium.
Stage Final temperature Ramp time Hold time
Test solution Use the solution described in the test for Cq (s) (s)
cadmium. Drying 110 10 20
Reference solution Prepare a solution of 0.100 ug/ml, of Pb Ashing 1000 20 30
by suitable dilutions of lead standardsolution (100 ppm Pb) R Atomisation 2300 0 5
with the blank solution.
Prepare mixtures of the test solution, the reference solution Loss on drying (2.2.32)
and the blank solution in the following proportions: Maximum 6.0 per cent, determined on 1.000 g by drying in
(1.0:0:1.0 VIVIV), (1.0:0.5:0.5 VIVIV), (1.0:1.0:0 VIVIV). an oven at 105 "C.
To each mixture add 50 JlL of the modifier solution and mix.
Microbial contamination
These solutions' contain respectively 0 ug, 0.025 ug and
TAMe: acceptance criterion 103 CFU/g (2.6.12).
0.05 J.l.g of lead per millilitre from the reference solution.
TYMC: acceptance criterion 102 CFU/g (2.6.12).
Source Lead hollow-cathode lamp.
Absence of Escherichia coli (2.6.13).
Wavelength 283.3 nm.
Absence of Salmonella (2.6.13).
Atomisation device Furnace.
Platform Pyrolytically coated with integrated tube. ASSAY
Aluminium
Operating conditions Use the temperature programme
To 0.250 g in a 250 mL conical flask add 20 mL of
recommended for lead by the GFAA manufacturer.
methanolR. and, slowly, 2 mL of sulfuric acid R. Heat the
An example of temperature parameters for GFAA analysis of
solution for 30 min under reflux on a water-bath, swirling
lead is shown below.
frequently. Allow to cool. Add 100 mL of water R and adjust
to about pH 1 by adding approximately 12 mL of dilute
Stage Final temperature Ramp time Hold time
eC) (s) (s) sodiumhydroxide solution R. Add 20.0 mL of 0.1 M sodium
edetate and adjust to between pH 5 and pH 6 by the addition
Drying 110 10 20
of sodium acetate R. Add 70 mg of xylenolorange triturate R
Ashing 450 10 30
and titrate immediately and quickly with 0.1 M zinc sulfate
Atomisation 2000 0 5
until the colour changes from yellow to pinkish-violet.
1 mL of 0.1 M sodium edetate is equivalent to 2.698 mg of
Nickel AI.
Maximum 5 ppm.
Stearic acid and palmitic acid
Atomic absorption spectrometry (2.2.23~ Method II).
Gas chromatography (2.2.28): use the normalisation
For the preparation of all aqueous solutions and for the rinsing of procedure.
glassware before use, employ water that has beenpassedthrough a
Test solution In a conical flask fitted with a reflux condenser,
strong-acid, strong-base, mixed-bedion-exchange resin before use.
dissolve 0.100 g of the substance to be examined in 5 mL of
Select all reagents to have as low a content of cadmium, lead and
boron trifluoride-methanol solution R. Boil under a reflux
nickel as practicable and storeall reagent solutions in containers of
condenser for 10 min. Add 4 mL of heptane R through the
borosilicate glass. Clean glassware before use by soakingin warm
condenser and boil again under a reflux condenser for
8 M nitric acidfor 30 min and by rinsing with deionised water.
10 min. Allow to cool. Add 20 mL of saturatedsodium
Blank solution Use the solution described in the test for chloride solution R. Shake and allow the layers to separate.
cadmium. Dry the organic layer over 0.1 g of anhydrous sodium sulfate R
Modifier solution. 'Dissolve 20 g of ammonium dihydrogen .,
phosphate R in water R and dilute to 100 mL with the same

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1-130 Aluminium Sulfate 2020

previously washed with heptaneR. Dilute 1.0 mL of the TESTS

solution to 10.0 mL with heptane R. Solution S
Reference solution Prepare the reference solution in the same Dissolve 2.5 g in water R and dilute to 50 mL with the same
manner as the test solution using 50.0 mg of palmitic solvent.
acid CRS and 50.0 mg of stearic acid CRS instead of the Appearance of solution
substance to be examined. Solution S is not more opalescent than reference
Column: suspension III (2.2.1) and is colourless (2.2.2, Method II).
- material: fused silica; pH (2.23)
- size: 1 = 30 m, 0 = 0.32 mm; 2.5 to 4.0.
- stationaryphase: macrogol20 000 R (film thickness
Dissolve 0.5 g in carbon dioxide-free water R and dilute to
0.5 urn).
25 mL with the same solvent.
Carrier gas .heliumfor chromatography R.
Alkali and alkaline-earth metals
Flow rate 2.4 mUmin. Maximum 0.4 per cent.
Temperature: To 20 mL of solution S add 100 mL of water R, heat and
add 0.1 mL of methyl red solution R. Add dilute ammonia Rl
Time Temperature until the colour of the indicator changes to yellow. Dilute to
(min) eC)
150 mL with water R, heat to boiling and filter. Evaporate
Column 0-2 70
75 mL of the filtrate to dryness on a water-bath and ignite.
2 - 36 70,-+ 240
The residue weighs a maximum of 2 mg.
36 - 41 240
Injection port 220
Ammonium (2.4.1)
Detector 260 Maximum 500 ppm.
Dilute 0.4 mL of solution S to 14 mL with water R.
Detection Flame ionisation. Iron (2.4.9)
Injection 1 ilL. Maximum 100 ppm.
Relative retention With reference to methyl stearate: methyl Dilute 2 mL of solution S to 10 mL with water R.
=
palmitate about 0.9. Use 0.3 mL of thioglycollic acid R in this test.
System suitability Reference solution: ASSAY
- resolution: minimum5.0 between the peaks due to methyl Dissolve 0.500.g in 20 mL of water R. Carry out the
palmitate and methyl stearate; complexometric titration of aluminium (2.5.11).
- repeatability: maximum relative standard deviation of 1 mL of 0.1 M sodium edetate is equivalent to 17.11 mg
3.0 per cent for the areas of the peaks due to methyl of Alz(S04h.
palmitate and methyl stearate after 6 injections; maximum
relative standard deviation of 1.0 per cent for the ratio of STORAGE
the areas of the peaks due to methyl palmitate to the areas In an airtight container.
of the peaks due to methyl stearate after 6 injections. _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur

Alverine Citrate
Aluminium Sulfate
(Ph. Bur. monograph 2156)
Aluminium Sulphate

(~H02H
(ph. Bur. monograph 0165)
A!z(SOJ3,xHzO 342.1
. (anhydrous substance) (OH
Preparation C02H
Aluminium Acetate Ear Drops
PhEur _ 473.6 5560-59-8
DEFINITION Action and use
Content Smooth muscle relaxant; antispasmodic.
51.0 per cent to 59.0 per cent of Alz(S04h.
Preparation
It contains a variable quantity of water of crystallisation. Alverine Capsules
CHARACTERS PhEur ~ '______'_ _
Appearance
Colourless, lustrous crystals or crystalline masses. DEFINITION
Solubility N-Ethyl-3-phenyl-N-(3-phenylpropyl)propan-1-amine
Soluble in cold water, freely soluble in hot water, practically dihydrogen 2-hydroxypropane-l,2,3-tricarboxylate.
insoluble in ethanol (96 per cent). Content
IDENTIFICATION· 99.0 per cent to 101.0 per cent (dried substance).
A. Solution S (see Tests) gives reaction (a) of sulfates (23.1).
B. Solution S gives the reaction of aluminium (2.3.1).

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2020 Alverine Citrate 1-131

CHARACTERS Detection Flame ionisation.

Appearance Injection 1 ilL.
White or almost white, crystalline powder.
I~tification of impurities Use the chromatogram obtained
Solubility ~lth r~ference solution (c) to identify the peak due to
Slightly soluble in water and in methylene chloride, sparingly impunty C; use the chromatogram obtained with reference
soluble in ethanol (96 per cent). solution (a) to identify the peak due to impurity D.
IDENTIFICATION Relativeretention With reference to alverine (retention
Infrared absorption spectrophotometry (2.2.24). time = about 18 min): impurity C = about 0.5;
Comparison alverine citrate CRS. =
impurity D about 0.97.
System suitabz7ity Reference solution (a):
TESTS - resolution: minimum 3.0 between the peaks due to
pH (2.2.3) impurity D and alverine.
3.5 to 4.5.
Limits:
Dissolve 0.250 g in carbon dioxide-free water R and dilute to - impurities C, D: for each impurity, maximum
50.0 mL with the same solvent. 0.15 per cent;
Related substances - unspecified impurities: for each impurity, maximum
Gas chromatography (2.2.28): use the normalisation 0.10 per cent;
procedure. Usefreshly prepared solutions. - total: maximum 0.5 per cent;
Test solution Dissolve 0.250 g of the substance to be - reporting threshold: 0.05 per cent (reference solution (b)).
examined in water R and dilute to 20 mL with the same Loss on drying (2.2.32)
solvent. Add 2mL of concentrated ammonia R and shake with Maximum 0.5 per cent, determined on 1.000 g by drying in
3 quantities, each of 15 mL, of methylene chloride R. To the an oven at 80°C for 2 h.
combined lower layers add anhydrous sodiumsulfate R, shake, Sulfated ash (2.4.14)
filter, and evaporate the filtrate by suitable means at a Maximum 0.1 per cent, determined on 1.0 g.
temperature not exceeding 30 "C. Take up the residue with
methylene chloride R and dilute to 10.0 mL with the same ASSAY
solvent. Dissolve 0.375 g in 50 mL of anhydrous acetic acid R. Titrate
Reference solution (a) Dissolve 5 mg of aluerine with O.lM perchloric acid, determining the end-point
impurity D CRS (impurity D citrate) in 5 mL of waterR, add potentiometrically (2.2.20).
1 mL of concentrated ammoniaR and shake with 3 quantities, 1 mL of 0.1 M perchloric acid is equivalent to 47.36 mg
each of 5 mL, of methylene chloride R. To the combined lower of C26H35N07'
layers add anhydrous sodium sulfate R, shake, filter, and STORAGE
evaporate the filtrate by suitable means at a temperature not Protected from light.
exceeding 30°C. Take up the residue with methylene
chloride R, add 0~2 mL of the test solution and dilute to IMPURITIES
2.0 mL with methylene chloride R. Specified impurities C, D.
Reference solution (b) Dilute 1.0 mL of the test solution to Otherdetectable impurities (the following substances would, if
100.0 mL with methylene chloride R. Dilute 1.0 mL of this . present at a sufficient level, be detected by one or otherof the tests
solution to 20.0 mL with methylene chloride R. in the monograph. They are limited by the general acceptance
Reference solution (c) Dissolve 20 mg of aluerine criterion for other/unspecified impurities and/or by the general
impurity C CRS in methylene chloride R and dilute to 20.0 mL monograph Substances for pharmaceutical use (2034). It is
with the same solvent. Dilute 1.0 mL of the solution to therefore not necessary to identify these impurities for
10.0 mL with methylene chloride R. demonstration of compliance. See also 5.1O. Control of impurities
in substances for pharmaceutical use) A, B, E.
Column:
- material: fused silica;
- size: 1 = 25 m, 0 = 0.32 mm;
- stationary phase: poly(dimethyl) (diphenyl)siloxane R (film ~CI
thickness 0.45 J.l.I11).
A. l-chloro- 3-phenylpropane,
Carner gas heliumfor chromatography R.
Flow rate 2.2 mUmin.
Split ratio 1:11.
Temperature:
~OH
B. 3-phenylpropan-l-ol,
Time Temperature
(min) eC)
Column 0-7 120
7- 13 120 -+ 240
13 - 21 240
2f - 24 240 -+ 290 e. N-ethyl-3-phenylpropan-l-amine,
24 - 39 290
Injection port 290
Detector 290

D. N-(3-cyc1ohexylpropyl)-N-ethyl-3-phenylpropan-l-amine,

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1-132 Amantadine Hydrochloride 2020

Acidity or alkalinity
Dilute 2 mL of solution S to 10 rnL with carbon dioxide-free
water R. Add 0.1 mL of methyl redsolution Rand 0.2 mL of
0.01 M sodium hydroxide. The solution is yellow. Add 0.4 mL
of 0.01 M hydrochloric acid. The solution is red.
Related substances
E. 3-phenyl-N,N-bis(3-phenylpropyl)propan-l-amine. Gas chromatography (2.2.28).
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur Internalstandard solution Dissolve 0.500 g of adamantane R
in methylene chloride R and dilute to 10.0 rnL with the same
solvent.
Test solution Weigh 0.5 g of the substance to be examined
Amantadine Hydrochloride into a centrifuge tube. Add 9 mL of methylene chloride Rand
10 mL of a 210 gIL solution of sodium hydroxide R. Shake for
(Ph. Bur. monograph 0463) 10 min. Discard the upper layer. Dry the lower layer over
anhydrous sodium sulfate R. Filter and collect the filtrate in a
hY.
lO NH
2

,Hel
volumetric flask. Add 0.1 mL of the internal standard
solution and dilute to 10.0 mL with methylene chloride R.
Reference solution Weigh 5 mg of amantadine
hydrochloride CRS into a centrifuge tube.: Add 9 mL of
187.7 665-66-7 methylene chloride Rand 10 mL of a 210 gIL solution of
sodium hydroxide R. Shake for 10 min. Discard the upper
Action and use layer. Dry the lower layer over anhydrous sodium sulfate R.
Viral replication inhibitor (influenza A); dopamine receptor Filter and collect the filtrate in a volumetric flask.
agonist; treatment of influenza and Parkinson's disease. Add 1.0 mL of the internal standard solution and dilute to
Preparations 100.0 mL with methylene chloride R.
Amantadine Capsules .Column:
Amantadine Oral Solution - material: fused silica;
PhEur ---,- _
- size: I = 30 m, 0 = 0.53 mm;
- stationary phase: base-deactivated poly(dimethyl)(diphenyl)
DEFINmON siloxane R (film thickness 1 J.Ul1).
Tricyclo[3.3.1.t3,7]decan-l-arnine hydrochloride. Carnergas helium for chromatography R.
Content Flow rate 4 mIJrnin.
98.5 percent to 101.0 per cent (anhydrous substance). Split ratio 1:50.
CHARACTERS Temperature:
Appearance
White or almost white, crystalline powder. Time Temperature
(min) eC)
Solubility
Column 0-5 70
Freely soluble in water and in ethanol (96 per cent).
5 - 23 70 ..... 250
It sublimes on heating. 23 - 40 250
IDENTIFICATION Injection port 220
First identification: A~ D. Detector 300
Second identification: B, C, D.
A. Infrared absorption spectrophotometry (2.2.24). Detection Flame ionisation.
Comparison amantadine hydrochloride CRS. Injection 1 ilL.
B. To 0.1 g add 1 rnL of pyridine R, mix and add 0.1 mL of Relative retention With reference to amantadine (retention
acetic anhydride R. Heat to boiling for about 10 s. Pour the time = about 14 min): internal standard = about 0.8.
hot solution into 10 mL of dilute hydrochloric acid R, cool to System suitability Reference solution:
5 DC and filter. The precipitate, washed with waterRand - resolution: minimum 5.0 between the peaks due to the
dried in vacuo at 60 DC for 1 h, melts (2.2.14) at 147 DC to internal standard and amantadine.
151 DC. Limits:
C. Dissolve 0.2 gin 1 mL of 0.1 M hydrochloric acid. - unspecified impurities: calculate the ratio (R 1) of the area of
Add 1 mL of a 500 gIL solution of sodium nitrite R. A white the peak due to amantadine to the area of the peak due to
precipitate is formed. the internal standard from the chromatogram obtained
D. 1 mL of solution S· (see Tests) gives reaction (a) of with the reference solution; from the chromatogram
chlorides (2.3.1). obtained with the test solution, calculate the ratio of the
area of any peak, apart from the principal peak and the
TESTS peak due to the internal standard, to the area of the peak
Solution S due to the internal standard: this ratio is not greater than
Dissolve 2.5 g in carbon dioxide-free waterR and dilute. to R 1 (0.10 per cent);
25 mL with the same solvent. - total: calculate the ratio (R 2 ) of 3 times the area of the
Appearance of solution peak due to amantadine to the area of the peak due to the
Solution S is clear (2.2.1) and not more intensely coloured internal standard from the chromatogram obtained with
than reference solutionY, (2.2.2, Method II).

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2020 Ambroxol Hydrochloride 1-133

the reference solution; from the chromatogram obtained

with the test solution, calculate the ratio of the sum of the
Ambroxol Hydrochloride
areas of any peaks, apart from the principal peak and the (Ph. Bur. monograph 1489)
peak due to the internal standard, to the area of the peak
due to the internal standard: this ratio is not greater than
R 2 (0.3 per cent);
- disregard limit: calculate the ratio (R3) of 0.5 times the
area of the peak due to amantadine to the area of the
peak due to the internal standard from the chromatogram
obtained with the reference solution; from the
chromatogram obtained with the test solution, calculate
the ratio of the area of any peak, apart from the principal 414.6 23828-92-4
peak and the peak due to the internal standard, to the
area of the peak due to the internal standard: disregard Action and use
any peak with a ratio less than R3 (0.05 per cent). Mucolytic expectorant.
Water (2.5.12) PhEur _
Maximum 0.5 per cent, determined on 2.00 g.
DEFINITION
Sulfated ash (2.4.14)
trans-4- [(2-Amino-3,5-dibromobenzyl)amino]cyclohexanol
Maximum 0.1 pet; cent, determined on 1.0 g.
hydrochloride.
ASSAY Content
Dissolve 0.150 s in a mixture of 5.0 mL of 0.01 M 99.0 per cent to 101.0 per cent (dried substance).
hydrochloric acid ang,50 mL of ethanol (96 percent) R. Carry
out a potentiometric titration (2.2.20), using O~l M sodium CHARACTERS
hydroxide.' Read the volume added between the 2 points of Appearance
inflexion. White or yellowish, crystalline powder.
1 mL of 0.1 M sodium hydroxide is equivalent to 18.77 mg of Solubility
ClOH18CIN~ Sparingly soluble in water, soluble in methanol, practically
insoluble in methylene chloride.
IMPURITIES
Other detectable impurities (thefollowing substances would, if IDENTIFICATION
present at a sufficient level, be. detected by oneorotherof the tests Firstidentification: B, D.
in the monograph. They are limited by the general acceptance Secondidentification: A, C, D.
criterion for other/unspecified impurities and/orby the general A. Ultraviolet and visible absorption spectrophotometry
monograph Substances for pharmaceutical use (2034), It is (2.2.25).
therefore not necessary to identify these impurities for
Test solution Dissolve 20.0 mg in dilute sulfuric acid R1 and
demonstration of compliance. See also5.10. Control of impurities
dilute to 100.0 mLwith the same acid. Dilute 2.0 mL of the
In substances for pharmaceutical use) A, B.
solution to 10.0 mL with dilute sulfuric acid R1.
Spectral range. 200-350 run.
~CI Absorption maxima At 245 run and 310 run.
Absorbance ratio. A245/A310 = 3.2 to 3.4.
B. Infrared absorption spectrophotometry (2.2.24).
A. l-chlorotricyc!0[3.3.1.1 3,7]decane, Comparison ambroxol hydrochloride CRS.
C. Thin-layer chromatography (2.2.27).
Testsolution Dissolve 50 mg of the substance to be
examined in methanolR and dilute to 5 mL with the same
solvent.
Reference solution Dissolve 50 mg of ambroxol
B. N-(tricyc!0[3.3.1.1 3,7]dec-l-yl)acetamide. hydrochloride CRS in methanolR and dilute to 5 mL with the
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur same solvent.
Plate TLC silica gel F 254 plate R.
Mobile phase concentrated ammoniaR, propanol R, ethyl
acetate R, hexaneR (1:10:20:70 V/V/V/V).
Application 10 ~L.
Development Over 2/3 of the plate.
Drying In air.
Detection Examine in ultraviolet light at 254 run.
Results The principal spot in the chromatogram obtained
with the test solution is similar in position and size to the
principal spot in the chromatogram obtained with the
reference solution.
D. Dissolve 25 mg in2.5 mL of waterR, mix with 1.0 ml, of
dilute ammonia Rl and allow to stand for 5 min. Filter and

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1-136 Amidotrizoic Acid Dihydrate 2020

IMPURITIES IDENTIFICATION
Other detectable impurities (the following substances uiould; if First identification: A.
present at a sufficient leoel, be detected by one or other of the tests Second identification: B, C.
in the monograph. They are limited by the general acceptance A. Infrared absorption spectrophotometry (2.2.24).
criterion for other/unspecified impurities and/or by the general
monograph Substancesfor pharmaceutical use (2034). It is Comparison amidotrizoic acid dihydrate CRS.
therefore not necessary to identify these impurities for B. Thin-layer chromatography (2.2.27).
demonstration of compliance. See also 5.10. Controlof impurities Test solution Dissolve 25 mg of the substance to be
in substances for pharmaceuticaluse) A~ B~ C, D. examined in a 3 per cent V/V solution of ammonia R in
methanol R and dilute to 5 mL with the same solution.

V
~CH3
H.... OH and enantiomer
Reference solution Dissolve 25 mg of amidotrizoic acid
dihydrate CRS in a 3 per cent V/V solution of ammonia R in
methanolR and dilute to 5 mL with the same solution.
A. (2RS)-1-phenylpropan-2-ol, Plate TLC silicagel GF254 plate R.
Mobile phase anhydrousformic acid R, methyl ethyl ketone R,
toluene R (20:25:60 V/V/V).
Application 2 ur,
Development Over 2/3 of the plate.
B. l-phenylpropan-2-one, Drying In air until the solvents have evaporated.
Detection In ultraviolet light at 254 nm.
Results The principal spot in the chromatogram obtained
with the test solution is similar in position and size to the
principal spot in the chromatogram obtained with the
reference solution.
C. Heat 50 mg gently in a small porcelain dish over a naked
C. (2S)-2-amino-1-phenylpropan-1-one (cathinone),
flame. Violet vapour is evolved.
~CHO TESTS

o Appearance of solution
The solution is clear (2.2.1) and colourless (2.2.2,
Method 11).
D. benzaldehyde. Dissolve 1.0 gin dilute sodium hydroxide solution R and dilute
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
to 20 mL with the same solution.
Related substances
Liquid chromatography (2.2.29).
Solvent mixture Dissolve 0.250 g of sodium hydroxide Rand
Amidotrizoic Acid Dihydrate 0.860 g of sodium dihydrogen phosphateR in 50 mL of water R
and dilute to 1000 mL with the same solvent.
(Ph. Bur. monograph 0873)
Test solution Dissolve 40.0 mg of the substance to be
examined in 10.0 mL of the solvent mixture with the aid of
ultrasound.
Reference solution (a) Dilute 1.0 mL of the test solution to
100.0 mL with the solvent mixture. Dilute 1.0 mL of this
solution to 10.0 mL with the solvent mixture.
Reference solution (b) Dilute 1.0 mL of reference solution (a)
650 50978-11-5 to 10.0 mL with the solvent mixture.
Reference solution (c) Dissolve the contents of a vial of
Action and use
amidotrizoic acid for system suitabz1ity CRS (impurities A, B, C
Iodinated contrast medium.
and D) in 1.0 mL of the solvent mixture.
Preparation Column:
Meglumine Amidotrizoate Injection - size: l = 0.25 m, (2) = 4.6 mm;
PhEur _ - stationary phase: end-cappedoctadecylsilyl silica gelfor
chromatography R (5 11m).
DEFINITION
Mobile phase Dissolve 3.4 g of tetrabutylammonium hydrogen
3,5- Bis (acetylamino)-2,4,6-triiodobenzoic acid dihydrate.
sulfate R in a mixture of 230 mL of acetonitrile Rand 770 mL
Content of waterR.
98.5 per cent to 101.0 per cent (dried substance). Flow rate 1.0 mIJrnin.
CHARACTERS Detection Spectrophotometer at 236 nm.
Appearance Injection 20 ilL.
White or almost white, crystalline powder.
Run time 4 times the retention time of amidotrizoic acid.
Solubility Identification of impurities Use the chromatogram supplied
Very slightly soluble in water and in ethanol (96 per cent). with amidotrizoic acid for system suitability CRS and the
It dissolves in dilute solutions of alkali hydroxides.

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2020 Amidotrizoic Acid Dihydrate 1-137

chromatogram obtained with reference solution (c) to identify through a sintered-glass filter (2.1.2) and wash the filter with
the peaks due to impurities A, B, C and D. several quantities of water R. Collect the filtrate and
Relative retention With reference to amidotrizoic acid washings. Add 40 mL of dilute sulfuric acid R and titrate
(retention time = about 5 min): impurity B = about 0.8; immediately with 0.1 M silvernitrate. Determine the
impurity C = about 0.9; impurity A = about 1.4; end-point potentiometrically (2.2.20).
impurity D = about 1.8. 1 mL of 0.1 M silver nitrate is equivalent to 20.47 mg of
System suisability: CIIHgI3Nz04.
- resolution: minimwn 1.5 between the peaks due to STORAGE
impurities Band C in the chromatogram obtained with Protected from light.
reference solution (c);
- signal-to-noise ratio: minimwn 25 for the principal peak in IMPURITIES
the chromatogram obtained with reference solution (b). Specified impurities A, B, D.
Limits: Other detectable impurities (the following substances would, if
- impurity B: not more than the area of the principal peak in present at a sufficientlevel, be detected by one or other of the tests
the chromatogram obtained with reference solution (a) in the monograph. They are limitedby the general acceptance
(0.1 per cent); criterion for other/unspecified impurities and/orby the general
- impurities A, D: for each impurity, not more than the area monograph Substances for pharmaceutical use (2034). It is
of the principal peak in the chromatogram obtained with therefore not necessary to identify these impurities for
reference solution (b) (0.01 per cent); demonstration of compliance. See also 5.10. Control of impurities
- unspecified impurities: for each impurity, not more than in substances for pharmaceutical use) C, E.
0.5 times the area of the principal peak in the
chromatogram obtained with reference solution (a)
(0.05 per cent);
- total: not more than 1.5 times the area of the principal
peak in the chromatogram obtained with reference
solution (a) (0.15 per tent);
- disregard limit: 0.3 times the area of the principal peak in
the chromatogram obtained with reference solution (a) A~ 3-(acetylamino)-5-amino-2,4,6-triiodobenzoic acid,
(0.03 per cent), except for the peaks due to impurities A
andD.
Halides expressed as chlorides (24.4)
Maximwn 150 ppm.
Dissolve 0.55 g in a mixture of 4 mL of dilute sodium
hydroxide solution Rand 15 mL of waterR. Add 6 mL of
dilute nitric acid R and filter.
B. 3,5-bis(acetylamino)-2,4-diiodobenzoic acid,
Free aromatic amines
Maintain the solutions and reagents in icedwater, protected from
bright light To 0.50 g in a 50 mL volumetric flask add
15 mL of water R. Shake and add 1 mL of dilute sodium
hydroxide solution R. Cool in iced water, add 5 mL of a
freshly prepared 5 gIL solution of sodium nitrite R and 12 mL
of dilute hydrochloric acid R. Shake gently and allow to stand
for exactly 2 min after adding the hydrochloric acid. C. 3,5-bis(acetylamino)-2,6-diiodobenzoic acid,
Add 10 mL of a 20 gIL solution of ammoniumsulfamate R.
Allow to stand for 5 min, shaking frequently, and add
0.15 mL of a 100 gIL solution of a-naphtholR in ethanol
(96 per cent) R. Shake and allow to stand for 5 min.
Add 3.5 ml, of buffersolution pH 10.9 R, mix and dilute to
50.0 mL with water R. The absorbance (2.2.25), measured
within 20 min at 485 nm using as the compensation liquid a
solution prepared at the same time and in the same manner
D.3-(acetylamino)-5-[(iodoacetyl)amino]-2,4,6-
but omitting the substance to be examined, is not greater
triiodobenzoic acid,
than 0.30.
Loss on drying (2.2.32)
4.5 per cent to 7 ~O per cent, determined on 0.500 g by
drying in an oven at 105°C.
Sulfated ash (2.4.14)
Maximwn 0.1 per cent, determined on 1.0 g.
ASSAY
To 0.150 g in a 250 mL round-bottomed flask add 5 mLof E. 3-(acetylamino)-5-(diacetylamino)-2,4,6-triiodobenzoic
strongsodium hydroxide solution R, 20 mLof water R, 1 g of acid.
zinc powderR and a few glass beads. Boil under a reflux _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _----,._ _ PhEur
condenser for 30 min. Allow to cool and rinse the condenser
with 20 mL of waterR, adding the rinsings to the flask. Filter

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1-138 Amikacin 2020

TESTS
Amikacin pH (2.2.3)
(Ph. Bur. monograph 1289) 9.5 to 11.5.
Dissolve 0.1 g in carbon dioxide-free waterR and dilute to
HO 10 mL with the same solvent.
o Specific optical rotation (2.2.7)
NH2 0 + 97 to + 105 (anhydrous substance).
Dissolve 0.50 g in water R and dilute to 25.0 mL with the
o OH)=<' H OH
same solvent.
OH HO--
Related substances
HO \ Liquid chromatography (2.2.29).
OH . 0 NH2
Test solution Dissolve 25 mg of the substance to be
examined in mobile phase A and dilute to 50.0 mL with
585.6 37517-28-5 mobile phase A.
Reference solution (a) Dilute 1.0 mL of the test solution to
Action and use
100.0 mL with mobile phase A.
Aminoglycoside antibacterial.
Reference solution (b) Dilute 1.0 mL of reference solution (a)
PhEur -'---_ to 10.0 mL with mobile phase A.
DEFINITION Reference solution (c) Dissolve 5 mg of amikacinfor system
6-0-(3-Amino-3-deoxy-et-D-glucopyranosyl)-4-0-(6-amino-6- suitability CRS (containing impurities A, B, F and H) in
deoxy-et-n-glucopyranosyl)-l-N-[(2S)-4-amino-2- mobile phase A and dilute to 10 mL with mobile phase A.
hydroxybutanoyl] -2-deoXY-D-streptamine. Reference solution (d) Dissolve 5.0 mg of amikacin
Antimicrobial substance obtained from kanamycin A. impurity I CRS in mobile phase A and dilute to 20.0 mL with
mobile phase A. Dilute 1.0 mL of the solution to 100.0 mL
Semi-synthetic product derived from a fermentation product.
with mobile phase A.
Content
Column:
96.5 per centro 102.0 per cent (anhydrous substance).
- size: I = 0.25 m, (2) = 4.6 mrn;
CHARACTERS - stationary phase: end-capped octadecylsilyl silica gelfor
Appearance chromatography R (5 urn);
White or almost white powder. - temperature: 40 "C.
Solubility Mobile phase:
Sparingly soluble in water, slightly soluble in methanol, - mobile phaseA: a mixture prepared with carbon dioxide-free
practically insoluble in acetone and in ethanol (96 per cent). water R, containing 1.8 gIL of sodium octanesulfonate R,
20 gIL of anhydrous sodium sulfate R1, 1.4 per cent V/Vof
IDENTIFICATION
tetrahydrofuran R, and 5 per cent V/Vof 0.2 M potassium
A. Infrared absorption spectrophotometry (2.2.24). dihydrogen phosphate R previously adjustedto pH 3.0 with
Comparison amikacin CRS. dilute phosphoric acid R; degas;
B. Thin-layer chromatography (2.2.27). - mobile phase B: a mixture prepared with carbon dioxide-free
Test solution Dissolve 25 mg of the substance to be water R, containing 1.8 gIL of sodium oaanesulfonate R,
examined in water R and dilute to 10 mL with the same 28 gIL of anhydrous sodium sulfate R1, 1.4 per cent V/Vof
solvent. tetrahydrofuran R, and 5 per cent V/Vof 0.2 M potassium
Reference solution (a) Dissolve 25 mg of amikacin CRS in dihydrogen phosphate R previously adjusted to pH 3.0 with
waterR and dilute to 10 mL with the same solvent. dilute phosphoric acid R; degas;
Reference solution (b) Dissolve 5 mg of kanamycin Time Mobile phase A Mobile phase B
monosulfate CRS in 1 mL of the test solution and dilute to (min) (per cent VIP) (per cent VIP)
10 mL with water R. 0-3 100 0
Plate TLC silica gelplate R. 3 - 38.0 100 -+ 30 0-->70
Mobile phase methylene chloride R, ammonia R, methanolR 38.0 - 38.1 30 --> 0 70 --> 100
(25:30:40 V/V/V). 38.1 - 68 0 100
Application 5 ~L.
Deoelopment Over 3/4 of the plate. Flow rate 1.0 mUmin.
Drying In air. Post-column solution Mixture of 1 volume of carbonate-free
sodium hydroxide solution Rand 24 volumes of previously
Detection Spray with ninhydrin solution R1 and heat at
degassed carbon dioxide-free waterR, which is added in a
110 -c for 5 min.
pulse1ess manner to the column effluent using a 375 JlL
System suitabz7ity Reference solution (b): polymeric mixing coil.
- the chromatogram shows 2 clearly separated spots.
Flow rate of post-column solution 0.3 mUmin.
Results The principal spot in the chromatogram obtained
Detection Pulsed amperometric detector or equivalent with a
with the test solution is similar in position, colour and size to
gold indicator electrode, a silver-silver chloride reference
the principal spot in the chromatogram obtained with
electrode, and a stainless steel auxiliary electrode which is the
reference solution (a).
cell body, held at respectively + 0.05 V detection, + 0.75 V

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 2020 Amikacin 1-139

oxidation and - 0.15 V reduction potentials, with pulse in the mobile phase; peak splitting may be observed when
durations according to the instrument used. the retention time becomes too short;
Injection 20 ilL. - repeatability: maximum relative standard deviation of
Identification of impurities Use the chromatogram supplied 1.5 per cent after 6 injections.
with amikacinfor system suitability CRS and the Calculate the percentage content of C22H43Ns013 taking into
chromatogram obtained with reference solution (c) to identify account the assigned content of amikacin CRS.
the peaks due to impurities A, B, F and H; use the IMPURITIES
chromatogram obtained with reference solution (d) to Specified impurities A~ B~ F~ H~ 1.
identify the peak due to impurity 1.
Other detectable impurities (thefollowing substances toould, if
Relativeretention With reference to amikacin (retention present at a sufficient leoel, be detected by one or other of the tests
time = about 28 min): impurity I = about 0.13; in the monograph. They are limited by the general acceptance
impurity F = about 0.92; impurity B = about 0.95; criterion for other/unspecified impurities and/or by the general
impurity A = about 1.62; impurity H = about 1.95. monograph Substances for pharmaceutical use (2034). It is
System suitability Reference solution (c): therefore not necessary to identify these impurities for
- peak-to-valley ratio: minimum 5, where Hp = height above demonstration of compliance. See also 5.10. Control of impurities
the baseline of the peak due to impurity B. and in substances for pharmaceutical use) C~ D, E, G.
H; = height above the baseline of the lowest point of the
curve separating this peak from the peak due to amikacin; HO
if necessary, adjust the volume of tetrahydrofuran in the
mobile phase.
Calculation of pert;entage contents:
for impurity I; use the concentration of impurity I in
OH~
-
reference solution (d); o·
OH. HO.-- .
- for impurities other than I, use the concentration of HO v: 0
amikacin in reference solution (a). OH 0 HN-<. .
Limits:
- impurities A, B~ F~ H~ I: for each impurity, maximum
H~
OH NH2
0.5 per cent;
- any other impurity: for each impurity, maximum A. 4-0-(3-amino-3-deoxy-a.-D-glucopyranosyl)-6-0-(6-amino-
0.5 per cent; 6-deoxy-a.-D-g1ucopyranosyn-1-lV-[(2~-4-amino-2­
- total: maximum 1.5 per cent; hydroxybutanoyl] -2-deoxy-L-streptamine,
- reporting threshold: 0.1 per cent.

H2N~HO)-NH.'0
Water (2.5.12)
Maximum 8.5 per cent, determined on 0.200 g.
Sulfated ash (2.4.14)
H~
0,'.
HN
1X
r
»<;

""'-/
.NH
2
Maximum 0.5 per cent, determined on 1.0 g.
ASSAY OH
° HO--
OH~' H "OH

Liquid chromatography (2.2.29). HO -, 0

Test solution Dissolve 50.0 mg of the substance to be OH 0 HN-<
examined in the mobile phase and dilute to 10.0 m.L with
H~
OH NH2
the mobile phase.
Reference solution Dissolve 50.0 mg of amikacin CRS in the
mobile phase and dilute to 10.0 mL with the mobil~ phase. B. 4-0-(3-amino-3-deoxy-a.-D-glucopyranosyl)-6-0-(6-amino-
6-deoxy-a.-D-glucopyranosyl)-1 ,3-lV-bis [(2~-4-amino-2­
Column: hydroxybutanoyl]-2-deoxy-L-streptamine,
- size: 1= 0.25 m, 0 = 4.6 mm;
- stationary phase: end-capped octadecylsilyl silica gelfor
chromatography R (5 urn);
- temperature: 40°C. ~N~O
Mobile phase A mixture prepared with carbon dioxide-free HO H
HN
~H
waterR, containing 1.8 gIL of sodium octanesulfonate R, 2~HO0 :NH
2
20 gIL of anhydrous sodium sulfate Rl, 5.8 per cent V/Vof o OHQ
OH HO--
acetonitrile Rl, and 5 per cent V/Vof 0.2 M potassium
HO . ,
dihydrogen phosphate R previously adjusted to pH 3.0 with
OH 0 'NH2
. dilute phosphoric acid R; degas.
Flow rate 1.0 mUmin.
C. 4-0-(6-amino-6-deoxy-a.-D-g1ucopyranosyl)-6-0-[3-[[(2~­
Detection Spectrophotometer at 200 nm. 4-amino-2-hydroxybutanoy~amino]-3-deoxy-a.-D­
Injection 20!J.L. glucopyranosyl]-2-deoxy-D-streptamine,
Run time 1.3 times the retention time of amikacin.
Retention time Amikacin = about 30 min.
System suitability Reference solution:
- symmetry factor: maximum 1.5 for the peak due to
amikacin; if necessary, adjust the amount of acetonitrile Rl

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1-140 Amikacin Sulfate 2020

I. (2S)-4-amino-2-hydroxybutanoic acid.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur

Amikacin Sulfate
D. 6-0-(3-amino-3-deoxy-ex-o-glucopyranosyl)-4-0-(6-amino-
6-deoxy-a.-o-glucopyranosyl)-2-deoxy-o-streptamine Amikacin Sulphate
(kanamycin), (Ph. Bur. monograph 1290)
HO HO

H'N~~EH~. o OH
0
::.20 0
OHp
HO-·
:N.H
2

OH
HO
o
.
. OHp'
HO-·
0 HN·.
H OH
,

HO " HO '.
OH 0 NH2 OH 0 NHz

E. 4-0-(3-amino-3-deoxy-ex-o-glucopyranosyl)-6-0-[6-[[ (2S)- 782 39831-55-5

4-amino~2-hydroxybutanoyl] amino] -6-deoxy-a.-o-
glucopyranosyl] -2-deoxy-L-streptamine, Action and use
Aminoglycoside antibacterial.
HO

~~~.~H~2 ~N~ 2
Preparation
Amikacin Injection
PhEur -'--
0 0 .. '. NH
o 0 OHp' H OH
DEFINITION
OH HO·· 6-0-(3-Arnino-3-deoxy-a.-o-glucopyranosyl)-4-0-(6-amino-6-
HO '. deoxy-a.-o-glucopyranosyl)-l-N- [(2S)-4-anaino-2-
OH 0 NH2 hydroxybutanoyl]-2-deoxy-o-streptamine sulfate.
Antimicrobial substance obtained from.kanamycin A.
F. 6-0-(3-amino-3-deoxy-a.-o-glucopyranosyl)-4-G- [6-[ (2S)-
Semi-synthetic product derived from a fermentation product.
4-amino-2-hydroxybutanoyl]amino-6-deoxy-a.-o-
glucopyranosyl] -l-N-[(2S)-4-amino-2-hydroxybutanoyl]-2- Content
deoxy-n-streptamine, 96.5 per cent to 102.0 per cent (dried substance).
CHARACTERS
Appearance
White or almost white powder.
Solubility
Freely soluble in water, practically insoluble in acetone and
in ethanol (96 per cent).
IDENTIFICATION
A. Infrared absorption spectrophotometry (2.2.24).
Comparison amikacin sulfate CRS.
Ci. 6-0-(3-amino-3-deoxy-ex-o-glucopyranosyl)-4-G-(6-amino- B. Thin-layer chromatography (2.2.27).
6-deoxy-a.-o-glucopyranosyl)-I-N- [(2R)-4-amino-2- Test solution Dissolve 25 nag of the substance to be
hydroxybutanoyl]-2-deoxy-o-streptamine, examined in water Rand dilute to 10 ml, with the sanae
solvent.
HO
Reference solution (a) Dissolve 25 nag of amikacin
sulfate CRS in water R and dilute to 10 ml, with the sanae
solvent.
HO 0 HN Reference solution (b) Dissolve 5 nag of kanamycin
o OHp" H 'OH monosulfate CRS in 1 ml, of the test solution and dilute to
OH HO--
10 ml, with water R.
HO .. '.
Plate TLC silica gel plate R.
NH2 0 NH2
Mobile phase methylene chloride R, ammonia R, methanol R
(25:30:40 V/VIV).
H. 6-0-(3-amino-3-deoxy-ex-o-glucopyranosyl)-1-N- [(2S)-4-
amino-2-hydroxybutanoyij-4-0-(2,6-dianaUno-2,6-dideoxy- Application 5 JlL.
a.-o-glucopyranosyl)-2-deoxy-o-streptamine, Development Over 3/4 of the plate.

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2020 Amikacin Sulfate 1-141

Drying In air. Post-eolumn solution Mixture of 1 volume of carbonate-free

Detection Spray with ninhydrin solution Rl and heat at sodium hydroxide solution Rand 24 volumes of previously
110°C for 5 min. degassed carbon dioxide-free water R, which is added in a
pulseless manner to the column effluent using a 375 IlL
System suitability Reference solution (b):
polymeric mixing coil.
- the chromatogram shows 2 clearly separated spots.
Flow rate of post-column solution 0.3 mUmin.
Results The principal spot in the chromatogram obtained
with the test solution is similar in position, colour and size to Detection Pulsed amperometric detector or equivalent with a
the principal spot in the chromatogram obtained with gold indicator electrode, a silver-silver chloride reference
reference solution (a). electrode, and a stainless steel auxiliary electrode which is the
cell body, held at respectively + 0.05 V detection, + 0.75 V
C. It gives reaction (a) of sulfates (2.3.1).
oxidation and - 0.15 V reduction potentials, with pulse
TESTS durations according to the instrument used.
pH (2.2.3) Injection 20 .IlL.
2.0 to 4.0.
Identification of impurities Use the chromatogram supplied
Dissolve 0.1 g in carbon dioxide-free water R and dilute to with amikacinfor system suitability CRS and the
10 mL with the same solvent. chromatogram obtained with reference solution (c) to identify
Specific optical rotation (2.2.7) the peaks due to impurities A, B, F and H; use the
+ 76 to + 84 (dried substance). chromatogram obtained with reference solution (d) to
Dissolve 0.50 g in waterR and dilute to 25.0 mL with the identify the peak due to impurity 1.
same solvent. Relative retention With reference to amikacin (retention
Related substances time = about 28 min): impurity I = about 0.13;
Liquid chromatography (2.2.29). = =
impurity F about 0.92; impurity B about 0.95;
impurity A = about 1.62; impurity H = about 1.95.
Test solution Dissolve 33 mg of the substance to be
examined in mobile phase A and dilute to 50.0 mL with System suitabilit» Reference solution (c):
mobile phase A. =
- peak-to-ualley ratio: minimum 5, where Hp height above
the baseline of the peak due to impurity Band
Reference solution (a) Dilute 1.0 mL of the test solution to
H; = height above the baseline of the lowest point of the
100.0 mL with mobile phase A.
curve separating this peak from the peak due to amikacin;
Reference solution (b) Dilute 1.0 mL of reference solution (a) if necessary, .adjust the volume of tetrahydrofuran in the
to 10.0 mL with mobile phase A. mobile phase.
Reference solution (c) Dissolve 5 mg of amikacinfor system Calculation of percentage contents:
suitability CRS (containing impurities A, B, F and H) in - for impurity I, use the concentration of impurity I in
mobile phase A and dilute to 10 mL with mobile phase A. reference solution (d);
Reference solution (d) Dissolve 6.6 mg of amikacin - for impurities other than I, use the concentration of
impurity I.CRS in mobile phase A and dilute to 20.0 mL with amikacin sulfate in reference solution (a).
mobile phase A. Dilute 1.0 mL of the solution to 100.0 mL Limits:
with mobile phase A. - impurities A, B, F, H, I: for each impurity, maximum
Column: 0.5 per cent;
~ size: 1= 0.25 m, 0 = 4.6 mm; - ~any otherimpurity: for each impurity, maximum
- stationary phase: end-capped octadecylsilyl silica gelfor 0.5 per cent;
chromatography R (5 urn); - total: maximum 1.5 per cent;
- temperature: 40°C. - reporting threshold: 0.1 per cent.
Mobilephase: Sulfate
- mobile phaseA: a mixture prepared with carbon dioxide-free 23.3 per cent to 25.8 per cent (dried substance).
water R, containing 1.8 gIL of sodium octanesulfonate R, Dissolve 0.250 g in 100 mL of waterR and adjust the
20 gIL of anhydrous sodium sulfate Rl, 1.4 per cent V/Vof solution to pH 11 using concentrated ammonia R.
tetrahydrofuran R, and 5 per cent V/Vof 0.2 M potassium Add 10.0 mL of 0.1 M barium chloride and about 0.5 mg of
dihydrogen phosphate R previously adjusted to pH 3.0 with phthalein purple R. Titrate with 0.1 M sodium edetate adding
dilute phosphoric acidR; degas; 50 mL of ethanol (96 per cent) R when the colour of the
- mobile phaseB: a mixture prepared with carbon dioxide-free solution begins to change and continue the titration until the
water R, containing 1.8 gIL of sodium octanesulfonate R, violet-blue colour disappears.
28 gIL of anhydrous sodium sulfate Rl, 1.4 per cent V/Vof
1 mL of 0.1 M barium chloride is equivalent to 9.606 mg of
tetrahydrofuran R, and 5 per cent V/Vof 0.2 M potassium
dihydrogen phosphate R previously adjusted to pH 3.0 with sulfate (S04)'
dilute phosphoric acidR; degas; Loss on drying (2.2.32)
Maximum 13.0 per cent, determined on 0.500 g by drying in
Time Mobile phase A Mobile phase B an oven at 105°C at a pressure not exceeding 0.7 kPa for
(min) (per cent VIJI) (per cent VIJI) 3 h.
0-3 100 o Pyrogens (2.6.8)
3 - 38.0 100 -+- 30 0->70 If intended for use in the manufacture of parenteral
38.0 - 38.1 30 -> 0 70 -> 100 preparations without a further appropriate procedure for the
38.1 - 68 o 100 removal of pyrogens,it complies with the test for pyrogens.
Inject per kilogram of the rabbit's mass 5 mL of a solution
Flow rate 1.0 mIJmin. containing 25 mg of the substance to be examined in water
for injections R.

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1-121:2 Amikacin Sulfate 2020

HO
ASSAY
Liquid chromatography (2.2.29).
Test solution Dissolve 50.0 mg of the substance to be
examined in the mobile phase and dilute to 10.0 mL with HO 0 HN ,
the mobile phase. o OHH,' H OH
Reference solution Dissolve 37.4 mg of amikacin CRS in the OH HO--
mobile phase and dilute to 10.0 mL with the mobile phase. HO " 0
Column:
. OH a HN---<

- size: 1 = 0.25 m, 0 = 4.6 IDID; H~

OH NH2
- stationaryphase: end-capped oetadecylsilyl silica gelfor
chromatography R (5 urn); B. 4-0-(3-amino-3-deoxy-cx-D-glucopyranosyl)-6-0-(6-amino-
- temperature: 40°C. 6-deoxy-cx-D-glucopyranosyl)-1,3-N-bis [(2S)-4-amino-2-
Mobile phase Mixture containing 1.8 gIL of sodium hydroxybutanoyl] -2-deoXY-L-streptamine,
octanesulfonate R, 20 gIL of anhydrous sodium sulfate Rl, .
5.8 per cent V/V of acetonitrile Rl, and 5 per cent V/Vof
0.2 M potassium dihydrogen phosphate R previously adjusted to
/pH 3.0 with dilute phosphoric acid R; degas.
Flow rate 1.0 mIJmin.
Detection Spectrophotometer at 200 nm.
Injection 20 ~L
Run time 1.3 times the retention time of amikacin.
Retention time Amikacin = about 30 min.
C. 4-0-(6-amino-6-deoxy-cx-D-glucopyranosyl)-6-0-[3-[[(2S)-
System suitabz7ity Reference solution:
- symmetryfactor: maximum 1.5 for the peak due to 4-amino-2-hydroxybutanoyl] amino]- 3-deoxy-cx-D-
amikacin; if necessary, adjust the amount of acetonitrile Rl glucopyranosyl]-2-deoxy-D-streptamine,
in the mobile phase; peak splitting may be observed when
the retention time becomes too short;
- repeatability: maximum relative standard deviation of
1.5 percent after 6 injections.
Calculate the percentage content of C22H47Ns02182 taking
into account the assigned content of amikacin CRS and a
correction factor of 1.335.
STORAGE
In an airtight container. If the substance is sterile, the
container is also sterile and tamper-proof. D. 6-0-(3-amino-3-deoxy-cx-D-glucopyranosyl)-4-0-(6-amino-
IMPURITIES 6-deoxy-cx-D-glucopyranosyl)-2-deoXY-D-streptamine
Specifiedimpurities A, B, F, H, 1. (kanamycin) ,
Other detectable impurities (thefollowing substances would, if

H~~~~~
present at a sufficientlevel, be detected by one or otherof the tests
in the monograph. They are limitedby the general acceptance
criterion for other/unspecified impurities and/or by the general
monograph Substances for pharmaceutical use (2034). It is "NH,
therefore not necessary to identify these impuritiesfor o 0
OH'
'OHQ
HO--
demonstration of compliance. See also 5.10. Controlof impurities
in substances for pharmaceutical use) C, D, E, G. HO OH
''NH
0 2

H'N~:~,N,H'
E. 4-0-(3-amino-3-deoXY-CX-D-glucopyranosyl)-6-0-[6-[[(2S)-
4-amino-2-hydroxybutanoyl] amino] -6-deoxy-cx-D-
glucopyranosyl]-2-deoXY-L-streptamine,
° 0,.• ,NH, HO

H'N~~~~~NH'
OH o OHH
HO--
HO -, 0
OH 0 HN--( ,,'
H~
OH NH 2

A. 4-0-(3-amino-3-deoxy-cx-D-glucopyranosyl)-6-0-(6-amino- OH 0 NH2
6-deoxy-cx-D-glucopyranosyD-1-lV-[(2S)-4-amino-2-
hydroxybutanoyl] -2-deoxy-L-streptamine, F. '6-0-(3-amino-3-deoxy-cx-D-glucopyranosyl)-4-0-[6- [(2S)-
4-amino-2-hydroxybutanoyl]amino-6-deoxy-cx-D-
glucopyranosyn-1-lV-[(2S)-4-amino-2-hydroxybutanoyn-2-
deoxy-n-streptamine,

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2020 Amiloride Hydrochloride Dihydrate 1-143

HO
Solubility
Slightly soluble in water and in anhydrous ethanol, practically
insoluble in heptane.
HO 0 HN . IDENTIFICATION
o O.Hp: H' OH First identification: A} C} D.
OH HO--
Second identification: B} C} D.
HO \
OH 0 NH2
A. Infrared absorption spectrophotometry (2.2.24).
Comparison amiloride hydrochloride CRS.
G.6-0-(3-amino-3-deoxy-a.-o-glucopyranosyl)-4-0-(6-amino- B. Thin-layer chromatography (2.2.27).
6-deoxy-ex-o-glucopyranosyl)-1-N-[(2R)-4-amino-2- Test solution Dissolve 5 mg ofthe substance to be examined
hydroxybutanoyl] -2-deoxy-o-streptamine, in methanol R and dilute to 10 mL with the same solvent.
HO
Reference solution .Dissolve 5 mg of amiloride
hydrochloride CRS in methanol R and dilute to 10 mL with the
same solvent.
Plate TLC silica gel F254 plate R.
HO 0 HN Mobz7e phase concentrated ammonia R, propanol R
OH O. O H..p
HO-- ' H 'em (30:70 VIV).
Application 5 ilL; the volume may be adapted according to
HO \
the type of plate used.
NH2 0 NH2
Development Over 2/3 of the plate.
.H~ 6-0-(3-amin9-:-3-deoxy-ex-o-glucopyranosyl)-1:-N-[(2S)-4- Drying In air.
amino-2-hydroxybutanoyl]-4-0-(2,6-diamino-2,6-dideoxy- Detection Examine in ultraviolet light at 254 nm.
ex-o-glucopyranosyl)-2-deoxy-o-streptamine, Results' The principal spot in the chromatogram obtained
with the test solution is similar in position and size to the
principal spot in the chromatogram obtained with the
reference solution.
e. Dissolve 25 mg of the substance to be examined in
I. (2S)-4-amino-2-hydroxybutanoic acid. waterR and dilute to 10 mL with the same solvent. 2 mL of
_..:...- PhEur the solution gives reaction (a) of chlorides (2.3.1);acidify
with 5 mL of dilute acetic acid R, instead of dilute nitric acidR.
D. Water (see Tests).
TESTS
Amiloride Hydrochloride Dihydrate Free acid
Dissolve 1.0 gin a mixture of 50 mL of methanol Rand
(Ph. Bur. monograph 0651) 50 mL of waterR and titrate with 0.1 M sodium hydroxide,
determining the end-point potentiometrically (2.2.20).
Not more than 0.3 mL of 0.1 M sodium hydroxide is required
to reach the end-point.
Related substances
Liquid chromatography (2.2.29).
Solution A Dissolve 2.76 g of sodium dihydrogen phosphate
302.1 17440-83-4 monohydrate R in 850 mL of waterfor chromatography R,
adjust to pH 3.0 with phosphoric acid R and dilute to 1.0 L
Action and use with water for chromatography R.
Sodium channel blocker; potassium-sparing diuretic.
Test solution Dissolve 20 mg of the substance to be
Preparations examined in 1 mL of methanol Rl and dilute to 10.0 mL
Amiloride Tablets with solution A.
Co-amilofruse Tablets Reference solution (a) Dissolve 2 mg of amiloride
Co-amilozide Oral Solution impurity A CRS in 0.5 mL of methanolRl, add 0.5 mL of the
Co-amilozide Tablets test solution and dilute to 10.0 mL with solution A.
PhEur _
Reference solution (b) Dissolve 4 mg of amiloride for peak
identification CRS (containing. impurity C) in 0.5 mL of
DEFINITION methanol Rl and dilute to 2.0 mL with solution A.
3,5-Diamino-6-chloro-N-(diaminomethylidene)pyrazine- 2- Reference solution (c) Dilute 1.0 mL of the test solution to
carboxamide hydrochloride dihydrate, 100.0 mL with solution A. Dilute ·1.0 mL of this solution to
Content 10.0 mL with solution A.
98.0 per cent to 101.0 per cent (anhydrous substance). Column:
--,---- size: 1 = 0.125 m, (2) = 4.0 mm;
CHARACTERS
- stationary phase: base-deactivated end-capped octylsilyl silica
Appearance
gelfor chromatography R (5 11m);
Pale yellow or greenish-yellow powder.
- temperature: 30 "C.

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1-144 Aminobenzoic Acid 2020

Mobz7e phase Dissolve 0.8 g of sodium hexanesulfonate

monohydrate R in a mixture of 80 mL of acetonitrile Rl and
920 mL of solution A.
Flow rate 1.5 mUmin.
B. 3,5-diamino-6-chloropyrazine-2-carboxylic acid,
Detection Spectrophotometer at 210 nrn.
Injection 20 ilL.
Run time Twice the retention time of.amiloride.
Identification of impurities Use the chromatogram obtained
with reference solution (a) to identify the peak due to
impurity A; use the chromatogram supplied with amiloride for
C. 3-amino-6-chloro-N-(diaminomethylidene)-5-
peak identification CRS and the chromatogram obtained with
hydroxypyrazine-2-carboxamide.
reference solution (b) to identify the peak due to impurity C.
_ _ _ _ _ _ _ _.,- PhEur
Relative retention With reference to amiloride (retention
time = about 10 min): impurity C = about 0.5;
=
impurity A about 0.8.
System suitability Reference solution (a):
- resolution: minimum 3.0 between the peaks due to Aminobenzoic Acid
impurity A and amiloride.
(4-Aminobenzoic Acid, Ph. Bur. monograph 1687)
Calculation of percentage contents:
--: for each impurity, use the concentration of amiloride
hydrochloride dihydrate in reference solution(c).
Limits:
- impurity C: maximum 0.2 per cent;
- unspecified impurities: for each impurity, maximum 137.1 150-13-0
0.10 per cent;
- total: maximum 0.4 per cent; Action and use
- reporting threshold: 0.05 per cent. Skin protective.
Water (2.5.12) PhEur .,- _
11.0 per cent to 13.0 per cent, determined on 0.200 g.
DEFINITION
Sulfated ash (2.4.14) 4-Aminobenzoic acid.
Maximum 0.1 per cent, determined on 1.0 g.
Content
ASSAY 99.0 per cent to 101.0 per cent (anhydrous substance).
Dissolve 0.200 g in a mixture of 5.0 mL of 0.01 M
CHARACTERS
hydrochloric acid and 50 mL of ethanol (96 per cent) R. Carry
out a potentiometric titration (2.2.20), using 0.1 M sodium Appearance
hydroxide. Read the volume added between the 2 points of White or slightly yellow, crystalline powder.
inflexion. Solubility
1mL of 0.1 M sodium hydroxide is equivalent to 26.61 mg Slightly soluble in water, freely soluble in alcohol. It dissolves
of C 6HgCI2N70. in dilute solutions of alkali hydroxides.

STORAGE IDENTIFICATION
Protected from light. First identification: B.
Second identification: A~ C.
IMPURITIES
Specified impurities C. A. Melting point (2.2.14): 186°C to 189 °C.
Other detectable impurities (thefollowing substances uiould, if B. Infrared absorption spectrophotometry (2.2.24).
present at a sufficient leoel, be detected by one or otherof the tests Comparison 4-aminobenzoic acid CRS.
in the monograph. They arelimitedby the general acceptance C. Thin-layer chromatography (2.2.27).
criterion for other/unspecified impurities and/or by the general Test solution Dissolve 20 mg of the substance to be
monograph Substances for pharmaceutical use (2034). It is examined in methanolR and dilute to 20 mL with the same
therefore not necessary to identify these impurities for solvent.
demonstration of compliance. See also 5.10. Controlof impurities
Reference solution (a) Dissolve 20 mg of 4-aminobenzoic
in substances for pharmaceutical use) A~ B.
acid CRS in methanolR and dilute to 20 mL with the same
solvent.
Reference solution (b) Dissolve 10 mg of 4-nitrobenzoic acid R
in 10 mL of reference solution (a).
Plate Suitable silica gel with a fluorescent indicator having
an optimal intensity at 254 nm as the coating substance.
A. methyl 3,5-diamino-6-chloropyrazine-2-carboxylate, Mobile phase glacial acetic acid R, hexane R, methylene
chloride R (5:20:75 V/V/V).
Application 1 ilL.
Development Over a path of 10 em.
Drying In air.

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2020 Aminobenzoic Acid 1-145

Detection Examine in ultraviolet light at 254 run. Test solution Dissolve 1.000 g of the substance to be
System suitabzlity The chromatogram obtained with examined in 10.0 mL of an 84 gIL solution of sodium
reference solution (b) shows 2 clearly separated spots, hydroxideR and extract with 2 quantities, each of 10 mL, of
Results The principal spot in the chroma.togram obtained methylenechloride R. Combine and wash with 5 mL of
with the test solution is similar in position and size to the water R; filter through anhydrous sodium sulfate R. Wash the
principal spot in the chromatogram obtained with reference filter with methylene chloride R. Evaporate in a water-bath at
50-60 DC to obtain a volume of about 1-5 ml., Add 1.0 mL
solution (a).
of the internal standard solution and dilute to 10.0 ml, with
TESTS methylene chloride R.
Appearance of solution Reference solution (a) Dissolve 20.0 mg of aniline R in
The solution is clear (2.2.1) and not more intensely coloured methylenechloride R and dilute to 100.0 ml, with the same
than reference solution Bs (2.2.2~ Method II). solvent.
Dissolve 1.0 g in alcohol R and dilute to 20 ml, with the Reference solution (b) Dissolve 20.0 mg ofp-toluidine R in
same solvent. methylene chloride R and dilute to 100.0 ml, with the same
Related substances solvent.
Liquid chromatography (2.2.29). Reference solution (c) Dilute 0.50 mL of reference
Test solution Dissolve 25.0 mg of the substance to be solution (a), 0.50 mL of reference solution (b) and 10.0 mL
examined in the mobile phase and dilute to 100.0 mL with of the internal standard solution to 100.0 mL with methylene
the mobile.phase: chloride R.
Reference solution Dissolve 25.0 mg of 4-nitrobenzoic acid R Column:
and 25.0 mgofb~nzocaine R in methanolR and dilute to - material: fused silica,
100.0 niL with the same solvent. Dilute 1.0 ml, to 50.0 ml, -'- size: I = 30 m, (2) = 0.32 mm,
with the mobile phase. Dilute 1.0 ml, of this solution to - stationaryphase: poly[methyl(95)phenyl(5)}siloxane R (film
10.0 mL with the mobile phase. thickness 0.5 J.U11).
Column: Carriergas helium for chromatography R.
- size: 1= 0.12 m, (2) = 4.0 mm, Flow rate 1.0 mUmin.
- stationaryphase: octylsilyl silica gelfor chromatography R Split ratio 1:10.
(5 1JlIl).
Temperature:
Mobile phase Mix 20· volumes of a mixture of 70 volumes of
acetonitrile Rand 80 volumes of methanolR, and 80 volumes Time Temperature
of a solution containing 1.5 gIL of potassium dihydrogen (min) ("C)
phosphate Rand 2.5 gIL of sodium octanesulfonate Radjusted Column 0-4 130
to pH 2.2 with phosphoric acid R. 4-6.5 130 -> 180
Flow rate 1.0 mUmin. 6.5 - 11.5 180
Detection Spectrophotometer at 270 nm. Injection port 280
Injection 20~. Detector 300
Run time 11 times the retention time of 4-aminobenzoic
acid. Detection Flame ionisation.
Relative retention With reference to 4-aminobenzoic acid Injection 2 ilL; inject the test solution and reference
(retention time = about 3 min): impurity A = about 4; solution (c).
impurity B = about 9. Retention time Internal standard = about 9.5 min.
Limits: Limits:
- impurity A: not more than the area of the corresponding - impurity C: calculate the ratio (R) of the area of the peak
peak in the chromatogram obtained with the reference due to impurity C to the area of the peak due to the
solution (0.2 per cent), internal standard from the chromatogram obtained with
- impurity B: not more than the area of the corresponding reference solution (c); calculate the ratio of the area of the
peak in the chromatogram obtained with the reference peak due to impurity C to the area of the peak due to the
solution (0.2 per cent), internal standard from the chromatogram obtained with
- any otherimpurity: not more than 0.5 times the area of the the test solution: this ratio isnot greater than R (10 ppm),
peak due to impurity A in the chromatogram obtained - impurity D: calculate the ratio (R) of the area of the peak
with the reference solution (0.1 per cent), due to impurity D to the area of the peak due to the
- total: not more than 2.5 times the area of the peak due to internal standard from the chromatogram obtained with
impurity A in the chromatogram obtained with the reference solution (c); calculate the ratio of the area of the
reference solution (0~5 per cent), peak due to impurity D to the area of the peak due to the
- disregard limit: 0.1 times the area of the peak due to internal standard from the chromatogram obtained with
impurity A in the chromatogram obtained with the the test solution: this ratio is not greater than R (10 ppm).
reference solution (0.02 per cent). Iron (2.4.9)
Impurity C and impurity D Maximum 40 ppm.
Gas chromatography (2.2.28). Dissolve 0.250 gin 3 mL of alcohol R and dilute to 10.0 mL
Internalstandard solution Dissolve 20.0 mg of lauric acid R in with water R.
methylenechloride R and dilute to 100.0 mL with the same Water (2.5.12)
solvent. Maximum 0.2 per cent, determined on 1.00 g.

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1-146 Aminocaproic Acid 2020

Sulfated ash (2.4.14) A. Examine by infrared absorption spectrophotometry -

Maximum 0.1 percent, determined on 1.0 g. (2.2.24), comparing with the spectrum obtained with
aminocaproic acid CRS. Examine the substances prepared as
ASSAY
discs.
Dissolve 0.100 g with heating in 50 mL of carbon dioxide-free
water R. Titrate with 0.1 M sodium hydroxide determining the B. Examine the chromatograms obtained in the test for
end-point potentiometrically (2.2.20). ninhydrin-positive substances. The principal spot in the
chromatogram obtained with the test solution (b) is similar in
1 mL of 0.1 M sodium hydroxide is equivalent to 13.71 mg of
position, colour and size to the principal spot in the
C 7H7NO z.
chromatogram obtained with reference solution (a).
STORAGE C. Dissolve 0.5 gin 4 mL of a mixture of equal volumes of
Protected from light. dilute hydrochloric acid R and waterR. Evaporate to dryness by
IMPURITIES heating on a water-bath. Dry the residue in a desiccator.
Dissolve the residue in about 2 mL of boiling ethanol R.
Allow to cool and maintain at 4 °C to 8 °C for 3 h. Filter
under reduced pressure. The residue washed with about
10 mL of acetone R and dried at 60°C for 30 min, melts
(2.2.14) at 131°C to 133 DC.
A. 4-nitrobenzoic acid, D. Dissolve about 5 mg in 0.5 mL of distilled water R.
Add 3 mL of dimethylformamide Rand 2 mL of ascorbic acid
solution R. Heat on a water-bath. An orange colour develops.
TESTS
Solution S
Dissolve 10.0 s in carbon dioxide-free water R and dilute to
B. ethyl 4-aminobenzoate (benzocaine), 50.0 mL with the same solvent.
Appearance of solution
Solution S is colourless (2.2.2, Method II) and remains clear
(2.2.1) on standing for 24 h.
pH (2.2.3)
The pH of solution S is 7.5 to 8.0.
C. aniline,
Absorbance (2.2.25)
A. The absorbance of solution S at 287 nm is not more
than 0.10 and at 450 nm is not more than 0.03.
B. Place 2.0 g in an even layer in a shallow dish 9 em in
diameter, cover and allow to stand at 98°C to 102°C for
D. 4-methylaniline (p-toluidine). 72 h. Dissolve in waterR and dilute to 10.0 mL with the
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur same solvent. The absorbance of the solution at 287 nm is
not more than 0.15 and at 450 nm is not more than 0.03.
Ninhydrin-positive substances
Examine by thin-layer chromatography (2.2.27), using a
Aminocaproic Acid suitable silica gel as the coating substance.
Test solution (a) Dissolve 0.10 g of the substance to be
(Ph. Bur. monograph 0874) examined in water R and dilute to 10 mL with the same
solvent.
Test solution (b) Dilute 1 mL of test solution (a) to 50 mL
with water R.
131.2 60-32-2
Reference solution (a) Dissolve 10 mg of aminocaproic
acid CRS in water R and dilute to 50 mL with the same
Action and use
solvent.
Antifibrinolytic.
Reference solution (b) Dilute 5mL of test solution (b) to
PhEur _
~

20 mL with water R.
DEFINITION Reference solution (c) Dissolve 10 mg of aminocaproic
Aminocaproic acid contains not less than 98.5 per cent and acid CRS and 10 mg of leucine CRS in water R and dilute to
not more than the equivalent of 101.0 per cent of 25 mL with the same solvent.
6-aminohexanoic acid, calculated with reference to the dried Apply separately to the plate 5 j.LL of each solution. Allow the
substance. plate to dry in air. Develop over a path of 15 em using' a
mixture of 20' volumes of glacial acetic acid R, 20 volumes of
CHARACTERS
water Rand 60 volumes of butanolR. Dry the plate in a
A white or almost white, crystalline powder or colourless
current of warm air. Spray with ninhydrin solution R and heat
crystals, freely soluble in water, slightly soluble in alcohol.
at 100°C to 105 DC for 15 min. Any spot in the
It melts at about 205°C with decomposition. chromatogram obtained with the test solution (a), apart from
IDENTIFICATION ' the principal spot, is not more intense than the spot in the
First identification: A. chromatogram obtained with reference solution (b)
Second identification: B, C, D. (0.5 per cent). The test is not valid unless the chromatogram

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 2020 Aminoglutethimide 1-147

obtained with reference solution (c) shows two clearly Mobile phase glacial acetic acidR, methanolR, ethyl- acetate R
separated principal spots. (0.5:15:85 VIVIV).
Loss on drying (2.2.32) Application· 5 jlL.
Not more than 0.5 per cent, determined on 1.000 gby Development Over 3/4 of the plate.
drying in an oven at 105 "C. Drying In air.
Sulfated ash (2.4.14) Detection Examine in ultraviolet light at 254 nm.
Not more than 0.1 per cent, determined on 1.0 g. System suitabz7ity Reference solution (b):
ASSAY - the chromatogram shows 2 clearly separed spots.
Dissolve 0.100 gin 20 mL of anhydrous acetic acid R. Using Results The principal spot in the chromatogram obtained
0.1 mL of crystal violetsolution R as indicator, titrate with with the test solution is similar in position and size to the
0.1 M perchloric acid until the colour changes from bluish- principal spot in the chromatogram obtained with reference
violet to bluish-green. solution (a).
1 mL of 0.1 M perchloric acid is equivalent to 13.12 mg of TESTS
C 6H13 N O z. Solution S
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur Dissolve 1.0 g in methanolR and dilute to 20.0 mL with the
same solvent.
Appearance of solution
Solution S is clear (2.2.1) and not more intensely coloured
Aniinoglutethimide than reference solution Y7 (2.2.2, Method II).

(Ph. Bur. monograph 1291) Optical rotation (2.2.7)

-0.100 to+ 0.10 0 , determined on solution S.
Related substances
Liquid chromatography (2.2.29).
and enantiomer
Solvent mixture methanolR, acetate buffersolution pH 5.0 R
(50:50 VITI);
Test solution Dissolve 0.100 g of the substance to be
examined in the solvent mixture and dilute to 50.0 mL with
232.3 125-84-8 the solvent mixture.
Action and use Reference solution (a) Dissolve 5.0 mg of aminoglutethimide
Inhibitor of adrenal corticosteroid synthesis; used in chemical impurity A CRS in the solvent mixture and dilute to 25.0 mL
adrenalectomy. with the solvent mixture.
Reference solution (b) Dilute 1.0 mL of reference solution (a)
PhEur ~ _
to 10.0 mL with the solvent mixture.
DEFINITION Reference solution (c) Dilute 1.0 mL of the test solution to
(3RS)-3-(4-Aminophenyl)-3-ethylpiperidine-2,6-dione. 100.0 mL with the solvent mixture.
. Content Reference solution (d) Dilute 1.0 mL of the test solution to
98.0 per cent to 101.5 per cent (dried substance). 10.0 mL with reference solution (a).
CHARACTERS Column:
- size: 1= 0.15 m, 0 = 3.9 mm;
Appearance
White or slightly yellow, crystalline powder. - stationary phase: octadecylsilyl silica gelfor chromatography R
(4 urn);
Solubility - temperature: 40 "C.
Practically insoluble in water, freely soluble in acetone,
Mobilephase Mix 27 volumes of methanol Rand 73 volumes
soluble in methanol.
of acetate buffersolution pH 5.0 R.
IDENI1FICATION Flow rate 1.3 mUmin.
First identification: B. Detection Spectrophotometer at 240 nrn.
Second identification: A, C. Injection 10 ul, of the test solution and reference
A. Melting point (2.2.14): 150°C to 154 -c. solutions (b), (c) and (d).
B. Infrared absorption spectrophotometry (2.2.24). Run time 4 times the retention time of amino glutethimide.
Comparison aminoglutethimide CRS. Identification of impurities Use the chromatogram obtained
C. Thin-layer chromatography (2.2.27). with reference solution (b) to identify the peak due to
Test solution Dissolve 25 mg of the substance to be impurity A.
examined in acetone R and dilute to 5 mL with the same Relative retention With reference to amino glutethimide
solvent. (retention time = about 9 min): impurity A = about 1.3.
Reference solution (a) Dissolve 25 mg of System suitability Reference solution (d):
aminoglutethimide CRS in acetone R and dilute to 5 mL with - resolution: minimum 2.0 between the peaks due to
the same solvent. aminoglutethimide and impurity A.
Reference solution (b) Dissolve 25 mg of Limits:
aminoglutethimide CRS and 25 mg of glutethimide CRS in - impUrity A: not more than twice the 'area of the principal
acetone R and dilute to 5 mL with the same solvent. peak in the chromatogram obtained with reference
Plate TLC silica gelF254 plate R. solution (b) (2.0 per cent);

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1-148 Aminophylline 2020

- unspecified impurities: for each impurity, not more than in the monograph. They arelimited by the general acceptance
0.1 times the area of the principal peak in the criterion for other/unspecified impurities and/or by the general
chromatogram obtained with reference solution (c) monograph Substances for pharmaceutical use (2034). It is
(0.10 per cent); therefore not necessary to identify these impurities for
- sum of impurities otherthan A: not more than the area of demonstration of compliance. See also 5.10. Control of impurities
the principal peak in the chromatogram obtained with in substances for pharmaceutical use) B, C.
reference solution (c) (1.0 per cent);
- total: maximum 2.0 per cent for the sum of the contents
of all impurities;
- disregard limit: 0.05 times the area of the principal peak in and enantiomer
the chromatogram obtained with reference solution (c)
(0.05 per cent).
ImpurityD
Liquid chromatography (2.2.29). Carry out the testprotected A. (3RS)-3-(3-aminophenyl)-3-ethylpiperidine-2,6-dione
from light. Use shaking, not sonication or heat, to dissolve the (3-aminoglutethimide),
reference substance and the substance to be examined.
Test solution Dissolve 0.100 g of the substance to be o ~ 0 N02
examined in dimethyl sulfoxide R and dilute to 100.0 mL with 'I" '(~K and enanfiomer
the same solvent.
Reference solution Dissolve 3.0 mg of aminoglutethimide ~ H3C
impurity D CRS in dimethylsulfoxide R and dilute to
100.0 mL with the same solvent. Dilute 1.0 mL of this
B. (3RS)- 3-ethyl- 3-(3-nitrophenyl)pip~ridine- 2,6-dione,
solution to 100.0 mL with dimethylsulfoxide R.
Column: H
- size: l = 0.12 m, 0 = 4 mm; o N 0

- stationaryphase: octadecylsilyl silica gelfor chromatography R 'I" '(~r> NO, and enantiorner
(5 urn).
Mobile phase Dissolve 0.285 g of sodium edetate R in
~
H3C
water R, add 7.5 mL of dilute acetic acid Rand 50 mL of
0.1 M potassium hydroxide and dilute to 1000 mL with C. (3RS)- 3-ethyl-3-(4-nitrophenyl)piperidine-2,6-dione,
water R; adjust to pH 5.0 with glacial acetic acid R; mix
350 mL of this solution with 650 mL of methanolR.
Flow rate 1.0 mUmin.
Detection Spectrophotometer at 328 nm.
Injection 10 ilL.
System suitability Test solution:
- number of theoretical plates: minimum 3300, calculated for
the principal peak; D. 3,3 '- [diazenediylbis(4, l-phenylene)]bis(3-ethylpiperidine-
- mass distribution ratio: 2.0 to 5.0 for the principal peak; 2,6-dione) (azoglutethimide).
- symmetryfactor: maximum 1.2 for the principal peak. _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
Limit:
- impurity D: not more than the area of the principal peak
in the chromatogram obtained with the reference solution
(300 ppm). Aminophylline
Sulfates (2.4.13)
Maximum 500 ppm. (Theophylline-Ethylenediamine, Ph. Eur. monograph
Dilute 6 mL of solution S to 15 mL with distilled water R. 0300)
Loss on drying (2.2.32)
Maximum 0.5 per cent, determined on 1.000 g by drying in
an oven at 105 "C.
Sulfated ash (2.4.14)
Maximum 0.1 per cent, determined on 1.0 g.
ASSAY
Dissolve 0.180 gin 50 rnL of anhydrous acetic acid Rand
420.4'· 317..34-0
titrate with 0.1 M perchloric acid, determining the end-point
potentiometrically (2.2.20). Action and use
1 mL of O.lM perchloric acid is equivalent to 23.23 mg Non-selective phosphodiesterase inhibitor; treatment of
of C13H16NzOz. reversible airways obstruction.
IMPURITIES Preparations
Specifiedimpurities A, D. Aminophylline Injection
Other detectable impurities (the following substances would, if Aminophylline Tablets
presentat a sufficientlevel, be detected by one or otherof the tests ~ophylline Prolonged-release Tablets

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2020 Aminophylline 1-149

PhEur _ solution and dilute to 100 mL with the mobile phase. Dilute
DEFINITION 5 mL of this solution to 50 mL with the mobile phase.
Content Column:
- theophylline (C 7HsN402; M r 180.2): 84.0 per cent to - size: 1 = 0.25 m, 0 = 4 mm;
87.4 per cent (anhydrous substance); - stationaryphase: octadecylsilyl silica gelfor chromatography R
- ethylenediamine (C zH sN 2; M r 60.1): 13.5 per cent to (7~).

15.0 per cent (anhydrous substance). Mobile phase Mix 7 volumes of acetonitrile for
chromatography Rand 93 volumes of a 1.36 gIL solution of
CHARACTERS
sodium acetate R containing 0.50 per cent V/V of glacial acetic
Appearance acid R.
White or slightly yellowish powder, sometimes granular,
hygroscopic. Flow rate 2.0 mUmin.
Detection Spectrophotometer at 272 run.
Solubility
Freely soluble in water (the solution becomes cloudy through Injection 20 ilL.
absorption of carbon dioxide), practically insoluble in Run time 3.5 times the retention time of theophylline.
anhydrous ethanol. Relative retention With reference to theophylline (retention
IDENI1FICATION time = about 6 min): impurity G = about 0.6,
First identification: B, C, E. System suitability Reference solution (b):
Second identification: A, C, D, E, F. - resolution: minimum 2.0 between the peaks due to
impurity G and theophylline.
Dissolve 1.0 gin 10 mL of water R and add 2 mL of dilute
hydrochloric acid R dropwise with shaking. Filter. Use the Limits:
precipitate for identification tests A, B, D and F and the ~ unspecified impurities: for each impurity, not more than the
filtrate for identification test C. area of the principal peak in the chromatogram obtained
with reference solution (a) (0.10 per cent);
A. Melting point (2.2.14): 270°C to 274 DC, determined
- total: not more than the area of the principal peak in the
after washing the precipitate with waterR and drying at
chromatogram obtained with reference solution (a)
105°C.
(0.10 per cent);
B. Infrared. absorption spectrophotometry (2.2.24). - disregard limit: 0.5 times the area of the principal peak in
Preparation Precipitate, washed with water R and dried at the chromatogram obtained with reference solution (a)
105°C. (0.05 per cent).
Comparison theophylline CRS. Water (2.5.12)
C. To the filtrate add 0.2 mL of benzoylchloride R, make Maximum 1.5 per cent, determined on 0.50 g.
alkaline with dilute sodium hydroxide solution R and shake Sulfated ash (2.4.14)
vigorously. Filter the precipitate, wash with 10 mL of Maximum 0.1 per cent, determined on 1.0 g.
water R, dissolve in 5 mL of hot ethanol (96 per cent) Rand
add 5 mL of waterR. A precipitate is formed, which, when ASSAY
washed and dried at 105°C, melts (2.2.14) at 248 °C to Ethylenediamine
252°C. Dissolve 0.250 gin 30 mL of waterR. Add 0.1 mL of
bromocresol green solution R. Titrate with 0.1 LVI hydrochloric
D. Heat about 10 mg of the precipitate with 1.0 mL of a
acid until a green colour is obtained.
360 g/L solution of potassium hydroxide R in a water-bath at
90°C for 3 min, then add 1.0 mL of diazotised sulfanilic acid 1 mL of 0.1 M hydrochloric acid is equivalent to 3.005 mg of
solutionR. A red colour slowly develops. Carry out a blank CzHsNz.
test. . Theophylline
E. Water (see Tests). Heat 0.200 g to constant mass in an oven at 135°C.
F. The precipitate gives the reaction of xanthines (2.3.1). Dissolve the residue with heating in 100 mL of waterR,
allow to cool, add 20 mL of 0.1 M silver nitrate and shake.
TESTS Add 1 mL of bromothymol bluesolution R1. Titrate with 0.1 M
Appearance of solution sodium hydroxide.
The solution is not more opalescent than reference 1 mL of 0.1 M sodium hydroxide is equivalent to 18.02 mg of
suspension II (2.2.1) and not more intensely coloured than C7HsN402 .
reference solution GY 6 (2.2.2, Method II).
STORAGE
Dissolve 0.5· g with gentle warming in 10 mL of carbon
dioxide-free water R. In an airtight container, protected from light.

Related substances IMPURITIES

Liquid chromatography (2.2.29). Other detectable impurities (thefollowing substances would, zf
Test solution Dissolve 47 mg of the substance to be present at a sufficient level; bedetected by one or otherof the tests
examined in the mobile phase and dilute to 20.0 mL with in the monograph. They are limitedby the general acceptance
the mobile phase. criterion for other/unspecified impurities and/or by the general
monograph Substances for pharmaceutical use (2034). It is
Reference solution (a) Dilute 1.0 mL of the test solution to therefore not necessary to identify these impurities for
100.0 mL with the mobile phase. Dilute 1~0 mL of this demonstration of compliance. See also 5.10. Control of impurities
solution to 10.0 mL with the mobile phase. in substances for pharmaceutical use) A, B, C, D, E, F, G.
Reference solution (b) Dissolve 10 mg of theobromine R
(impurity G) in the mobile phase, add 5 mL of the test

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1-150 Aminophylline Hydrate 2020

Aminophylline Hydrate
(Theophylline-Ethylenediamine Hydrate, Ph. Bur.
monograph 0301)

A. 1,3,7-trirnethyl-3,7-dihydro-1H-purine-2,6-dione
(caffeine),

C 16H z4NlO04,xHzO 420.4 72487-55-9

(anhydrous substance)

B. 3-methyl-3,7 -dihydro-1H-purine-2,6-dione, Action and use

Non-selective phosphodiesterase inhibitor; treatment of
reversible airways obstruction.
Preparations
Aminophylline Injection
Aminophylline Tablets
Aminopylline Prolonged-release Tablets
C. N-(6-amino-1,3-dimethyl-2,4-dioxo-1,2,3,4- PhEur _
tetrahydropyrimidin-5-yl)fonnamide,
DEFINITION
Content
- theophylline (C 7HsN4 0 Z; M r 180.2): 84.0 per cent to
87.4 per cent (anhydrous substance);
- ethylenediamine (CzHsNz; M r 60.1): 13.5 per cent to
15.0 per cent (anhydrous substance).
It contains a variable quantity of water.
D. N-methyl-5-(methylamino)-lH-imidazole-4-carboxamide, CHARACTERS
Appearance
White or slightly yellowish powder, sometimes granular.
Solubility-
Freely soluble in water (the solution becomes cloudy through
absorption of carbon dioxide), practically insoluble in
anhydrous ethanol.

E. 1,3-dimethyl-7,9-dihydro-1H-purine-2,6,8 (3H)-trione, IDENTIFICATION

First identification: B~ C~ E.

HC ·:J:°~OH
Second identification: A, C~ D~ B~
F.
3 'N N Dissolve 1.0 g in 10 mL of water R and add 2 mL of dilute

o
A I> N N
hydrochloric acid R dropwise with shaking. Filter. Use the
precipitate for identification tests A, B, D and F and the
CH I
3 filtrate for identification test C.
A. Melting point (2.2.14): 270°C to 274 °C, determined
F. 7-(2-hydroxyethyl)-1,3-dimethyl-3,7 -dihydro-1H-purine- after washing the precipitate with water R and drying at
2,6-dione (etofylline), 105°C.
B. Infrared absorption spectrophotometry (2.2.24).
Preparation Precipitate, washed with water R and dried at
105°C.
Comparison theophylline CRS.
C. To the filtrate add 0.2 mL of benzoyl chloride R, make
alkaline with dilutesodium hydroxide solution R and shake
G.3,7-dimethyl-3,7-dihydro-1H-purine-2,6-dione vigorously. Filter the precipitate, wash with 10 mL of
(theobromine). water R, dissolve in 5 mL of hot ethanol (96 per cent) Rand
_ _ _ _ _ _ _ _ _ _----'- PhEur add 5 mL of water R. A precipitate is formed which, when
washed and dried at 105°C, melts (2.2.14) at 248 °C to
252°C.
D. Heat about 10 mg of the precipitate with 1.0 mL of a
360 gIL solution of potassium hydroxide R in a water-bath at
90°C for 3 min, then add 1.0 mL of diazotised sulfanilic acid

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2020 Aminophylline Hydrate 1-151

solution R. A red colour slowly develops. Carry out a blank Theophylline

test. Heat 0.200 g to constant mass in an oven at 135°C.
E. Water (see Tests). Dissolve the residue with heating in 100 mL of water R,
allow to cool, add 20 mL of 0.1 M silvernitrate and shake.
F. The precipitate gives the reaction of xanthines (2.3.1).
Add 1 mL of bromothymol blue solution Rl. Titrate with 0.1 M
TESTS sodium hydroxide.
Appearance of solution 1 mL of 0.1 M sodium hydroxide is equivalent to 18.02 mg of
The solution is not more opalescent than reference C7HsN 402 •
suspension II (2.2.1) and not more intensely coloured than
reference solution GY6 (2.2.2, Method II). STORAGE
In a well-filled, airtight container, protected from light.
Dissolve 0.5 g with gentle warming in 10 mL of carbon
dioxide-free water R. IMPURITIES
Related substances Other detectable impurities (the following substances would, if
Liquid chromatography (2.2.29). present at a sufficient level, be detected by one or otherof the tests
in the monograph. They are limitedby the general acceptance
Test solution Dissolve 50 mg of the substance to be
criterion for other/unspecified impurities and/or by the general
examined in the mobile phase and dilute to 20.0 mL with
monograph Substances for pharmaceutical use (2034). It is
the mobile phase.
therefore not necessary to identify these impurities for
Reference solution (a) Dilute 1.0 mL of the test solution to demonstration of compliance. See also 5. LO. Controlof impurities
100.0 mL with the mobile phase. Dilute 1.0 mL of this in substances for pharmaceutical use) A) B, C, D, E, F, G.
solution to 10.0 mL with the mobile phase.
Reference solution (b) Dissolve 10 mg of theobromine R
(impurity G) in the mobile phase, add 5 mL of the test
solution and dilute to 100 mL with the mobile 'phase, Dilute
5 mL of this solution to 50 mL with the mobile phase.
Column:
- size: 1= 0.25 m, (2) = 4 mID;
- stationaryphase: octadecylsilyl silica gelfor chromatography R A. 1,3,7-trimethyl-3,7-dihydro-1H-purine-2,6-dione
(7 urn). (caffeine),
Mobile phase Mix 7 volumes of acetonitrile for
chromatography R and 93 volumes of a 1.36 gIL solution of
sodium acetate R containing 0.50 per cent V/Vof glacial acetic
acid R.
Flow rate 2.0 mlJmin.
Detection Spectrophotometer at 272 nm.
Injection 20 JlL.
Run time 3.5 times the retention time of theophylline. B. 3-methyl-3,7-dihydro-1H-purine-2,6-dione,
Relative retention. With reference to theophylline (retention
time = about 6 min): impurity G = about 0.6.
System suitability Reference solution (b):
- resolution: minimum 2.0 between the peaks due to
impurity G and theophylline.
Limits:
- unspecified impurities: for each impurity, not more than the
area of the principal peak in the chromatogram obtained C. N-(6-amino-l,3-dimethyl-2,4-dioxo-l,2,3,4-
with reference solution (a) (0.10 per cent); tetrahydropyrimidin-5-yl)formamide,
- total: not more than the area of the principal peak in the
chromatogram obtained with reference solution (a)
(0.1 per cent);
- disregard limit: 0.5 times the area of the principal peak in
the chromatogram obtained with reference solution (a)
(0.05 per cent).
Water (2.5.12)
3.0 per cent to 8.0 per cent, determined on 0.50 g. D. N-methyl-5-(methylamino)-IH-imidazole-4-carboxamide,
Sulfated ash (2.4.14)
Maximum 0.1 per cent, determined on 1.0 g.
ASSAY
Ethylenediamine
Dissolve 0.250 gin 30 mL of water R. Add 0.1 mL of
bromocresol green solution R. Titrate with 0.1 M hydrochloric
acid until a green colour is obtained. E. 1,3-dimethyl-7,9-dihydro-lH-purine-2,6,8(3H)-trione,
1 mL of 0.1 M hydrochloric acid is equivalent to 3.005 mg of
C 2HsN2 ·

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1-152 Amiodarone Hydrochloride 2020

pH (2.2.3)
3.2 to 3.8.
Dissolve 1.0 gin 'carbon dioxide-free water R, heating at 80 DC,
cool and dilute to 20 mL with the same solvent.
ImpurityH
Thin-layer chromatography (2.2.27). Preparethe solutions
F. 7-(2-hydroxyethyl)-1,3-dimethyl-3,7-dihydro-1H-purine-
immediately before use and keep protected from bright light.
2,6-dione (etofylline),
Test solution Dissolve 0.500 g of the substance to be
examined in methylene chloride R and dilute to 5.0 mL with
the same solvent.
Reference solution (a) Dissolve 10.0 mg of (2-chloroethyl)
diethylamine hydrochloride R (impurity H) in methylene
chloride R and dilute to 50.0 mL with the same solvent.
Dilute 2.0 mL of the solution to 20.0 mL with methylene
G. 3,7-dimethyl-3,7-dihydro-1H-purine-2,6-dione chloride R.
,(theobromine). ' Reference solution (b) Mix 2.0 mL of the test solution and
________ ~ PhEur 2.0 mL of reference solution (a).
Plate TLC silica gel F 254 plate R.
Mobile phase anhydrous formic acid R, methanol R, methylene
chloride R (5:10:85 V/V/V).
Amiodarone Hydrochloride Application 50 ilL of the test solution and reference
solution (a); 100 IlL of reference solution (b).
(Ph. Bur. monograph 0803)
Development Over 2/3 of the plate.
Drying In a current of cold air.
Detection Spray with potassium iodobismuthate solution R1 and
then with dilute hydrogen peroxidesolution R; examine
immediately in daylight.
System suitability .Reference solution (b):
- the spot due to. impurity H is clearly visible.
682 19774-82-4 Limit:
- impurity H: any spot due to impurity H is not more
Action and use intense than the spot in the chromatogram obtained with
Potassium channel blocker; class TIl antiarrhythmic. reference solution (a) (0.02 per cent).
Preparations Related substances
Amiodarone Intravenous Infusion Liquid chromatography (2.2.29).
Amiodarone Oral Suspension Buffer solution pH 4.9 To 800 mL of water R add 3.0 mL of
Amiodarone Tablets glacial acetic acid R, adjust to pH 4.9 with dilute ammonia R1
PhEur _
and dilute to 1000 mL with water R.
Test solution Dissolve 0.125 g of the substance to be
DEFINITION examined in a mixture of equal volumes of acetonitrile Rand
(2-Butylbenzofuran-3-yl) [4-[2-(diethylamino)ethoxy]-3,5- water R and dilute to 25.0 mL with the same mixture of
diiodophenyl] methanone hydrochloride. solvents.
Content Reference solution Dissolve 5 mg of amiodarone
98.5 per cent to 101.0 per cent (dried substance). impurity D CRS, 5 mg of amiodarone impurity E CRS and
CHARACTERS 5.0 mg of amiodarone hydrochloride CRS in methanol Rand
dilute to 25.0 mL with the same solvent. Dilute 1.0 mL of
Appearance
the solution to 20,0 mL with a mixture of equal volumes of
White or almost white, fine, crystalline powder.
acetonitrile R and water R.
Solubility
Column:
Very slightly soluble in water, freely soluble in methylene
- size: 1 = 0.15 m, 0 = 4.6 mm;
chloride, soluble in methanol, sparingly soluble in ethanol
- stationary phase: end-capped octadecylsilyl silica gel for
(96 per cent).
chromatography R (5 urn);
IDENTIFICATION - temperature: 30 DC.
A. Infrared absorption spectrophotometry (2.2.24). Mobile phase Buffer solution pH 4.9, methanol R,
Comparison amiodarone hydrochloride CRS. acetonitrile R (30:30:40 V/V/V).
B. It gives reaction (b) of chlorides (2.3.1). Flow rate 1 mIlmin.
TESTS Detection Spectrophotometer at 240 om.
.Appearance of solution Injection 10 ilL.
The solution is clear (2.2.1) and not more intensely coloured Run time Twice the retention time of amiodarone.
than reference solution GYs or BYs (2.2.2, Method Il). Relative retention With reference to amiodarone (retention
Dissolve 1.0 g in methanol R and dilute to 20 mL with the =
time about 24 min): impurity A about 0.26; =
same solvent.

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2020 Amiodarone Hydrochloride 1-153

impurity D = about 0.29; impurity E =about 0.37;

and enantiomer

impurity B = about 0.49; impurity C = about 0.55;

impurity G = about 0.62; impurity F = about 0.69.
System suitability Reference solution:
- resolution: minimum 3.5 between the peaks due to
impurities D and E.
Limits: A. (2-butylbenzofuran-3-yl) [4-[2-(diethylamino)ethoxy]
- impurities A., B., C., D., E., P., G: for each impurity, not phenyl]methanone,
more than the area of the peak due to amiodarone in the
chromatogram obtained with the reference solution and enantiomer

(0.2 per cent);

- unspecified impurities: for each impurity, not more than
0.5 times the area of the peak due to amiodarone in the
chromatogram obtained with the reference solution
(0.10 per cent);
- total: not more than 2.5 times the area of the peak due to
amiodarone in the chromatogram obtained with the B. (2-butylbenzofuran-3-yl)[4-[2-(ethylamino)ethoxy]-3,5-
reference solution (0.5 per cent); diiodophenyl] methanone,
- disregard limit: 0.25 times the area of the peak due to and enantiomer
amiodarone in the chromatogram obtained with the
reference solution (0.05 per cent).
Iodides
Maximum 150 ppm.
Prepare the test and 'reference solutions simultaneously.
SolutionA Add 1.50 g of the substance to be examined to
40 mL of waterR at 80 DC and shake until completely C. (2-butylbenzofuran-3-yl)[4-[2-(diethylamino) ethoxy]-3-
iodophenyl] methanone,
dissolved. Cool and dilute to 50.0 mL with waterR.
Test solution To 15.0 mL of solution A add 1.0 mL of
0.1 M hydrochloric acid and 1.0 mL of 0.05 M potassium
iodate. Dilute to 20.0 mL with water R. Allow to stand
protected from light for 4 h. OH
Reference solution To 15.0 mL of solution A add 1.0 mL of
0.1 M hydrochloric acid, 1.0 mL of an 88.2 mg/L solution of
potassium iodideR and 1.0 mL of 0.05 M potassium iodate.
D. (2-butylbenzofuran-3-yD(4-hydroxy-3,5-
Dilute to 20.0 mL with waterR. Allow to stand protected
diiodophenyl)methanone,
from light for 4 h.
Measure the absorbances (2.2.25) of the solutions at 420 nm,
using a mixture of 15.0 mL of solution A and 1.0 mL of
0.1 M hydrochloric acid diluted to 20.0 mL with water R as
the compensation liquid. The absorbance of the test solution OH
is not greater than half the absorbance of the reference
solution.
Loss on drying (2.2.32) E. (2-butylbenzofuran-3-yl)(4-hydroxyphenyl)methanone,
Maximum 0.5 per cent, determined on 1.000 g by drying at
50°C at a pressure not exceeding 0.3 kPa for 4 h.
Sulfated ash (2.4.14)
Maximum 0.1 per cent, determined on 1.0 g.
OH
ASSAY
Dissolve 0.600 g in a mixture of 5.0 mL of
0.01 M hydrochloric acid and .75 mL of ethanol (96 per cent) R.
Carry out a potentiometric titration (2.2.20), using F. (2-butylbenzofuran-3-yl)(4-hydroxy-3-iodophenyl)
0.1 M sodium hydroxide. Read the volume added between the methanone,
2 points of inflexion.
1 mL of 0.1 M sodium hydroxide is equivalent to 68.18 mg of
and enantiomer
C25H30CII2N03'
STORAGE
Protected from light, at a temperature not exceeding 30 DC.
IMPURITIES
Specified impurities A., B, C, D, E., P, G, H.
G. [4-[2-(diethylamino)ethoxy]-3,5-diiodophenyl] [2-[(lRS)-
l-methoxybutyllbenzofuran-3-yl]methanone,

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1-154 Amisulpride 2020

Plate TLC silica gel G plate R.

Mobilephase 50 per cent VIV solution of concentrated
ammonia R, anhydrous ethanol R, di-isopropyl etherR
(10:25:65 VIVIV); use the upper layer obtained aftershaking
H. 2-cWoro-N,N-diethylethanamine (2-chlorotriethylamine, the mixture.
(2-chloroethyl) diethylamine).
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
Application 10 ilL.
Development Over 2/3 of the plate.
Drying In air.
Detection Spray with ninhydrinsolution R and heat at
Amisulpride 100-105 DC for 15 min.
Retardation factors Impurity A = about 0.2;
(Ph. Bur. monograph 1490) =
amisulpride about 0.5.
System suitability The chromatogram obtained with
reference solution (b) shows 2 clearly separated spots.
and enantiomer Limit:
- impurityA: any spot due to impurity A is not more
intense than the corresponding spot in the chromatogram
obtained with reference solution (a) (0.1 per cent).
369.5 71675-85-9 Related substances
Liquid chromatography (2.2.29).
Action and use Solvent mixture acetonitrile R, methanolR, mobile phase A
Dopamine receptor antagonist; neuroleptic.
(12:16:72 VIVIV).
Preparations Test solution Dissolve 0.100 g of the substance to be
'Amisulpride Oral Solution examined in 16 mL of methanolR, add 12 mL of
Amisulpride Tablets acetonitrile R and dilute to 100.0 mL with mobile phase A.
PhEur ~ _ Reference solution (aJ Dilute 1.0 mL of the test solution to
100.0 mL with the solvent mixture. Dilute 1.0 mL of this
DEFINITION solution to 10.0 mL with the solvent mixture.
4-Amino-N-[[(2RS)-1-ethylpyrrolidin-2-yl]methyl]-5- Reference solution (b) Dissolve the contents of a vial of
(ethylsulfonyl)-2-methoxybenzamide. amisulpride for system suitability CRS (containing impurity B)
Content in 1 mL of the solvent mixture.
99.0 per cent to 101.0 per cent (dried substance). Column:
CHARACTERS - size: 1 = 0.25 m, (2) = 4.6 mm;
Appearance - stationary phase: base-deactivated octylsilyl silica gelfor
White or almost white, crystalline powder. chromatography R (5 11m);
- temperature: 40 DC.
Solubility
Practically insoluble in water, freely soluble in methylene Mobilephase:
chloride, sparingly soluble in anhydrous ethanol. - mobile phaseA: dissolve 0.7 g of sodium octanesulfonate R in '
930 mL of waterfor chromatography R and add 45.0 mL
mp of a 5 per cent VIV solution of dilute sulfuric acid R; adjust
About 126 ClC.
to pH 2.3 with dilute sulfuric acid R and dilute to
IDENTIFICATION 1000 mL with waterfor chromatography R;
Infrared absorption spectrophotometry (2.2.24). - mobile phaseB: methanol R1 ;
Comparison amisulpride CRS. - mobile phase C: acetonitrile for chromatography R;
TESTS Time Mobile phase A Mobile phase B Mobile phase C
Appearance of solution (min) (per cent VflI) (per cent VflI) (per cent VflI)
The solution is clear (2.2.1) and not more intensely coloured 0-18 72 16 12
than reference solution Y6 (2.2.2, Method II). 18 - 35 72 ..... 50 16 ..... 38 12
Dissolve 1.0 gin 3 mL of a mixture of 1 volume of acetic
acid R and 4 volumes of waterR, and dilute to 20 mL with Flow rate 1.5 mLlmin.
water R.
Detection Spectrophotometer at 225 nm.
Impurity A Injection 10!JL.
Thin-layer chromatography (2.2.27).
Identification ofimpurities Use the chromatogram obtained
Test solution Dissolve 0.20 g of the substance to be with reference solution (b) to identify the peak due to
examined in methanolR and dilute to 10 mL with the same impurity B.
solvent.
Relative retention With reference to amisulpride (retention
Reference solution (a) Dissolve 5 mg of sulpiride time = about 17 min): impurity B = about 1.1.
impurityA CRS (amisulpride impurity A) in methanol Rand
dilute to 25 mL with the same solvent. Dilute 2 mL of the System suitability Reference solution (b):
solution to 20 mL with methanolR. ---..:.. peak-to-valley ratio: minimum 2.0, where Hp = height
above the baseline of the peak due to impurity Band
Reference solution (b) Dilute 1 mL of the test solution to H; = height above the baseline of the lowest point of the
10 mL with reference solution (a).

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2020 Amitriptyline Hydrochloride 1-155

curve separating this peak from the peak due to

amisulpride.
Calculation of percentage contents:
- for each impurity, use the concentration of amisulpride in
reference solution (a).
E. 4-amino-5-(ethylsulfonyl)-2-methoxybenzoic acid,
Limits:
- unspecified impurities: for each impurity, maximum
0.10 per cent;
- total: maximum 0.3 per cent; and enantiomer
- reporting threshold: 0.05 per cent.
Loss on drying (2.2.32)
Maximum 0.5 per cent, determined on 1.000 g by drying in F. 4-amino-N-[[(2RS)-I-ethyl-I-oxidopyrrolidin-2-yl]
an oven at 105°C for 3 h. methyl]-5-(ethylsulfonyl)-2-methoxybenzamide,
Sulfated ash (2.4.14)
Maximum 0.1 per cent, determined on 1.0 g.
ASSAY
Dissolve 0.300 g with shaking in a mixture of 5 mL of acetic
anhydride Rand 50 mL of anhydrous acetic acid R. Titrate
with 0.1 M perchloric acid, determining the end-point
G.4-amino-N-[(3RS)-I-ethylpiperidin-3-yl]-5-(ethylsulfonyl)-
potentiometrically (2.2.20).
2-methoxybenzamide,
I mL of 0.1 M perchloric acid is equivalent to 36.95 mg of
C17H27N304S,
IMPURITIES
Specifiedimpurities A.
H3
C
:'sd
H2N
0#'

~
I
0 ~
~"
. CH 3H
OCH 3
N
l
and enantiomer

Other detectable impurities (the following substances would, if . CH 3

presentat a sufficientlevel, be detected by one or otherof the tests
in the monograph. They are limitedby the general acceptance H. 4-amino-N-[[(2RS)-I-ethylpyrrolidin-2-yl]methyl]-5-
criterion for other/unspecified impurities and/orby the general (ethylsulfonyl)-2-methoxy-N-methylbenzamide.
monograph. Substances for pharmaceutical use (2034). It is _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
therefore not necessary to identify these impurities for
demonstration of compliance. See also 5.10. Controlof impurities
in substances for pharmaceutical use) B, C, D,· B, F, G, H.
Amitriptyline Hydrochloride
H2N~
H' N and enantlomer (Ph. Bur. monograph 0464)
l.CH 3

A. [(2RS)-I-ethylpyrrolidin-2-yl] methanamine, N-,CH 3

I , HCI
CH3

H3C :5¥/do~ I
O.NAlo..,/\
A
H' H N) and enantiomer
. H2N ~ OH l CH3
313.9 549-18-8

Action and use

B. 4-amino-N- [[(2RS)-I-ethylpyrrolidin-2-yl] methyl] -5-
Monoamine reuptake inhibitor; tricyclic antidepressant.
(ethylsulfonyl)-2-hydroxybenzamide,
Preparations
Amitriptyline Tablets
Amitriptyline Oral Solution
and enantiomer
PhEur _

DEFINITION
3-(I 0,11-Dihydro-Sff-dibenzo [a,d] [7] annulen-5-ylidene )-N,
C. 4-amino-N-[[(2RS)-I-ethylpyrrolidin-2-yl] methyl]-5-iodo-
N-dimethylpropan"l-amine hydrochloride.
2-methoxybenzamide,
Content
99.0 per cent to 101.0 per cent (dried substance).
CHARACTERS
and enaritiomer
Appearance
White or almost white powder or colourless crystals.
Solubility
D. 4-amino-N- [[(2RS)-I-ethylpyrrolidin-2-yl] methyl] -2- Freely soluble in water,· in ethanol (96 per cent) and in
methoxy-5-(methylsulfonyl)benzamide, methylene chloride.

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1-156 Amitriptyline Hydrochloride 2020

IDENTIFICATION - disregard limit: 0.5 times the area of the peak due to -
A. Infrared absorption spectrophotometry (2.2.24). amitriptyline in the chromatogram obtained with reference
Comparison amitriptyline hydrochloride CRS. solution (b) (0.05 per cent).
B. 20 mg gives reaction (a) of chlorides (2.3.1). Loss on drying (2.2.32)
Maximum 0.5 per cent, determined on 1.000 g by drying in
TESTS an oven at 105°C for 2 h.
Appearance of solution
The solution is clear (2.2.1) and not more intensely coloured Sulfated ash (2.4.14)
than reference solution B7 (2.2.2, Method II). Maximum 0.1 per cent, determined on 1.0 g.
Dissolve 1.25 g in waterR and dilute to 25 mL with the ASSAY
same solvent. . Dissolve 0.250 gin 30 mL of ethanol (96 per cent) R. Titrate
Acidity or alkalinity with 0.1 M sodium hydroxide, determining the end-point
Dissolve 0.20 g in carbon dioxide-free water R and dilute to potentiometrically (2.2.20).
10 mL with the same solvent. Add 0.1 mL of methyl red 1 mL of 0.1 M sodiumhydroxide is equivalent to 31.39 mg of
solution Rand 0.2 mL of 0.01 M sodium hydroxide. C ZOHZ4ClN.
The solution is yellow. Add 0.4 mL of 0.01 M hydrochloric STORAGE
acid. The solution is red. Protected from light.
Related substances IMPURITIES
Liquid chromatography (2.2.29).
Specifiedimpurities A, B.
Test solution Dissolve 50.0 mg of the substance to be
Other detectable impurities (the following substances would, if
examined in the mobile phase and dilute to 50.0 mL with
present at a sufficient level, be detected by one or otherof the tests
the mobile phase.
in the monograph. They are limited by the general acceptance
Reference solution (a) Dissolve 5.0 mg of criterion for other/unspecified impurities and/or by the general
dibenzosuberone CRS (impurity A) and 5.0 mg of monograph Substances for pharmaceutical use (2034). It is
cyclobenzaprine hydrochloride CRS (impurity B) in 5.0 mL of therefore not necessary to identify these impurities for
the test solution and dilute to 100.0mL with the mobile demonstration of compliance. See also 5.10. Controlof impurities
phase. in substances for pharmaceutical use) C, D, E, F, G.
Reference solution (b) Dilute 1.0 mL of reference solution (a)
to 50.0 mL with the mobile phase.
Column:
- size: 1= 0.15 m, 0 = 4.6 mm;
- stationary phase: end-capped polar-embedded octadecylsilyl
amorphous organosilica polymer R (5 urn);
- temperature: 40°C.
Mobile phase Mix 35 volumes of acetonitrile Rand
65 volumes of a 5.23 gIL solution of dipotassium hydrogen A. 10, I1-dihydro-5H-dibenzo [a,d] [7]annulen-5-one
phosphate R previously adjusted to pH 7.0 with phosphoric (dibenzosuberone),
acid R.
Flow rate 1.2 mUmin.
Detection Spectrophotometer at 220 nm.
Injection 10 ilL.
Run time 3 times the retention time of amitriptyline.
Relative retention With reference to amitriptyline (retention
time = about 14 min): impurity B = about 0.9;
impurity A = about 2.2. B. 3-(5H-dibenzo [a,d] [7]annulen-5-ylidene)-N,N-
System suitability Reference solution (a): dimethylpropan-l-amine (cyclobenzaprine),
~ resolution: minimum 2.0 between the peaks due to
impurity B and amitriptyline.
Limits:
- impurity B: not more than the area of the corresponding
peak in the chromatogram obtained with reference
solution (b) (0.1 per cent); ,
- impun'tyA: not more than 0.5 times the area of the
corresponding peak in the chromatogram obtained with
reference solution (b) (0.05 per cent); C. 3-(10,II-dihydro-5H-dibenzo[a,d] [7]annulen-5-ylidene)-
- unspecified impurities: for each impurity, not more than the N-methylpropan-l-amine (nortriptyline),
area of the peak due to amitriptyline in the chromatogram
obtained with reference solution (b) (0.10 per cent);
~ total: not more than 3 times the. area of the peak due to
amitriptyline in the chromatogram obtained with reference
solution (b) (0.3 per cent);

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2020 Amlodipine Besilate 1-157

C(r;
~. .
3C'N_CH PhEur _
H 3
"..--:;: OH DEFINITION
3-Ethyl 5-methyl (4RS)-2-[(2-aminoethoxy)methyl]-4-(2-
chlorophenyl)-6-methyl-l ,4-dihydropyridine-3,5-dicarboxylate
~ ~ benzenesulfonate.
:::::,....
Content
97.0 per cent to 102.0 per cent (anhydrous substance).
D. 5-[3-(dimethylamino)propyl] -10, II-dihydro-5H-dibenzo
[a,dJ [7]annulen-5-o1,
PRODUCTION
It is considered that alkyl benzenesulfonate esters are
genotoxic and are potential impurities in amlodipine besilate.
The manufacturing process should be developed taking into
consideration the principles of quality risk management,
together with considerations of the quality of starting
materials, process capability and validation. The general
method 2.5.41. Methyl, ethyl and isopropyl benzenesuljonate in
active substances is available to assist manufacturers.
E. N,N-dimethyl-'3-(1,2,3,4,4a,10, 11,11a-octahydro-5H- CHARACTERS
dibenzo [a,dJ [7]annulen-5-ylidene)propan-l-amine, Appearance
White or almost white powder.
Solubility
3
Slightly soluble in water, freely soluble in methanol, sparingly
N --CH its (E)-isomer and
I their enantiomers
soluble in anhydrous ethanol, slightly soluble in 2-propanoI.
CH3
IDENTIFICATION
Infrared absorption spectrophotometry (2.2.24).
Comparison amlodipine besilate CRS.
F. (5BZ,10RS)-5-[3-(dimethylamino)propylidene]-1O,11- TESTS
dihydro-5H-dibenzo [a,dJ [7]annulen-l 0-01,
Optical rotation (2.2.7)
-0.100 to + 0.10 0 •

Q;
~
Dissolve 0.250 g in methanolR and dilute to 25.0 mL with
the same solvent.
-: O.H
Related substances
~ ~ Liquid chromatography (2.2.29). Carry out the test protected
:::::,.... from light.
Test solution (a) Dissolve 50.0 mg of the substance to be
G. 10, ll-dihydro-5H-dibenzo [a,dJ [7]annulen-5-o1 examined in the mobile phase and dilute to 50.0 mL with
(dibenzosuberol). the mobile phase.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur Test solution (b) Dilute 5.0 mL of test solution (a) to
100.0 mL with the mobile phase.
Reference solution (a) Dilute 1.0 mL of test solution (a) to
10.0 mL with the mobile phase. Dilute 1.0 mL of this
Amlodipine Besilate solution to 100.0 mL with the mobile phase.
Reference solution (b) Dissolve 2.5 mg of amlodipine
(Ph. Bur. monograph 1491) impurity B CRS and 2.5 mg of amlodipine impurity G CRS in
the mobile phase and dilute to 25.0 mL with the mobile
phase. Dilute 1.0 mL of the solution to 10.0 mL with the
mobile phase.
Reference solution (c) Dissolve 2.5 mg of amlodipine for peak
identification CRS (containing impurities D, E and F) in
5 mL of the mobile phase.
Reference solution (d) Dissolve 5.0 mg of amlodipine
impurity A CRS in acetonitrile R and dilute to 5.0 mL with
the same solvent. Dilute 1.0 mL of the solution to 100.0 mL
567.1 111470-99-6 with the mobile phase. Dilute 1.0 mL of this solution to
10.0 mL with the mobile phase.
Action and use Reference solution (e) Dissolve 50.0 mg of amlodipine
Calcium channel blocker. besilate CRS in the mobile phase and dilute to 50.0 mL with
Preparations the mobile phase. Dilute 5.0 mL of the solution to 100.0 mL
Amlodipine Oral Solution with the mobile phase. '
Amlodipine Besilate Tablets Column:
=
- size: 1 0.25 m, 0 = 4.0 mm;

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1-158 Amlodipine Besilate 2020

- stationaryphase: octadecylsilyl silica gelfor chromatography R criterion for other/unspecified impurities and/orby the general
(5 11m); monograph Substances for pharmaceutical use (2034). It is
- temperature: 30°C. therefore not necessary to identify these impurities for
Mobilephase 2.3 gIL solution of ammonium acetate R, demonstration of compliance. See also 5.10. Control of impurities
methanol R (30:70 VIV). in substances for pharmaceutical use) B, G, H.
Flow rate 1.5 mIJrnin.
Detection Spectrophotometer at 237 urn.
Injection 20 ilL of test solution (a) and reference
solutions (a), (b), (c) and (d).
.
H3CyN~o~N
H'CO~O,-/CH'
H 0t? 0
\ I
.

Run time Twice the retention time of amlodipine.

Identification of impurities Use the chromatogram supplied o : 0
with amlodipine for peak identification CRS and the ~CI
chromatogram obtained with reference solution (c) to identify
the peaks due to impurities D, E and F; use the V and enantiomer

chromatogram obtained with reference solution (d) to

identify the peak due to impurity A. A. 3-ethyl 5-methyl (4RS)-4-(2-chlorophenyl)-2-[[2-(1,3-
dioxo-1,3-dihydro-2H-isoindol-2-yl)ethoxy]methyl]-6-
Relative retention With reference to amlodipine (retention
methyl-1,4-dihydropyridine-3,5-dicarboxylate,
time = about 20 min): impurity G = about 0.21;
= =
impurity B about 0.25; impurity D about 0.5;
H3C'NH
impurity F = about 0.8; impurity E = about 1.3.

~ O~~)o
System suitability Reference solution (b):
- resolution: minimum 2.0 between the peaks due to
impurities G and B. H:&C I I
Limits: H3CO. O <:> CH3 0
- correction factors: for the calculation of content, multiply : H
the peak areas of the following impurities by the o : 0

()
~
corresponding correction factor: impurity D 1.7; = CI

impurity F 0.7;= I #
and enantiomer

~ impurity D: not more than 3 times the area of the

principal peak in the chromatogram obtained with
B. 3-ethyl 5-methyl (4RS)-4-(2-chlorophenyl)-6-methyl-2-
reference solution (a) (0.3 per cent);
[[2-[[2-(methyIcarbamoyl)benzoyl]amino]ethoxy]methyl]-
- impurity A: not more than 1.5 times the area of the
1,4-dihydropyridine-3,5-dicarboxylate,
corresponding peak in the chromatogram obtained with
reference solution (d) (0.15 per cent);
- impurities E, F: for each impurity, not more than 1.5 times
the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.15 per cent);
- unspecified impurities: for each impurity, not more than the
area of the principal peak in the chromatogram obtained
with reference solution (a) (0.10 per cent);
- total: maximum 0.8 per cent;
- disregard limit: 0.5 times the area of the principal peak in D.3-ethyl 5-methyl 2-[(2-aminoethoxy)methyl]-4-(2-
the chromatogram obtained with reference solution (a) chlorophenyl)-6-methylpyridine-3,5-dicarboxylate,
(0.05 per cent); disregard any peak due to benzene
=
sulfonate (relative retention about 0.14). H
H:&
N C '~NH2
Water (2.5.12) I I .0
Maximum 0.5 per cent, determined on 1.000 g. H3C............... O .: O <:> CH3
Sulfated ash (2.4.14) : H
o : 0

~I~
Maximum 0.2 per cent, determined on 1.0 g.
ASSAY V CI
and enantiomer

Liquid chromatography (2.2.29) as described in the test for

related substances with the following modification.
E. diethyl (4RS)-2-[(2-aminoethoxy)methyl]-4-(2-
Injection Test solution (b) and reference solution (e). chlorophenyl)-6-methyl-1 ,4-dihydropyridine- 3,5-
Calculate the percentage content of CZ6H31CINzOaS taking dicarboxylate,
into account the assigned content of amlodipine besilate CRS.
STORAGE
In an airtight container, protected from light.
IMPURITIES
Specifiedimpurities A, D, E, F.
Other detectable impurities (the followingsubstances would, if
present at a sufficientlevel, be detectedby One or otherof the tests
in the monograph. They are limited by the general acceptance

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2020 Ammonia 1-159

H
changes from red to yellow. Add 1 mL of sodium cobaitinitrite
H 0 & C . N O~NH2
solution R. A yellow precipitate is formed.
H3CO I I OCH3
: H
TESTS
o : 0 Solution S
~1.~Cl
V and enantiomer
Evaporate 220 mL almost to dryness on a water-bath. Cool,
add 1 mL of dilute acetic acid R and dilute to 20 mL with
distilled water R.
Appearance of solution
F. dimethyl (4RS)-2-[(2-aminoethoxy)methyl]-4-(2-
cWorophenyl)-6-methyl-l ,4-dihydropyridine-3,5- The solution is clear (2.2.1) and colourless (2.2.2,
Method II).
dicarboxylate,
To 2 mL add 8 mL of waterR.
Oxidisable substances
Cautiously add, whilst cooling, 8.8 mL to 100 mL of dilute
sulfuric acid R. Add 0.75 mL of 0.002 M potassium
permanganate. Allow to stand for 5 min. The solution
remains faintly pink.
Pyridine and related substances
Maximum 2 ppm, calculated as pyridine.
Measure the absorbance (2.2.25) at 252 nm using water R as
G. dimethyl 4-(2-chlorophenyl)-2,6-dimethyl-1,4- the compensation liquid. The absorbance is not greater
dihydropyridine-3,5-dicarboxylate, than 0.06.
Carbonates
H0:;OC ~ Maximum 60 ppm.
H H I To 10 mL in a test-tube with a ground-glass neck add
H0&
N C I I O~N· #
10 mL of calcium hydroxide solution R. Stopper immediately
H3CO 0'-.,./CH3 0
and mix. Any opalescence in the solution is not more intense
: H than that in a standard prepared at the same time and in the
o : 0

U
~ ·CI same manner using 10 mL of a 0.1 gIL solution of anhydrous
I #
and enantiomer sodium carbonate R.
Chlorides (2.4.4)
Maximum 1 ppm.
H.2-[[2-[[(4RS)-4-(2-chlorophenyl)-3-(ethoxycarbonyl)-5-
Dilute 5 mL of solution S to 15 mL with water R.
(methoxycarbonyl)-6-methyl-l,4-dihydropyridin-2-yl]
methoxy] ethyl] carbamoyl]benzoic acid. Sulfates (2.4.13)
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur Maximum 5 ppm.
Dilute 3 mL of solution S to 15 mL with distilled waterR.
Iron (2.4.9)
Maximum 0.25 ppm.
Strong Ammonia Solution Dilute 4 mL of solution S to 10 mL with water R.
Residue on evaporation
(Ammonia Solution, Concentrated, Ph. Bur. Maximum 20 mgIL.
monograph 0877)
Evaporate 50 mL to dryness on a water-bath and dry at
NH 3 17.03 100-105 DC for 1 h. The residue weighs a maximum of
Preparation 1 mg.
Dilute Ammonia Solution ASSAY
PhEur ~ _ _-'-- _ Weigh accurately a flask with a ground-glass neck containing
50.0 mL of 1 M hydrochloric acid. Add 2 mL of the substance
DEFINITION
to be examined and re-weigh. Add 0.1 mL of methyl red
Content
solution R as indicator. Titrate with 1 M sodium hydroxide
25.0 per cent m/m to 30.0 per cent mlm.
until the colour changes from red to yellow.
CHARACTERS 1 mL of 1 M hydrochloric acid is equivalent to 17.03 mg of
Appearance NH3 ·
Clear, colourless liquid, very caustic.
STORAGE
Solubility Protected from air, at a temperature not exceeding 20 DC.
Miscible with water and with ethanol (96 per cent).
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
IDENTIFICATION
A. Relative density (2.2.5): 0.892 to 0.910.
B. It is strongly alkaline (2.2.4).
C. To 0.5 mL add 5 mL of water R. Bubble air through the
solution and lead the gaseous mixture obtained over the
surface of a solution containing 1 mL of 0.1 M hydrochloric
acid and 0.05 mL of methyl red solution R. The colour

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1-160 Ammonio Methacrylate Copolymer 2020

Monomers
Ammonia Methacrylate .Copolymer Liquid chromatography (2.2.29).
(Type A) Solution A Dissolve 3.5 g of sodium perchlorate R in water R
(ph. Eur. monograph 2081) and dilute to 100 mL with the same solvent.
Test solution Dissolve 5.00 g of the substance to be
examined in methanol R and dilute to 50.0 mL with the same
solvent. To 10.0 mLofthis solution add 5.0 mL of
solution A, dropwise, while continuously stirring. Remove the
precipitated polymer by centrifugation. Use the clear
supernatant solution.
Reference solution Dissolve 50.0 mg of ethyl acrylate R and
10.0 mg of methyl methacrylate R in methanol R and dilute to
50.0 mL with the same solvent. Dilute 1.0 mL of the
solution to 100.0 mL with methanol R. Add 10 mL of this
solution to 5 mL of solution A.
Action and use Column:
Excipient. =
- size: 1 0.12 m, (0 =4.6 mrn;
PhEur _ - stationary phase: irregular end-capped octadecylsilyl silica gel
for chromatography R (7 urn).
DEFINITION
Mobile phase Mix 20 volumes of methanol R2 and
Poly [ethyl propenoate-co-methyl 2-methylprop-2-enoate-co-N, 80 volumes of water for chromatography R previously ajusted
N,N-trimethyl-2- [(2-methylprop:-2-enoyl)oxy] ethan-1- to pH 2.0 with phosphoric acid R.
aminium chloride] having a mean relative molecular mass of
Flow rate 2.0 mLlmin.
about 150 000.
Detection Spectrophotometer at 202 nm.
The ratio of ethyl acrylate (ethyl propenoate) groups to
methyl methacrylate (methyl 2-methylprop-2-enoate) groups Injection 50 J.LL.
to ammonio methacrylate (N,N,N-trimethyl-2-[(2- System suitabil£ty Reference solution:
methylprop-2-enoyl)oxy]ethan-1-aminium chloride) groups is - resolution: minimum 1.5 between the peaks due to
about 1:2:0.2. impurity A and impurity B.
Content of ammonio methacrylate groups 8.9 per cent to Limits:
12.3 per cent (dried substance). - impurity A: not more than the area of the corresponding
CHARACTERS peak in the chromatogram obtained with the reference
solutiou·(100 ppm);
Appearance
- impurity B: not more than 2.5 times the area of the
Colourless to white or almost white granules or powder.
corresponding peak in the chromatogram obtained with
Solubility the reference solution (50 ppm).
Practically insoluble in water, freely soluble in anhydrous
Methanol (2.4.24, System A)
ethanol and in methylene chloride giving clear to cloudy
Maximum 1.5 per cent.
solutions. Due to the polymeric nature of the substance, a
stirring time of up to 5 h may be necessary. Loss on drying (2.2.32)
Maximum 3.0 per cent, determined on 1.000 g by drying in
IDENTIFICATION vacuo at 80 DC for 5 h.
~~ Infrared absorption spectrophotometry (2.2.24).
ASSAY
Comparison ammonio methacrylate copolymer CRS.
Dissolve 1.000 gin 75 mL of glacial acetic acid Rat about
B. Viscosity (see Tests). 50 DC within about 30 min. Allow to cool to room
C. It complies with the limits of the assay. temperature and add 25 mL of a 6 gIL solution of copper
TESTS acetate R in glacial.acetic acid R. Titrate with 0.1 M perchloric
Solution S acid, determining the end-point potentiometrically (2.2.20).
Dissolve a quantity of the substance to be examined 1 mL of 0.1 M perchloric acid is equivalent to 20.77 mg
corresponding to 12.5 g of the dried substance in a mixture of C 9 H 1S0 2NCI (ammonio methacrylate groups).
of 35.0 g of acetone Rand 52.5 g of 2-propanol R. IMPURITIES
Viscosity (2.2.10) Specified impurities A, B.
Maximum 15 ml'a-s, determined on solution S.
Apparatus Rotating viscometer.
Dimensions:
- spindle: diameter = 25.15 mm; height = 90.74 mm; shaft
=
diameter 4.0 mm; A. ethyl propenoate (ethyl acrylate),
- cylinder: 'diameter = 27.62 mm; height = 0.135 m.
Stirring speed 30 r/min.
Volume of solution 16 mL of solution S.
Temperature 20 -c.
Appearance of a film
Spread 2 mL of solution S evenly on a glass plate. Upon B. methyl 2-methylprop-2-enoate (methyl methacrylate).
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
drying a clear film is formed.

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2020 Ammonio Methacrylate Copolymer 1-161

Monomers
Ammonia Methacrylate Copolymer Liquid chromatography (2.2.29).
(Type B) Solution A· Dissolve 3.5 g of sodium perchlorate R in water R
(Ph. Bur. monograph 2082) and dilute to 100 mL with the same solvent.
Test solution Dissolve 5.00 g of the substance to be
examined in methanol R and dilute to 50.0 mL with the same
solvent. To 10.0 mL of this solution add 5.0 mL of
solution A, dropwise, while continuously stirring. Remove the
precipitated polymer by centrifugation. Use the clear
supernatant solution.
Reference solution Dissolve 50.0 mg of ethyl acrylate Rand
10.0 mg of methyl methacrylate R in methanolR and dilute to
50.0 mL with the same solvent. Dilute 1.0 mL of the
solution to 100.0 mL with methanolR. Add·1 0 mL of this
solution to 5 mL of solution A.
Action and use Column:
Excipient. - size: 1= 0.12 m, 0 = 4.6 mm;
PhEur _ _ -= _ - stationary phase: irregular end-capped octadecylsilyl silicagel
for chromatography R (7 11m).
DEFINITION Mobile phase Mix 20 volumes of methanolR2 and
Poly [ethyl propenoate-co-methyl 2-methylprop-2-enoate-co-N, 80 volumes of waterfor chromatography R previously ajusted
N,N-trimethyl-2-[(2-methylprop-2-enoyl)oxy]ethan-1- to pH 2.0 with phosphoric acid R.
aminiumchloride] having a mean relative molecular mass of
Flow rate 2.0 mIJmin.
about 150 000.
The ratio of ethyl acrylate (ethyl propenoate) groups to Detection Spectrophotometer at 202 nm.
methyl methacrylate (methyl 2-methylprop-2-enoate) groups Injection 50 IlL.
to ammonio methacrylate (N,N,N-trimethyl-2-[(2- System suitability Reference solution:
methylprop-2-enoyl)oxy]ethan-1-aminium chloride) groups is - resolution: minimum 1.5 between the peaks due to
about 1:2:0.1. impurity A and impurity B.
Content of ammonio methacrylate groups 4.5 per cent to Limits:
7.0 per cent (dried substance). - impurity A: not more than the area of the corresponding
peak in the chromatogram obtained with the reference
CHARACTERS
solution (l00 ppm);
Appearance
- impurity B: not more than 2.5 times the area of the
Colourless to white or almost white granules or powder.
corresponding peak in the chromatogram obtained with
Solubility the reference solution (50 ppm).
Practically insoluble in water, freely soluble in anhydrous
Methanol (2.4.24, System A)
ethanol and in methylene chloride giving clear to cloudy
Maximum 1.5 per cent.
solutions. Due to the polymeric nature of the substance, a
stirring time of up to 5 h may be necessary. Loss on drying (2.2.32)
Maximum 3.0 per cent, determined on 1.000 g by drying in
IDENTIFICATION vacuo at 80°C" for 5 h.
A. Infrared absorption spectrophotometry (2.2.24).
ASSAY
Comparison ammonio methacrylate copolymer CRS.
Dissolve 2.000 gin 75 mL of glacial acetic acid R at about
B. Viscosity (see Tests). 50°C within about 30 min. Allow to cool to room
C. It complies with the limits of the assay. temperature and add 25 mL of a 6 gIL solution of copper
TESTS acetate R in glacial acetic acid R. Titrate with 0.1 M perchloric
Solution S acid, determining the end-point potentiometrically (2.2.20).
Dissolve a quantity of the substance to be examined 1 mL of 0.1 M perchloric acid is equivalent to 20.77 mg
corresponding to 12.5 g of the dried substance in a mixture of C 9H1S 0 2N CI (ammonio methacrylate groups).
of 35.0 g of acetone R and 52.5 g of 2-propanol R. IMPURITIES
Viscosity (2.2.10) Specifiedimpurities A, B.
Maximum 15 mf'a-s, determined on solution S.
Apparatus Rotating viscometer.
o
Dimensions: H2CJO ............... CH3
= =
- spindle: diameter 25.15 mID; height 90.74 mm; shaft
diameter = 4.0 mm; A. "ethyl propenoate (ethyl acrylate),
- cylinder. diameter = 27.62 mm; height = 0.135 m.
Stirring speed 30 r/min,
Volume of solution 16 mL of solution S.
Temperature 20 -c.
Appearance of a film
Spread 2 mL of solution S evenly on a glass plate. Upon B. methyl 2-methylprop-2-enoate (methyl methacrylate).
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
drying a clear film is formed.

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1-162 Ammonium Bicarbonate 2020

Ammonium Bicarbonate Ammonium Bromide

(Ammonium Hydrogen Carbonate, Ph. Bur. (Ph. Bur. monograph 1389)
monograph 1390)
NHJk 97.9 12124-97-9
NH 4HC03 79.1 1066-33-7 PhEur _

Action and use DEFINITION

Expectorant. Content
Preparations 98.5 per cent to 101.0 per cent (dried substance).
Aromatic Ammonia Solution CHARACTERS
Strong Ammonium Acetate Solution Appearance
Aromatic Ammonia Spirit White or almost white, crystalline powder or colourless
crystals, hygroscopic.
PhEur ...,.-- _
Solubility
DEFINITION Freely soluble in water, sparingly soluble in ethanol
.Content (96 per cent).
98.0 per cent to 101.0per cent. It becomes yellow when exposed to light or air.
CHARACTERS IDENTIFICATION
Appearance A. It gives reaction (a) of bromides (2.3.1).
Fine, white or almost white, crystalline powder or white or B. 10 mL of solution S (see Tests) gives the reaction of
almost white crystals, slightly hygroscopic. ammonium salts (2.3.1).
Solubility TESTS
Freely soluble in water, practically insoluble in ethanol
Solution S
(96 per cent).
Dissolve 10.0 g in carbon dioxide-free water R and dilute to
It volatilises rapidly at 60°C. The volatilisation takes place 100 mL with the same solvent.
slowly at ambient temperatures if the substance is slightly
Appearance of solution
moist.. It is in a state of equilibrium with ammonium
Solution S is clear (2.2.1) and colourless (2.2.2, Method If).
carbamate.
Acidity or alkalinity
IDENTIFICATION To 10 mL of solution S add 0.05 mL of methyl red solution R.
A. It gives the reaction of carbonates and bicarbonates Not more than 0.5 mL of 0.01 M hydrochloric acid or 0.01 M
(2.3.1). sodium hydroxide is required to change the colour of the
B. Dissolve 50 mg in 2 mL of waterR. The solution gives the indicator.
reaction of ammonium salts (2.3.1). Bromates
TESTS To 10 mL of solution S add 1 mL of starch solution R,
Solution S 0.1 mL of a 100 gIL solution of potassium iodide Rand
Dissolve 14.0 gin 100 mL of distilled water R. Boil to remove 0.25 mL of 0.5 M sulfuric acid and allow to stand protected
the ammonia, allow to cool and dilute to 100.0 mL with from light for 5 min. No blue or violet colour develops.
distilled waterR. Chlorides and sulfates
Chlorides (2.4.4) Liquid chromatography (2.2.29)..
Maximum 70 ppm. Test solution (a) Dissolve 0.400 g of the substance to be
Dilute 5 mL of solution S to 15 mL with waterR. examined in 50 mL of waterfor chromatography R and dilute
Sulfates (2.4.13) to 100.0 mL with the same solvent.
Maximum 70 ppm, determined on solution S. Testsolution (b) Dilute 25.0 mL of test solution (a) to
50.0 mL with waterfor chromatography R. .
Iron (2.4.9)
Maximum 40 ppm. Reference solution (a) To 25.0 mL of test solution (a) add
1.0 mL of sulfate standard solution (10 ppm S04J Rand
Dilute 1.8 rnL of solution S to 10 mL with waterR.
12.0 mL of chloride standard solution (50 ppm Cl) R and dilute
ASSAY to 50.0 mL with waterfor chromatography R.
Dissolve cautiously 0.500 gin 50 mL of carbon dioxide-free Reference solution (b) Dilute 10.0 mL of test solution (a) to
water R. Titrate with 1 M hydrochloric acid, determining the 100.0 mL with waterfor chromatography R. To 2.0 mL of this
end-point potentiometrically (2.2.20). Read the volume solution add 8.0 mL of chloride standard solution
added at the 2n d point of inflection, or at the point of (50 ppm Cl) R and dilute to 20.0 mL with waterfor
inflection if only 1 point is detected. chromatography R.
1 mL of 1 M hydrochloric acid is equivalent to 79.1 mg Blank solution waterfor chromatography R.
ofNH4HC03 .
Column:
STORAGE - size: 1= 0.25 m, 0 = 2 mm;
In an airtight container. - stationary phase: strongly basic anion-exchange resin for
_ _ _ _ _ _----,- PhEur chromatography R (13 urn).
Mobile phase Dissolve 0.600 g of potassium hydroxide R in
waterfor chromatography R and dilute to 1000.0 mL with the
same solvent.

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2020 Ammonium Chloride 1-163

Flow rate 0.4 mIJmin.

Ammonium Chloride
Detection Conductivity detector equipped with a suitable ion
suppressor. (Ph. Eur. monograph 0007)
Injection 50 ~ of test solution (b), reference solutions (a) ~CI 53.49 12125-02-9
and (b) and the blank solution.
Run time 2.5 times the retention time of bromide. Action and use
Retention time Chloride = about 5 min; bromide = about Used for the acidification of urine and to correct metabolic
8 min; sulfate = about 16 min. alkalosis.
System suitability Reference solution (b): Preparation
- resolution: minimum 8.0 between the peaks due to Ammonium Chloride Mixture
chloride and bromide. PhEur _
Calculation ofpercentage contents:
- for chlorides, use the concentration of chloride in DEFINITION
reference solution (a); correct the area of the peak due to Content
chloride in the chromatogram obtained with reference 99.0 percent to 100.5 per cent (dried substance).
solution (a) by subtracting the area of the peak due to CHARACTERS
chloride in the chromatogram. obtained with test Appearance
,solution (b); White or almost white, crystalline powder or colourless
~:Jor sulfates, use the concentration of sulfate in reference crystals.
, solution (a); correct the area of the peak due to sulfate in
Solubility
.the chromatogram obtained with reference solution (a) by
Freely soluble in water.
subtracting the area of the peak due to sulfate in the
chromatogram obtained with test solution (b). IDENTIFICATION
Limi~: A. It gives the reactions of chlorides (2.3.1).
~ chlorides: maximum 0.6 per cent; B. 10 mL of solution S (see Tests) gives the reaction of
- sulfates: maximum 0.01 per cent. ammonium salts (2.3.1).
Iodides 'TESTS
To 5 rnL of solution S add 0.15 mL offerric chloride SolutionS
solution Rl and 2 mL of methylene chloride R. Shake and allow Dissolve 10.0 g in carbon dioxide-free water R prepared from
to separate. The lower layer is colourless (2.2.2~ Method l). distilled water R and dilute to 100 mL with the same solvent.
Iron (2.4.9) Appearance of solution
Maximum 20 ppm. Solution S is clear (2.2.1) and colourless (2.2.2~ Method II).
Dilute 5 mL of solution S to 10 mL with waterR. Acidity or alkalinity
Magnesium and alkaline-earth metals (2.4.7) To 10 mL of solution S add 0.05 mL of methyl red solution R.
Maximum 200 ppm, calculated as Ca. Not more than 0.5 mL of 0.01 M hydrochloric acid or 0.01 M
10.0 g complies with the test for magnesium and alkaline- sodium hydroxide is required to change the colour of the
earth metals. The volume of 0.01 M sodium edetate used does indicator.
not exceed 5.0 mL. Bromides and iodides
Loss on drying (2.2.32) To 10 mL of solution S add 0.1 mL of dz7ute hydrochloric
Maximum 1.0 per cent, determined on 1.000 g by drying in acid Rand 0.05 mL of chloramine solution R. After 1 min, add
an oven at 105°C. 2 mL of chloroform R and shake vigorously. The chloroform
layer remains colourless (2.2.2, Method I).
Sulfated ash (2.4.14)
Maximum 0.1 per cent, determined on 1.0 g. Sulfates (2.4.13)
Maximum 150 ppm.
ASSAY
Dilute 10 mL of solution S to 15 mL with distilled water R.
Dissolve 80.0 mg in water R, add 5 mL of dilute nitric acid R
and dilute to 50 mL with waterR. Titrate with 0.1 M silver Calcium (2.4.3)
nitrate, determining the end-point potentiometrically (2.2.20). Maximum 200 ppm.
1 mL of 0.1 M silvernitrate is equivalent to 9.794 mg of Dilute 5 mL of solution S to 15 mL with distilled water R.
N}4Br. Iron (2.4.9)
Calculate the percentage content of NH4Br using the Maximum 20 ppm.
following expression: Dilute 5 mL of solution S to 10 mL with water R.
Loss on drying (2.2.32).
a-2.763 b
MaximUlll1.0 per cent; .determined on 1.00g by drying in
a percentage content of NI4Br and NH 4Cl obtained in the assay an oven at 105°C for 2 h.
and calculated as NHJ3r; Sulfated ash (2.4.14)
percentage content of Cl obtained in the test for chlorides.
Maximum 0.1 per cent, determined on 2.0 g.

STORAGE ASSAY
In an airtight container, protected from light. Dissolve 1.000 g in 20 mL of waterR and add a mixture of
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur 5 mL of formaldehyde solution R, previously neutralised to
phenolphthalein solution R, and 20 mL of water R. After

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1-164 Ammonium Glycyrrhizinate 2020

1-2 min, titrate slowly with 1 AlI sodium hydroxide, using a Related substances
further 0.2 mL of the same indicator. Liquid chromatography (2.2.29).
1 mL of 1 M sodium hydroxide is equivalent to 53.49 mg Test solution Dissolve 0.100 g of the substance to be
ofNH4 Cl. examined in the mobile phase and dilute to 100,0 mL with
____ ~ PhEur the mobile phase.
Reference solution (a) Dilute 1.0 mL of the test solution to
20.0 mL with the mobile phase.
Reference solution (b) Dissolve 50 mg of ammonium
Ammonium Glycyrrhizinate glycyrrhizate CRS in the mobile phase and dilute to 50.0 mL
with the mobile phase. Dilute 1.0 mL of the solution to
(Ammonium Glycyrrhizate, Ph. Bur. monograph 20.0 mL with the mobile phase.
1772) Column:
- size: 1= 0.25 m, 0 = 4.0 mm,
- stationary phase: octadecylsilyl silica gelfor chromatography R
(5-10 11m).

tE~\
Mobile phase glacial acetic acid R, acetonitrile R, waterR
(6:380:614 V/V/V).
Flow rate 1.2 mUmin.
H~:;-(6 , Detection Spectrophotometer at 254 nm.

H~ H~'Cand
Injection 10 ilL.
epimer at C" , NH, Run time 3 times the retention time of 18~-glycyrrhizic acid.
Relative retention With reference to 18~-glycyrrhizic acid
OH (retention time = about 8 min): impurity A = about 0.8;
18cx.-glycyrrhizic acid = about 1.2.
C42H6SN016 840 53956-04-0 System suitability Reference solution (b):
PhEur .,.,..- _ -resolution: minimum 2.0 between the peaks due to 18~­
glycyrrhizic acid and 18cx.-glycyrrhizic acid.
DEFINITION
Limits:
Mixture of ammonium 18cx.- and 18~-glycyrrhizate
- 18rx-glycyrrhizic acid: not more than twice the sum of the
(ammonium salt of (20~)-3~-[[2-0-(~-D- .
areas of the peaks in the chromatogram obtained with
glucopyranosyluronic acidj-c-n-giucopyranosyluronic acid]
reference solution (a) (10.0 per cent),
oxy]-1l-oxoolean-12-en-29-oic acid), the 18~-isomer being
- impurityA: not more than the sum of the areas of the
the main component.
peaks in the chromatogram obtained with reference
Content solution (a) (5.0 per cent),
98.0 per cent to 102.0 per cent (anhydrous substance). - any otherimpurity: for each impurity, not more than
CHARACTERS 0.4 times the sum of the areas of the peaks in the
Appearance chromatogram obtained with reference solution (a)
White or yellowish-white, hygroscopic powder. (2.0 per cent),
- sum of otherimpurities: not more than 1.4 times the sum of
Solubility the areas of the peaks in the chromatogram obtained with
Slightly soluble in water, very slightly soluble in anhydrous reference solution (a) (7.0 per cent),
ethanol, practically insoluble in acetone. It dissolves in dilute - disregard limit: 0.04 times the sum of the areas of the
solutions of acids and of alkali hydroxides. peaks in the chromatogram obtained with reference
IDENTIFICATION solution (a)' (0.2 per cent).
A. Infrared absorption spectrophotometry (2.2.24). Water (2.5.12)
Comparison ammonium glycyrrhizate CRS. Maximum 6.0 per cent, determined on 0.250 g.
B. Dissolve 0.1 gin 20 mL of water R, add 2 mL of dilute Sulfated ash (2.4.14)
sodium hydroxide solution R and heat cautiously. On heating, Maximum 0.2 per cent, determined on 1.0 g.
the solution gives off vapours that may be identified by the
ASSAY
alkaline reaction of wet litmus paper (2.3.1).
Dissolve 0.600 gin 60 mL of anhydrous acetic add R heating
TESTS at 80°C if necessary, Cool. Titrate with 0.1 M perchloric acid,
Appearanceofswution determining the end-point potentiometrically (2.2.20).
The solution is clear (2.2.1) and not more intensely coloured 1 mL of 0.1 M perchloric acid is equivalent to 84.0 mg
than reference solution BY7 (2.2.2~ Method 1). of C42H6SN016'
Dissolve 1.0 g in ethanol (20 per cent V/"V,> R and dilute to
STORAGE
100.0 mL with the same solvent.
In an airtight container.
Specific optical rotation (2.2.7)
+ 49.0 to + 54.0 (anhydrous substance).
Dissolve 0.5 gin ethanol (50 per cent V/"V,> R and dilute to
50.0 mL with the same solvent.

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2020 Amobarbital 1-165

IMPURITIES Reference solution Dissolve 0.1 g of amobarbital CRS in

alcohol R and dilute to 100 mL with the same solvent.
Apply separately to the plate 10 ul, of each solution. Develop
over a path of 18 em using the lower layer from a mixture of
5 volumes of concentrated ammonia R, 15 volumes of alcohol R
and 80 volumes of chloroform R. Examine immediately in
ultraviolet light at 254 urn. The principal spot in the
chromatogram obtained with the test solution is similar in
position and size to the principal spot in the chromatogram
obtained with the reference solution.
D. It gives the reaction of non-nitrogen substituted
barbiturates (2.3.1).
TESTS
Appearance of solution
A. (4~,20P)-3P-[[2-0-(P-D-glucopyranosyluronic acidj-e-n-
Dissolve 1.0 g in a mixture of 4 mL of dilute sodium hydroxide
glucopyranosyluronic acid] oxy]-23-hydroxy-11-oxoolean-
solution Rand 6 mL of waterR. The solution is clear (2.2.1)
12-en-29-oic acid (24-hydroxyglycyrrhizinic acid).
and not more intensely coloured than reference solution Y 6
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
(2.2.2, Method II).
Acidity or alkalinity
To 1.0 g add 50 mL of waterR and boil for 2 min. Allow to
*** cool and filter, To 10 mL of the filtrate add 0.15 mL of
Amobarbital
** ** methyl red solution Rand 0.1 mL of 0.01 M sodium hydroxide.
The solution is yellow. Add 0.2 mLof 0.01 M hydrochloric
(lJl,I;:.Bur. monograph 0594) *****
acid. The solution is red. .
Related substances
o, ,~~, ° Examine by thin-layer chromatography (2.2.27), using silica
;g;,
H3C I
NH gel GF254 R as the coating substance.
Test solution Dissolve 1.0 g of the substance to be examined
H3C
CH3
° in alcohol R and dilute to 100 mL with the same solvent.
Reference solution Dilute 0.5 mL of the test solution to
100 mL with alcohol R.
226.3 57-43-2 Apply separately to the plate 20 IlL of each solution. Develop
over a path of 15 em using the lower layer from a mixture of
Action and use 5 volumes of concentrated ammonia R, 15 volumes of alcohol R
Barbiturate. and 80 volumes of chloroform R. Examine the plate
PhEur _ immediately in ultraviolet light at 254 nm, Any spot in the
chromatogram obtained with the test solution, apart from the
DEFINITION principal spot, is not more intense than the spot in the
Amobarbital contains not less than99.0 per cent and not chromatogram obtained with the reference solution. Spray
more than the equivalent of 101.0 per cent of 5-ethyl-5-(3- with diphenylcarbazone mercuric reagent R. Allow the plate, to
methylbutyl)pyrimidin-2,4,6(lH,3H,5H)-trione, calculated dry in air and spray with freshly prepared alcoholic potassium ,
with reference to the dried substance. hydroxide solution R diluted 1 in 5 with aldehyde-free alcohol R.
CHARACTERS Heat at 100 DC to 105 DC for 5 min and examine
A white or almost white, crystalline powder, very slightly immediately. Any spot in the chromatogram obtained with
soluble in water, freely soluble in alcohol, soluble in the test solution, apart from the principal spot, is not more
methylene chloride. It forms water-soluble compounds with intense than the spot in the chromatogram obtained with the
alkali hydroxides and carbonates and with ammonia. reference solution (0.5 per cent).
Loss on drying (2.2.32)
IDENTIFICATION
Not more than 0.5 per cent, determined on 1.000 g by
First identification: A, B.
drying in an oven at 105 DC.
Second identification: A, C, D.
Sulfated ash (2.4.14)
A. Determine the melting point (2.2.14) of the substance to
Not more than 0.1 per cent, determined on 1.0 g.
be examined. Mix equal parts of the substance to be
examined and amobarbital CRS and determine the melting ASSAY
point of the mixture. The difference between the melting Dissolve ,0.100 g in 5 mL of pyridine R. Add 0.5 mL of
points (which are about 157 DC) is not greater than 2 DC. thymolphthalein solution Rand 10 mL of silvernitrate solution
B. Examine by infrared absorption spectrophotometry in pyridine R. Titrate with 0.1' M ethanolic sodium hydroxide
(2.2.24), comparing with the spectrum obtained with until a pure bluecolouris obtained. Carry out a blank
titration.
amobarbital CRS.
C. Examine by thin-layer chromatography (2.2.27), using 1 mL of 0.1 M ethanolic sodium hydroxide is equivalent to
silicagel GF254 R as the coating substance. 11.31 mg of CllHlSNz03'
--- --:.. Ph Eur
Test solution Dissolve 0.1 g of the substance to be examined
in alcohol R and dilute to 100 mL with the same solvent. '

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 1-166 Amobarbital Sodium 2020

TESTS
Amobarbital Sodium Solution S
(Ph. Eur. monograph 0166) Dissolve 5.0 g in alcohol (50 per cent VIV) R and dilute to
50 mL with the same solvent.

~
o ~yONa Appearance of solution
Solution S is clear (2.2.1) and not more intensely coloured
H3C I
N than reference solution Y7 (2.2.2, Method II).
pH (2.2.3)
H3C 0
Dissolve 5.0 g in carbon dioxide-free water R and dilute to
CH3
50mL with the same solvent. Disregard any slight residue.
The pH of the solution is not more than 11.0.
248.3 64-43-7 Related substances
Examine by thin-layer chromatography (2.2.27), using silica
Action and use
gel GF254 R as the coating substance.
Barbiturate.
Test solution Dissolve 1.0 g of the substance to be examined
_ PhEur --'--_ _ in alcohol R and dilute to 100 mL with the same solvent.
DEFINITION Reference solution Dilute 0.5 mL of the test solution to
Amobarbital sodium contains not less than 98.5 per cent and 100 mL with alcohol R.
not more than the equivalent of 102.0 per cent of sodium Apply separately to the plate 20 ul, of each solution. Develop
derivative of 5-ethyl-5-(3-methylbutyl)pyrimidin-2,4,6 over a path of 15 cm using the lower layer of a mixture of
(IH,3H,5H)-trione, calculated with reference to the dried 5 volumes of concentrated ammonia R, 15 volumes of alcohol R
substance. and 80 volumes of chloroform R. Examine the plate
immediately in ultraviolet light at 254 nm.Spray with
CHARACTERS
diphenylcarbazone mercuric reagent R. Allow the plate to dry in
A white or almost white, granular powder, hygroscopic, very
air and spray with freshly prepared alcoholic potassium
soluble in carbon dioxide-free water (a small fraction may be
hydroxide solution R diluted 1 in 5 with aldehyde-free alcohol R.
insoluble), freely soluble in alcohol.
Heat at 100 "C to 105 "C for 5 min and examine
IDENTIFICATION immediately. When examined in ultraviolet light and after
First identification: A, B, E. spraying, any spot in the chromatogram obtained with the
Second identification: A, C, D, E. test solution, apart from the principal spot, is not more
intense than the spot in the chromatogram obtained with the
A. Acidify 10 mL of solution S (see Tests) With dilute
reference solution (0.5 per cent). Disregard any spot at the
hydrochloric add R and shake with 20 mL of etherR. Separate
point of application.
the ether layer, wash with 10 mL of waterR, dry over
anhydrous sodium sulfateR and filter. Evaporate the filtrate to Loss on drying (2.2.32)
dryness and dry the residue at 100°C to 105 "C (test Not more than 3.0 per cent, determined on 0.50 g by drying
residue). Repeat the operations using 0.1 g of amobarbital in an oven at 130 "C.
sodium CRS (reference residue). Determine the melting point ASSAY
(2.2.14) of the test residue. Mix equal parts of the test Dissolve 0.200 ginS mL of ethanol R. Add 0.5 mL of
residue and the reference residue and determine the melting thymolphthalein solution Rand 10 mL of silver nitrate solution
point of the mixture. The difference between the melting in pyridine R. Titrate with 0.1 M ethanolic sodium hydroxide
points (which are about 157°C) is not greater than 2°C. until a pure blue colour is obtained. Carry out a blank
B. Examine by infrared absorption spectrophotometry titration.
(2.2.24), comparing the spectrum obtained with the reference 1 mL of 0.1 M ethanolic sodium hydroxide is equivalent to
residue prepared from amobarbital sodium CRS with that 24.83 mg of CuH17N2Na03'
obtained with the test residue (see identification test A).
C. Examine by thin-layer chromatography (2.2.27), using
STORAGE
silica gel GF254 R as the coating substance. Store in an airtight container.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _- - - - - PhEur
Test solution Dissolve 0.1 g of the substance to be examined
in alcohol R and dilute to 100 mL with the same solvent.
Reference solution Dissolve 0.1 g of amobarbital sodium CRS
in alcohol R and dilute to 100 mL with the same solvent.
Apply separately to the plate 10 ul, of each solution. Develop
over a path of 18 em using the lower layer of a mixture of
5 volumes of concentrated ammonia R, 15 volumes of alcohol R
and 80 volumes of chloroform R. Examine immediately in
ultraviolet light at 254 nm. The principal spot in the
chromatogram obtained with the test solution is similar in
position and size to the principal spot in the chromatogram
obtained with the reference solution.
D. It gives the reaction of non-nitrogen substituted
barbiturates (2.3.1).
E. It gives reaction (a) of sodium (2.3.1).

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2020 Amorolfine Hydrochloride 1-167

Mobilephase:
Amorolfine Hydrochloride - mobile phase A: acetonitrile Rl, buffer solution, methanolR2
(Ph. Bur. monograph 2756) (5:35:60 VIVIV);
- mobile phase B: buffer solution, acetonitrile Rl, methanolR2
(10:30:60 VIVIV);

Hel Time Mobile phase A Mobile phase B

(min) (per cent VIJI) (per cent VIV)
0-2 90 10
2 - 25 90 -> 0 10 -> 100

354.0 78613-38-4 Flow rate 1.5 mUmin.

Action and use Detection Spectrophotometer at 214 nm.
Antifungal. Injection 20 ilL.
PhEur _ Identification of impurities· Use the chromatogram supplied
with amorolfine for system suitabz1ity CRS and the
DEFINITION chromatogram obtained with reference solution (a) to
(2RS,6SR)~2,6-Dimethyl-4-[(2RS)-2-niethyl-3-[4-(2­ identify the peaks due to impurities D, E, I and J; use the
methylbut~-2-yl)phenyl]propyl]morpholine
hydrochloride. chromatogram supplied with amorolfine for peak
Content identification CRS and the chromatogram obtained with
99.0 per cent to 101.0 per cent (dried substance). reference solution (c) to identify the peaks due to impurity M
(peaks 1 and 2).
CHARACTERS
Relative retention With reference to amorolfine (retention
Appearallce
time = about 15 min): impurity M(peak 1) = about 0.56;
"White or almost white, crystalline powder.
impurity M (peak 2) = about 0.60; impurity D = about 0.85;
Solubility impurity J = about 0.97; impurity I = about 1.05; impurity E
Slightly soluble in water, soluble in methanol and in (peak 1) = about 1.14; impurity E (peak 2)= about 1.17.
methylene chloride. System suitability:
IDENTIFICATION - resolution: minimum 2.0 between the peaks due to
A. Infrared absorption spectrophotometry (2.2.24). impurity J and amorolfine in the chromatogram obtained
Comparison amorolfine hydrochloride CRS. with reference solution (a); (
- signal-to-noise ratio: minimum 20 for the principal peak in
B. To 20 mg add 4.0 mL of waterR, and acidify with dilute
the chromatogram obtained with reference solution (b).
nitric acid R. A precipitate is formed. Centrifuge, and to
2 mL of the supernatant add 0.4 mL of silvernitrate Calculation of percentage contents:
solution Rl. A curdled, white precipitate is formed. Centrifuge - for each impurity, use the concentration of amorolfine
and wash the precipitate with 3 quantities, each of 1 mL, of hydrochloride in reference solution (b).
water R. Suspend the precipitate in 2 mL of waterR and add Limits:
1.5 mL of ammonia R. The precipitate dissolves easily with - impurity D: maximum 0.2 per cent;
the possible exception of a few large particles which dissolve - impurity B: maximum 0.2 per cent for the sum of the
slowly. areas of the 2 peaks;
- impurity I: maximum 0.15 per cent;
TESTS
- impurityM : maximum 0.15 per cent for each peak;
Related substances - unspecified impurities: for each impurity, maximum
Liquid chromatography (2.2.29). 0.10 per cent;
Buffer solution Dissolve 3.5 g of dipotassium hydrogen - total: maximum 0.4 per cent;
phosphate R in 1000 mL of waterfor chromatography Rand - reporting threshold: 0.05 per cent.
adjust to pH 7.0 with phosphoric acid R.
Loss on drying (2.2.32)
Test solution Dissolve 20 mg of the substance to be Maximum 1.0 per cent, determined on 1.000 g by drying in
examined in mobile phase A and dilute to 20.0 mL with an oven at 105°C for 3 h.
mobile phase A.
Sulfated ash (2.4.14)
Reference solution (a) Dissolve 4 mg of amorolfine for system Maximum 0.1 per cent, determined on 1.0 g.
suitabz1ity CRS (containing impurities D, E, I and D in
mobile phase A and dilute to 5 mL with mobile phase A. ASSAY
Reference solution (b) Dilute 1.0 mL of the test solution to Dissolve 0.250 g in a mixture of 10.0 mL of 0.01 M
100.0 mL with mobile phase A. Dilute 1.0 mL of this hydrochloric acid and 40 mL of ethanol (96 per cent) R. Carry
solution to 10.0 mL with mobile phase A. out a potentiometric titration (2.2.20), using 0.1 M sodium
hydroxide. Read the volume added between the 2 points of
Reference solution (c) Dissolve 4 mg of amorolfine for peak
inflexion.
identification CRS (containing impurity M) in mobile phase A
and dilute to 5 mL with mobile phase A. ' 1 mL of 0.1 M sodium hydroxide is equivalent to 35.40 mg of
C 2 1H36ClNO.
Column:
- size: 1= 0.15 m, 0 = 4.6 mm; IMPURITIES
- stationary phase: end-capped amidohexadecylsilyl silica gelfor Specified impurities D, B} I~ M.
chromatography R (3J.lITI). Other detectable impurities (thefollowz'ng substances would) if
present at a sufficientlevel, be detected by one or otherof the tests

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1-168 Amorolfine Hydrochloride 2020

in the monograph. They are limited by the general acceptance o

criterion for other/unspecified impurities and/or by the general
monograph Substances for pharmaceutical use (2034). It is ~c~rJ~ .
therefore not necessary to identify these impurities for ~ ~d ~.~C~
demonstration of compliance. See also 5.10. Control of impurities H CH3 H3C CH3
in substances for pharmaceutical use) A, B~ C, F~ G, H~ J~ K~ and enantiomer

L~ O.
G. (ZRS)-3-[ (2RS,6SR)-2,6-dimethylmorpholin-4-yl] -1-[4-(Z-
methylbutan-2-yl)phenyl]-Z-methylpropan-1-one,

A. (ZRS,48,6SR)-2,6-dimethyl-4-[(ZRS)':2-methyl-3-[4-(2-
methylbutan-Z-yl)phenyl]propyl]morpholine 4-oxide,
H. Z- [(2RS)- 3- [(2RS,6SR)-2,6-dimethylmorpholin-4-yl]-Z-
methylpropyl] -5-(Z-methylbutan-Z-yl)phenol,

its epimer at C* and their enantiomers

B. mixture of (2RS)-1-[N-[(ZR)-2-methyl-3-[4-(Z-
methylbutan-Z-yl)phenyl]propyl]formamido]propan-Z-yl
acetate and (ZRS)-1-[N-[(ZS)-2-methyl-3-[4-(Z- I. (ZRS,6SR)-2,6-dimethyl-4-[(2RS)-Z-methyl-3-[4-[(Z8)-3-
methylbutan-Z-yljphenyl]propyl]formamido] propan-Z-yl methylbutan-2-yl]phenyl]propyl] morpholine,
acetate,

H
H ~H3C
CH3 CH

H'CQ~
H3C"~N.
oy .:. .
H" CH3
~
~ I
3

H CH3 and enantiomer

H CH3 and enantiomer

C. (ZRS,6SR)-2,6-dimethyl-4- [(2RS)-2-methyl-3- J. (ZRS,6SR)-Z,6-dimethyl-4-[(2RS)-Z-methyl-3-[3-(Z-

phenylpropyl]morpholine, , methylbutan-2-yl)phenyl]propyl] morpholine,

H H
H3C"~N~
oy H CH'~CH' ~c-rJ~. I ..
y ". ". ""'3.~CH3
H CH3 H3C CH3 H CH3 .. H3C CH3
and enantiomer and enantiomer

D. (ZRS,6SR)-Z,6-dimethyl-4-[(ZRS)-3-(4-tert-butylphenyl)- K. (ZRS,6SR)-Z,6-dimethyl-4-[(2RS)-2-methyl-3-[4-(3-
Z-methylpropyl] morpholine, methylpentan- 3-yl)pheI~.yI]propyl]morpholine,

CH3
H
CH3
H3C"~N
oy
H CH3
its epimer at C* and their enantiomers
and enantiomer CH3
E. mixture of (ZRS,6RS)-Z,6-dimethyl-4-[(ZR)-Z-methyl-3-
[4-(Z-methylbutan-2-yl)phenyl] propyl] morpholine and L. (ZRS,6SR)-4- [(2RS)- 3-[3,5-bis(Z-methylbutan-2-yl)
(ZRS,6RS)-Z,6-dimethyl-4-[(2S)-2-methyl-3-[4-(Z- phenyl] -Z-methylpropyl]-2,6-dimethylmorpholine,
methylbutan-Z-yl)phenyl] propyl] morpholine,

H3C~.
. . ~ .I . ....
~" CH
3
H3C CH3

F. 1-[4-(Z-methylbutan-2-yl)phenyl] propan-1-one,

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 2020 Amoxicillin Sodium 1-169

H ~H
... OH 10 min in iced water. Filter the crystals and wash with

H'C-r;H,iH:
y
I
CH3
2-3 mL of a mixture of 1 volume of water Rand 9 volumes
of acetone R, then dry in an oven at 60 DC for 30 min.
Comparison amoxicillin trihydrate CRS.
H b~ ~C C~
B. Thin-layer chromatography (2.2.27).
its epimer at C* and their enantiomers
Test solution Dissolve 25 mg of the substance to be
examined in 10 mL of sodium hydrogen carbonate solution R.
M.mixture of (IRS,2RS)-3-[(2RS,6SR)-2,6-
dimethylmorpholin-4-yl] -2-methyl-l-[4-(2-methylbutan-2- Reference solution (a) Dissolve 25 mg of amoxicillin
yljphenyljpropan-l-ol and (IRS,2SR)-3-[(2RS,6SR)-2,6- trihydrate CRS in 10 mL of sodium hydrogen carbonate
dimethylmorpholin-4-yl] -2-methyl-l-[4-(2-methylbutan-2- solution R.
yl)phenyl]propan-l-ol, Reference solution (b) Dissolve 25 mg of amoxicillin
trihydrate CRS and 25 mg of ampicillin trihydrate CRS in
H 10 mL of sodium hydrogen carbonate solution R.
H3C"~NXY]
oy H- CH, ~CH'
Plate TLC silanised silica gelplateR.
Mobilephase Mix 10 volumes of acetone Rand 90 volumes
H CH3 - CH3 of a 154 gIL solution of ammonium acetate R previously
and enantiomer adjusted to pH 5.0 with glacial acetic acid R.
Application 1 J.Ll...
O.-(2RS,6SR)-2,6-dimethyl-4-[(2RS)-2-methyl-3-[4-(propan- Development Over a path of 15 cm.
2-yl)phenyl]propyl] morpholine.
Drying In air.
---,- ---,- -'- PhEur
Detection Expose to iodine vapour until the spots appear
and examine in daylight.
System suitability Reference solution (b):
- the chromatogram shows 2 clearly separated spots.
Amoxicillin Sodium Results The principal spot in the chromatogram obtained
(Ph. Bur. monograph 0577) with the test solution is similar in position, colour and size to
the principal spot in the chromatogram obtained with
reference solution (a).
C. Place about 2 mg in a test-tube about 150mm long and
about 15 mm in diameter. Moisten with 0.05 mL of waterR
and add 2 mL of sulfuric acid-formaldehyde reagent R. Mix the
contents of the tube by swirling; the solution is practically
colourless. Place the test-tube in a water-bath for 1 min;
387.4 34642-77-8 a dark yellow colour develops.
D. It gives reaction (a) of sodium (2.3.1).
Action and use
TESTS
Penicillin antibacterial.
Appearance of solution
Preparations The solution is not more opalescent than reference
Amoxicillin Injection suspension II (2.2.1), it may show an initial, but transient,
Co-amoxiclav Injection pink colour, and after 5 min, its absorbance (2.2.25) at
PhEur --'-
430 nm is not greater than 0.20.
Dissolve 1.0 g in waterR and dilute to 10.0 mL with the
DEFINITION same solvent. Examine immediately after dissolution.
Sodium (2S,5R,6R)-6-[[(2R)-2-amino-2-(4- pH (2.2.3)
. hydroxyphenyl) acetyl]amino]-3,3-dimethyl-7-oxo-4-thia-l-
8.0 to 10.0.
azabicyclo[3.2.0]heptane-2-carboxylate.
Dissolve 2.0 g in carbon dioxide-free water R and dilute to
Semi-synthetic product derived from a fermentation product.
20 mL with the same solvent.
Content Specific optical rotation (2.2: 7)
89.0 per cent to 102.0 per cent (anhydrous substance).
+ 240 to + 290 (anhydrous substance).
CHARACTERS Dissolve 62.5 mg in a 4 gIL solution of potassium hydrogen
Appearance phthalate R and dilute to 25.0 mL with the same solution.
White or almost white, very hygroscopic, powder.
Related substances
Solubility Liquid chromatography (2.2.29).
Very soluble in water, sparingly soluble in anhydrous ethanol, Test solution (aJ Dissolve 30.0 mg of the substance to be
very slightly soluble in acetone. examined in mobile phase A and dilute to 50.0 mL with
IDENTIFICATION mobile phase A.
First identification: A, D. Test solution (b) Dissolve 30.0 mg of the substance to be
Secondidentification: B, C, D. examined in mobile phase A and dilute to 20.0 mL with
A. Infrared absorption spectrophotometry (2.2.24). mobile phase A. Prepare immediately before use.
Preparation Dissolve 0.250 g in? mL of waterR, add
0.5 mL of dilute acetic acid R, swirl and allow to stand for

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1-170 Amoxicillin Sodium 2020

Reference solution (a) Dissolve 30.0 mg of amoxicillin Limits:

trihydrate CRS in mobile phase A and dilute to 50.0 mL with - impurity J (n = 1): not more than 3 times the area of the
mobile phase A. principal peak in the chromatogram obtained with
Reference solution (b) Dissolve 4.0 mg of cefadroxil CRS in reference solution (c) (3 per cent);
mobile phase A and dilute to 50 mL with mobile phase A. - any otherimpurity: for each impurity, not more than twice
To 5.0 mL of this solution add 5.0 mL of reference the area of the principal peak in the chromatogram
solution (a) and dilute to) 00 mL with mobile phase A. obtained with reference solution (c) (2 per cent);
- total: not more than 9 times the area of the principal peak
Reference solution (c) Dilute 2.0 mL of reference solution (a)
in the chromatogram obtained with reference solution (c)
to 20.0 mL with mobile phase A. Dilute 5.0 mL of this
(9 per cent);
solution to 20.0 mL with mobile phase A.
- disregard limit: 0.1 times the area of the principal peak in
Reference solution (d) To 0.20 g of amoxicillin trihydrate R the chromatogram obtained with reference solution (c)
add 1.0 mL of water R. Shake and add dropwise dilute sodium (0.1 per cent).
hydroxide solution R to obtain a solution. The pH of the
solution is about 8.5. Store the solution at room temperature
N,N-Dimethylaniline (2.4.26, Method A or B)
for 4 h. Dilute 0.5 mL of this solution to 50.0 mL with Maximum 20 ppm.
mobile phase A. 2-Ethylhexanoic acid (2.4.28)
Column: Maximum 0.8 per cent m/m.
=
- size: I 0.25 m, 0 =
4.6 mm; Water (2.5.12)
- stationary phase: octadecylsilyl silica gelfor chromatography R Maximum 3.0 per cent, determined on 0.400 g.
(5 urn). . Bacterial endotoxins (2.6.14)
Mobile phase: Less than 0.25IU/mg, if intended for use in the manufacture
- mobile phaseA: mix 1 volume of acetonitrile Rand of parenteral preparations without a further appropriate
99 volumes of a 25 per cent V/V solution of 0.2 M procedure for the removal of bacterial endotoxins.
potassium dihydrogen phosphate R adjusted to pH 5.0 .with
ASSAY
dilute sodium hydroxide solution R;
Liquid chromatography (2.2.29) as described in the test for
- mobile phase B: mix 20 volumes of acetonitrile R and
related substances with the following modifications.
80 volumes of a 25 per cent V/V solution of 0.2 M
potassium dihydrogen phosphate R adjusted to pH 5.0 with Mobilephase Initial composition of the mixture. of mobile
dilute sodium hydroxide solution R; phases A and B, adjusted where applicable.
Injection Test solution (a) and reference solution (a).
Time Mobile phase A Mobile phase B System suitabzlity Reference solution (a):
(min) . (per cent VIV) (per cent VIV)
- repeatability: maximum relative standard deviation of
0- tR 92 8 1.0 per cent after 6 injections.
tR - (tR + 25) 92 -> 0 8 -+ 100
Calculate the percentage content of amoxicillin sodium by
(tR + 25) - (tR + 40) 0 100
multiplying the percentage. content of amoxicillin by 1.060.
(tR + 40) - (tR + 55) 92 8
STORAGE
tR = retention time of amoxicillin determined with reference solution (c)
In an airtight container. If the substance is sterile, store in a
If the mobile phase has been adjusted to achieve the required sterile, airtight, tamper-proof container.
resolution, the adjusted composition will apply at time zero IMPURITIES
in the gradient and in the assay.
Flow rate 1.0 mUmin.
Detection Spectrophotometer at 254 run.
Injection 50 J,tL of reference solutions (b) and (c) with
isocratic elution at the initial mobile phase composition and
50 J,tL of test solution (b) and reference solution (d)
A. (2S,5R,6R)-6-amino-3,3-dimethyl-7-oxo-4-thia-l-
according to the elution gradient described under Mobile
azabicydo[3.2.0]heptane-2-carboxylic acid
phase; inject mobile phase A as a blank according to the
(6-aminopenicillanic acid),
elution gradient described under Mobile phase.
Identification of impurities Use the chromatogram obtained
with reference solution (d) to identify the 3 principal peaks
eluted after the main peak corresponding to impurity C,
amoxicillindimer (impurity J; n = 1) and amoxicillin trimer
(impurity J; n = 2).
Relative retention With reference to amoxicillin:
=
impurity C about 3.4; impurity J (n 1) about 4.1; = = B. (2S,5R,6R)-6-[[(2S)-2-amino-2-(4-hydroxyphenyl)acetyl]
impurity J (n = 2) = about 4.5.
amino] -3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]
System suitability Reference solution (b): heptane-Z-carboxylic acid (t-amoxicillin),
- resolution: minimum 2.0 between the peaks due to
amoxicillin and cefadroxil; if necessary, adjust the ratio A:
B of the mobile phase. .

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2020 Amoxicillin Trihydrate 1-171

HN/H ;/o,H]
[HO~~S
HN, )<CH 3

. CH, n

HN 0 ~.,
C0 2H

dr 1+
C. (4S)-2- [5-(4-hydroxyphenyl)-3,6-dioxopiperazin-2-yl]';'5,5- CH3
H. H
dimethylthiazolidine-4-carboxylic acid (amoxicillin ~. N-· S CH3
diketopiperazines), I ' H H
HO # °
J. co-oligomers of amoxicillin and penicilloic acids of
amoxicillin,

D. (4S)-2- [[[(2R)-2-amino- 2-(4-hydroxyphenyl) acetyl]

'1.,+amino] carboxymethyl]-5,5-dimethylthiazolidine-4-
carboxylic acid (penicilloic acids of amoxicillin),

~ C0 2H

',,.. ° H NH
2
HNX--CH3
~ . . . . . ·f's
L. )<CH 3

dr
and epimer at C*
~ '
K. oligomers of penicilloic acids of amoxicillin.
HO
I # H
_ _ _ _ _ _ _ _ _ _ _---'----, - - PhEur

E. (2RS,4S)-2-[[[(2R)-2-amino-2-( 4-hydroxyphenyl)
acetyl]amino] methyl]-5,5-dimethylthiazolidine-4-
carboxylic acid (penilloic acids of amoxicillin),
Amoxicillin. Trihydrate
(ph. Bur. monograph 0260)

F. 3-(4-hydroxyphenyl)pyrazin-2-01,

HO,
-
' :
-o-t 1+~.
H
NH
0
2

0,
H
C0 2H 61336-70-7
, , ' HN H CH3

~°
H
~ , N-- S CH3 Action and use
I H H Penicillin antibacterial.
HO # Preparations
Amoxicillin Capsules
G. (2S,5R,6R)-6-[[(2R)-2-[[(2R)-2-amino-2-(4-
hydroxyphenyl) acetyl] amino] 2-( 4-hydroxyphenyl) acetyl] Amoxicillin Oral Suspension
amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0] Co-amoxiclav Oral Suspension
heptane2-carboxylic acid (0-(4-hydroxyphenyl) Co-amoxiclav Tablets
glycylamoxicillin) , Co-amoxiclav Dispersible Tablets
PhEur _

DEFINITION
(2S,5R,6R)-6-[ [(2R)-2-Amino-2-( 4-hydroxyphenyl)
acetyl]amino] -3,3-dimethyl-7-oxo-4-thia-1-azabicyclo [3.2.0]
heptane-2-carboxylic acid trihydrate.
Semi-synthetic product derived from a fermentation product.
H. (2R)-2-[ (2,2-dimethylpropanoyl)amino]-2-(4-
Content
hydroxyphenyl) acetic acid,
95.0 per cent to 102.0 per cent (anhydrous substance).
CHARACTERS
Appearance
White or almost white, crystalline powder.

I. (2R)-2-amino-2-(4-hydroxyphenyl)acetic acid,

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1-172 Amoxicillin Trihydrate 2020

Solubility Reference solution (a) Dissolve 30.0 mg of amoxicillin

Slightly soluble in water, very slightly soluble in ethanol trihydrate CRS in mobile phase A and dilute to 50.0 ml, with
(96 per cent), practically insoluble in fatty oils. It dissolves in mobile phase A .
dilute acids and dilute solutions of alkali hydroxides. Reference solution (b) Dissolve 4.0 mg of cefadroxil CRS in
IDENTIFICATION mobile phase A and dilute to 50 mL with mobile phase A.
First identification: A. To 5.0 mL of this solution add 5.0 mL of reference
solution (a) and dilute to 100 mL with mobile phase A.
Second identification: B, C.
Reference solution (c) Dilute 2.0 mL of reference solution (a)
A. Infrared absorption spectrophotometry (2.2.24).
to 20.0 mL with mobile phase A. Dilute 5.0 mL of this
Comparison amoxicillin trihydrate CRS. solution to 20.0 mL with mobile phase A.
B. Thin-layer chromatography (2.2.27). Column:
Test solution Dissolve 25 mg of the substance to be - size: l = 0.25 m, 0 4.6 mm; =
examined in 10 mL of sodium hydrogen carbonate solution R. - stationary phase: octadecylsilyl silica gelfor chromatography R
Reference solution (a) Dissolve 25 mg of amoxicillin (5 1J.IIl).
trihydrate CRS in 10 mL of sodium hydrogen carbonate Mobilephase:
solution R. - mobile phase A: acetonitrile R, buffer solution pH 5.0
Reference solution (b) Dissolve 25 mg of amoxicillin (1:99 V/V);
trihydrate CRS and 25 mg of ampicillin trihydrate CRS in - mobile phase B: acetonitrile R, buffer solution pH 5.0
10 rnL of sodium hydrogen carbonate solution R. (20:80 V/V);
Plate TLC silanised silicagel plate R.
Time Mobile phase A Mobile phase B
Mobile phase Mix 10 volumes of acetone Rand 90 volumes (min) (per cent VIJI) (per cent VIJI)
of a 154 gIL solution of ammonium acetate R previously
0- tR 92 8
adjusted to pH 5.0 with glacial acetic acid R.
tR - (tR + 25) 92 -+ 0 8 --> 100
Application 1 J.LL. (tR + 25) - (tR + 40) 0 100
Development Over a path of 15 em. (tR + 40) - (tR + 55) 92 8
Drying In air. tR = retention time of amoxicillin determined with reference solution (c)
Detection Expose to iodine vapour until the spots appear
and examine in daylight. If the mobile phase composition has been adjusted to achieve
System suitability Reference solution (b): the required resolution, the adjusted composition will apply
- the chromatogram shows 2 clearly separated spots. at time zero in the gradient and in the assay.
Results The principal spot in the chromatogram obtained Flow rate 1.0 mL/min.
with the test solution is similar in position, colour and size to Detection Spectrophotometer at 254 nm.
the principal spot in the chromatogram obtained with Injection 50 J.LL of reference solutions (b) and (c) with
reference solution (a). isocratic elution at the initial mobile phase composition and
C. Place about 2 mg in a test-tube about 150 mm long and 50 J.LL of test solution (b) according to the elution gradient
about 15 mm in diameter. Moisten with 0.05 mL of water R described under Mobile phase; inject mobile phase A as a
and add 2 mL of sulfuric acid-formaldehyde reagent R. Mix the blank according to the elution gradient described under
contents of the tube by swirling; the solution is practically Mobile phase.
colourless. Place the test-tube in a water-bath for 1 min; System suitability Reference solution (b):
a dark yellow colour develops. - resolution: minimum 2.0 between the peaks due to
TESTS amoxicillin and cefadroxil; if necessary, adjust the ratio A:
B of the mobile phase.
Solution S
With the aid of ultrasound or gentle heating, dissolve 0.100 g Limit:
in carbon dioxide-free water R and dilute to 50.0 mL with the - any impurity: for each impurity, not more than the area of
same solvent. the principal peak in the chromatogram obtained with
reference solution (c) (1 per cent).
pH (2.2.3)
3.5 to 5.5 for solution S. N,N-Dimethylaniline (2.4.26, Method A or B)
Maximum 20 ppm.
Specific optical rotation (2.2.7)
+ 290 to + 315 (anhydrous substance), determined on Water (2.5.12)
solution S. 11.5 per cent to 14.5 per cent, determined on 0.100 g.
Related substances Sulfated ash (2.4.14)
Liquid chromatography (2.2.29). Maximum 1.0 per cent, determined on 1.0 g.
Buffer solution pH 5.0 To 250 rnL of 0.2 M potassium ASSAY
dihydrogen phosphateR add dilute sodium hydroxide solution R Liquid chromatography (2.2.29) as described in the test for
to pH 5.0 and dilute to 1000.0 mL with water R. related substances with the following modifications.
Test solution (a) Dissolve 30.0 mg of the substance to be Mobile phase Initial composition of the mixture of mobile
examined inmobile phase A and dilute to 50.0 mL with phases A and B, adjusted where applicable.
mobile phase A: Injection Test solution (a) and reference solution (a).
Test solution (b) Dissolve 30.0 mg of the substance to be System suitability Reference solution (a):
examined in mobile phase A and dilute to 20.0 mL with - repeatability: maximum relative standard deviation. of
mobile phase A. Prepare immediately before use. 1.0 per cent after 6 injections.

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2020 Amoxicillin Trihydrate 1-173

~
Calculate the percentage content of C16H19N30SS taking H -, NH20 H
into account the assigned content of amoxicillin ~ 0 ' C~H
~
trihydrate CRS.
STORAGE
In an airtight container.
H0i:(r'H
I
-:::?
I. H NH 1+--<--CH3
N--
H H
S)< CH3

HO ~ 0
IMPURITIES
G. (2S,5R,6R)-6-[[(2R)-2-[ [(2R)-2-amino-2-(4-
hydroxyphenyl)acetyl] amino] -2-(4-hydroxy-phenyl) acetyl]
amino]-3,3-dimethyl-7-oxo-4-thia-l-azabicyc10[3.2.0]
heptane-2-carboxylic acid (n-(4-hydroxyphenyl)
glycylamoxicillin) ,

A. (2S,5R,6R)-6-amino-3,3-dimethyl-7-oxo-4-thia-l-
azabicyclo[3.2.0]heptane-2-carboxylic acid
(6-aminopenicillanic acid),

H. (2R)-2- [(2,2-dimethylpropanoyl)amino] -2-(4-

hydroxyphenyl)acetic acid,

B.(2S,5R,6R)-6-[ [(2S)-2-amino-2-(4-hydroxyphenyl) acetyl] .

aDlUno]-3,3-dimethyl-7-oxo-4-thia-l-azabicyclo[3.2.0]
heptane-2-carboxylic acid (t-amoxicillin),

H
)(C0 2H 1. (2R)-2-aDlUno-2-(4-hydroxyphenyl) acetic acid,
HN, )<CH3

~~s
HO 8
~

~
0

I
.•..
NO'
H
CH,

C. (4S)-2-[5-(4-hydroxyphenyl)-3,6-dioxopiperazin-2-yl]-5,5-
dimethylthiazolidine-I-carboxylic acid (amoxici1lin
diketopiperazines),

J,co-oligomers of amoxicillin and of penicilloic acids of

amoxicillin,

D. (4S)-2-[[[(2R)-2-amino-2-(4-hydroxyphenyl) acetyl] amino]

carboxymethyl]-5,5-dimethylthiazolidine-4-carboxylic acid
(penicilloic acids of amoxicillin),

HO m'
.~

1.#
H NH
2

0
~ C0 2H
HN'-:<- -CH3
~ . . . . . ·r-s
H
l.. )<CH
3
and eptrnerat C"

K. oligomers of penicilloic acids of amoxicillin,

E. (2RS,4S) -2-[[[(2R)-2-amino-2-(4-hydroxyphenyl)
acetyl] amino] methyl]-5,5-dimethylthiazolidine-4-
carboxylic acid (penilloic acids of amoxicillin),

N~
~N
HO
~ ·i>H
F. 3-( 4-hydroxyphenyl)pyrazin-2-01,

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1-174 Amphotericin 2020

IDENTIFICATION
First identification: B, D.
Second identification: A, C.
A. Ultraviolet and visible absorption spectrophotometry
(2.2.25).
Test solution Dissolve 25 mg in 5 mL of dimethyl sulfoxide R
and dilute to 50 mL with methanol R. Dilute 2 mL of the
solution to 200 mL with methanol R.
L. (2S,5R,6R)-6-[[(2S,5R,6R)-6-[[(2R)-2-amino-2-(4- Spectral range 300-450 nm.
hydroxyphenyl)acetyl] amino]-3,3-dimethyl-7-oxo-4-thia-1- Absorption maxima At 362 nm, 381 nm and 405 nm.
azabicyc1o[3.2.0]heptane-2-carbonyl]amino]-3,3-dimethyl- Absorbance ratios:
7-oxo-4-thia-1-azabicyc1o[3.2.0]heptane-2-carboxylic acid - A362/A381 = 0.57 to 0.61;
(6-APA amoxicillin amide). - A381/A405 = 0.87 to 0.93.
- - - - - -__- - - - - - - - - - - - - PhEur B. Infrared absorption spectrophotometry (2.2.24).
Comparison amphotericin B CRS.
If the spectra obtained show differences, dry the substance to
be examined and reference substance at 60 DC at a pressure
Amphotericin not exceeding 0.7 kPa for 1 h and record new spectra.
(Amphotericin B~ Ph. Eur. monograph 1292) C. To 1 mL of a 0.5 gIL solution in dimethyl sulfoxide R, add
5 mL of phosphoric acid R. to form a lower layer, avoiding
mixing the 2 liquids. A blue ring is immediately produced at
the junction of the liquids. Mix, an intense blue colour is
produced. Add 15 mL of water R and mix; the solution
becomes pale yellow.
D. Examine the chromatograms obtained in the test for
related substances.
Results The principal peak in the chromatogram obtained
with the test solution at 383 nm is similar in retention time
to the principal peak in the chromatogram obtained with
reference solution (a).
TESTS
Related substances
Liquid chromatography (2.2.29). Protect the solutions from light
924 1397-89-3 and use within 24 h of preparation, except for reference
solution (c) which should be injected immediately after its
Action and use preparation.
AntifungaL
Solvent mixture 10. gIL solution of ammonium. acetate R, N-
Preparation methylpyrrolidone R, methanol R (1:1:2 V/V/V).
Amphotericin for Infusion
Test solution Dissolve 20.0 mg of the substance to be
PhEur _ examined in 15 mL of N-methylpyrrolidone R and within 2 h
dilute to 50.0 mL with the solvent mixture. Dilute 5.0 mL of
DEFINITION this solution to 25.0 mL with thesolvent mixture.
Mixture of antifungal polyenes produced by the growth of
Reference solution (a) Dissolve 20.0 mgofc,
certain strains of Streptomyces nodosus or obtained by any
amphotericin B CRS in 15 mL of N~methy/:pyn'olidone Rand··
other means. It consists mainly of amphotericin B Which is
within 2 h dilute to 50.0 mL with the solvent mixture. Dilute
(lR,3S,5R,6R,9R,11R,15S,16R,17R,18S,19E,21E,23E,-
5.0 mL of this solution to 25.0 mL with the solvent mixture.
25E,27B,29E,31E,33R,35S,36R,37S)-33-[(3-amino-3,6-
Reference solution (b) Dilute 1.0 mL of reference solution (a)
dideo:xy-~-D-mannopyranosyl)oxy]-l,3,5,6,9,ll,17,37­
octahydroxy-Ifi,16, 18-trimethyl-13-oxo-14,39-dioxa-bicyc1o to 100.0 mL with the solvent mixture.
[33.3.1]nonatriaconta-19,21,23,25,27,29,31-heptaene-36- Reference solution (c) Dissolve 20.0 mg of nystatin CRS in
carboxylic acid. 15 mL of N-methylpyrrolidone R and within 2 h dilute to
50.0 mL with the solvent mixture. Dilute 5.0 mL of the
Content
solution to 25.0 mL with reference solution (a). Dilute
Minimum 750 ill/mg (dried substance).
2.0 mL of this solution to 100.0 mL with the solvent
CHARACTERS mixture.
Appearance Reference solution (d) In order to prepare impurities Band
Yellow or orange, hygroscopic powder. C, dissolve 10 mg of the substance to be examined in 5 mL
Solubility of N-methylpyrrolidone R and within 2 h add 35 mL of a
Practically insoluble in water, soluble in dimethyl sulfoxide mixture of 1 volume of methanol Rand 4 volumes of
and in propylene glycol, slightly soluble in anhydrous ethanol R. Add 0.10 mL of dilute hydrochloric
dimethylformamide, very slightly soluble in methanol, acid R, mix and incubate at 25 DC for 2.5 h. Add 10 mL of
practically insoluble in ethanol (96 per cent). 10 gIL solution of ammonium acetate R and mix.
It is sensitive to light in dilute solutions.

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2020 Amphotericin 1-175

Reference solution (e) Dissolve 4 mg of amphotericin B for - disregard limit at 303 nm: 0.05 times the area ofthe
peak identification CRS (containing impurities A and B) in principal peak in the chromatogram obtained. with
5 mL of N-methylpyrrolidone R and within 2 h dilute to reference solution (c) (0.1 percent);
50 mL with the solvent mixture. - disregard limitat 383 nm: 0.1 times the area of the
Blank solution The solvent mixture. principal peak in the chromatogram obtained with
reference solution (b) (0.1 per cent).
Column: .
=
- size: 1= 0.15 m, {O 4.6 mm; Loss on drying (2.2.32)
- stationary phase: base-deactiuated end-capped octadecylsilyl Maximum 5.0 per cent, determined on 1.000 g by drying in
silica gelfor chromatography R (3 urn); an oven at 60 DC at a pressure not exceeding 0.7 kPa.
- temperature: 20 DC. Sulfated ash (2.4.14)
Mobilephase: Maximum 3.0 per cent, determined on 1.0 g; if intended for
- mobile phase A: mix 1 volume of methanolR, 3 volumes of use in the manufacture of parenteral preparations: maximum
acetonitrile Rand 6 volumes of a 4.2 gIL solution of citric 0.5 per cent.
acid monohydrate R previously adjusted to pH 4.7 using Bacterial endotoxins (2.6.14)
concentrated ammonia R; Less than 1.0 IV/mg, if intended for use in the manufacture
-. mobile phase B: mix 12 volumes of methanolR, 20 volumes of parenteral preparations without a further appropriate
of a 4.2 gIL solution of citric acid monohydrate R previously procedure for the removal of bacterial endotoxins.
adjusted to pH 3.9 using concentrated ammonia Rand
68 volumes of acetonitrile R; ASSAY
Protect all solutions from light throughout the assay Dissolve
Time Mobile phase A Mobile phase B 25.0 mg in dimethyl sulfoxide R and dilute, with shaking, to
(min) (per cent VIl') (per cent V/JI) 25.0 mL with the same solvent. Under constant stirring of
0-3 100 o this stock solution, dilute with dimethylsulfoxide R to obtain
3 - 23 100 -> 70 0->30 solutions of appropriate concentrations (the following
43 - 33 70 -> 0 30 -> 100 concentrations have been found suitable: 44.4, 66.7 and
33 - 40 o 100 100 IV/mL). Prepare final solutions by diluting 1:20 with
0.2 M phosphate buffer solution pH 10.5 so that they all
contain 5 per cent V/V of dimethyl sulfoxide. Prepare the
Flow rate 0.8 mUmin.
reference and the test solutions simultaneously. Carry out the
Detection Spectrophotometer: microbiological assay of antibiotics (2.7.2).
- at 303 nm:detection of tetraenes;
- at 383 nm: detection of heptaenes. STORAGE
Protected from light, at a temperature of 2 DC to 8 DC in an
Injection 20 J.tL of the test solution and reference
solutions (b), (c), (d) and (e). airtight container. If the substance is sterile, store in a sterile,
tamper-proof container.
Identification of impurities Use the chromatograms supplied
with amphotericin B for peak identification CRS and the LABELLING
chromatograms obtained with reference solution (e) to The label states, where applicable, that the substance is
identify the peaks due to impurities A and B. suitable for use in the manufacture of parenteral
Relative retention With reference to amphotericin B preparations.
(retention time = about 16 min): impurity B = about 0.75; IMPURITIES
=
impurity A = about 0.8; nystatin about 0.85. Specified impurities A, B.
System suitability at 383 nm Reference solution (d): Other detectable impurities (the following substances would, if
- resolution: minimum 1.5 between the 2 peaks presenting a present at a sufficient leuel, be detected by one or otherof the tests
relative retention of about 0.7. in the monograph. They are limited by the general acceptance
Limits: criterion for other/unspecified impurities and/or by the general
- impurity A atl03 nm: not more than 2.5 times the area of monograph Substances for pharmaceutical use (2034). It is
the principal peak in the chromatogram obtained with therefore not necessary to identify these impurities for
reference solution (c) (5.0 per cent); if intended for use in demonstration of compliance. See also 5.10. Control of impurities
the manufacture of parenteral preparations: not more than in substances for pharmaceutical use) C.
the area of the principal peak in the chromatogram
obtained with reference solution (c) (2.0 percent);
- any other impurity at 303 nm: for each impurity, not more
than 0.5 times the area of the principal peak in the
chromatogram obtained with reference solution (c)
(1.0 per cent);
- impurity B at 383 nm: not more than 4 times the area of
the principal peak in the chromatogram obtained with
reference solution (b) (4.0 per cent);
- any other impurity at 383 nm: for each impurity, not more
than 2 times the area of the principal peak in the
chromatogram obtained with reference solution (b)
(2.0 per cent);
- total at 303 and 383 nm: maximum 15.0 percent;
A. amphotericin A (28,29-dihydro-amphotericin B),

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1-176 Ampicillin 2020

Solubility
Sparingly soluble in water, practically insoluble in acetone, ill
ethanol (96 per cent) and in fatty oils. It dissolves in dilute
solutions of acids·and of alkali hydroxides.
It shows polymorphism (5.9).
IDENTIFICATION
First identification: A, D.
Secondidentification: B, C, D.
A. Infrared absorption spectrophotometry (2.2.24).
Preparation Discs of potassium bromide R.
Comparison anhydrous ampicillin CRS.
B. amphotericin Xl (13-0-methyl-amphotericin B), B. Thin-layer chromatography (2.2.27).
Test solution Dissolve 25 mg of the substance to be
examined in 10 mL of sodium hydrogen carbonate solution R.
Reference solution (a) Dissolve 25 mg of anhydrous
ampicillin CRS in 10 mL of sodium hydrogen carbonate
solution R.
Reference solution (b) Dissolve 25 mg of amoxicillin
trihydrate CRS and 25 mg of anhydrous ampiczllin.CRS in
10 mL of sodium hydrogen carbonate solution R.
Plate TLC silanised silica gelplate R.
Mobilephase Mix 10 volumes of acetone Rand 90 volumes
of a 154 gIL solution of ammonium acetate R previously
adjusted to pH 5.0 with glacial acetic acid R.
Application 1~.
C. amphotericin X2 (13-0-ethyl-amphotericin B). Development .Over a path of 15 em.
_______________ ~ PhEur Drying In air.
Detection Expose to iodine vapour until the spots appear
and examine in daylight.
System suitability Reference solution (b):
Ampicillin - the chromatogram shows 2 clearly separated spots.
Results The principal spot in the chromatogram obtained
(Ph. Eur. monograph 0167)
with the test solution is similar in position, colour and size to
the principal spot in the chromatogram obtained with
reference solution (a).
C. Place about 2 mg in a test-tube about 150 mm long and
about 15 mm in diameter. Moisten with 0.05 mL of water R
and add 2 mL of sulfuric acid-formaldehyde reagent R. Mix the
contents of the tube by swirling; the solution is practically
colourless. Place the test-tube in a water-bath for 1 min;
349.4 69-53-4 a dark yellow colour develops.
Action and use D. Water (see Tests).
Penicillin antibacterial. TESTS
Preparations Appearance of solution
Ampicillin Capsules The solutions are not more opalescent than reference
Ampicillin Oral Suspension suspension IT (2.2.1).
Dissolve 1.0 gin 10 mL of 1 M hydrochloric acid. Separately
PhEw _
dissolve 1.0 gin 10 mL of dilute ammonia R2. Examine
DEFINITION immediately after dissolution.
(2S,5R,6R)-6-[[(2R)-2-Amino-2-phenylacetyl]amino]-3,3- pH (2.2.3)
dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2- 3.5 to 5.5.
carboxylic acid. Dissolve 0.1 g in carbon dioxide-free waterR and dilute to
Semi-synthetic product derived from a fermentation product. 40 mL with the same solvent.
Content Specific optical rotation (2.2.7)
96;0 per cent to 102.0 per cent (anhydrous substance). + 280 to + 305 (anhydrous substance).
CHARACTERS Dissolve 62.5 mg in waterR and dilute to 25.0 mL with the
Appearance same solvent.
White or alinost white, crystalline powder. Related substances
Liquid chromatography (2.2.29).

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2020 Ampicillin 1-177

Test solution (a) Dissolve 27.0 mg of the substance to be Mobile phase Initial composition of the mixture ofmobile
examined in mobile phase A and dilute to 50.0 mL with phases A and B, adjusted where applicable.
mobile phase A. Injection Test solution (a) and reference solution (a).
Test solution (b) Prepare immediately before use. Dissolve System suitability Reference solution (a):
27.0 mg of the substance to be examined in mobile phase A - repeatability: maximum relative standard deviation of
and dilute to 10.0 mL with mobile phase A. 1.0 per cent after 6 injections.
Reference solution (a) Dissolve 27.0 mg of anhydrous Calculate the percentage content of C16H19N304S from the
ampicillin CRS in mobile phase A and dilute to 50.0 mL with declared content of anhydrous ampicillin CRS.
mobile phase A.
STORAGE
Reference solution (b) Dissolve 2.0 mg of cefradine CRS in
In an airtight container, at a temperature not exceeding
mobile phase A and dilute to 50 mL with mobile phase A. 30 DC.
To 5.0 mL of this solution add 5.0 mL of reference
solution (a). IMPURITIES
Reference solution (c) Dilute 1.0 mL ofreference solution (a)
to 20.0 mL with mobile phase A.
Column:
- size: 1= 0.25 m, 0.= 4.6 mID;
- .stationary phase: octadecylsilyl silica gelfor chromatography R
(5 urn).
A. (2S,5R,6R)-6-amino- 3,3-dimethyl-7-oxo-4-thia-1-
Mobile phase: azabicyclo[3.2.0]heptane-2-carboxylic acid
- mobile phaseA: mix 0.5 mL of dilute aceticacid R, 50 mL (6-aminopenicillanic acid),
of 0.2 M potassium dihydrogen phosphate Rand 50 mL of
acetonitrile R, then dilute to 1000 mL with waterR; H

·cti
. .
o . C0 2H
-mobile phase B: mix 05 mL of dilute acetic acid R, 50 mL
of 0.2 M potassium dihydrogen phosphate Rand 400 mL of
H ,NH'H )-~.J(--CH,
N--H--s)<CH 3
acetonitrile R, then dilute to 1000 mL with waterR;
I H H

Time Mobile phase A Mobile phase B

~ °
(min) (per cent V/JI) (per cent VIJI)
B. (2S,5R,6R)-6-[[ (2S)-2-amino-2-phenylacetyl] amino] -3,3-
0- tR 85 15
dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-
tR - (tR + 30) 85 -., 0 15 -+ 100
carboxylic acid (t-ampicillin),
(tR + 30) - (tR + 45) o 100
(tR + 45) - (tR + 60) 85 15 H

tR =retention time of ampicillin determined with reference solution (c)

J(C0 2H
HN, )<CH 3
0
.~rs

cY
If the mobile phase composition has been adjusted to achieve CH,
the required resolution, the adjusted composition will apply
at time zero in the gradient and in the assay. ~ I ~ 0
~
Flow rate 1.0 mUmin.
Detection Spectrophotometer at 254 nm.
C. (4S)-2-(3,6-dioxo-5-phenylpiperazin-2-yl)-5,5-
Injection 50 JlL of reference solutions (b) and (c) with dimethylthiazolidine-4-carboxylic acid (diketopiperazines
isocratic elution at the initial mobile phase composition and of ampicillin),
50 JlL of test solution (b) according to the elution gradient

,
described under Mobile phase; inject mobile phase A as a
~ C0 2H
blank according to the elution gradient described under HW~-<--CH3

orr
H. NH

i"**-
Mobile phase. 2
-:;:? , H_
N S)< CH3
System suitability Reference solution (b):
- resolution: minimum 3.0 between the peaks due to ~ I ° C02H
ampicillin and cefradin; if necessary, adjust the ratio A:B
of the mobile phase. D. (4S)-2-[[[(2R)-2-amino-2-phenylacetyl]
Limit: amino] carboxymethyl] -5,5-dimethyl-l ,3-thiazolidine-4-
- any impurity: for each impurity, not more than the area of carboxylic acid (penicilloic acids of ampicillin),
the principal peak in the chromatogram obtained with
reference solution (c) (1.0 per cent). o H~C02H
N,N-Dimethylaniline (2.4.26, Method B) . .~ , .f? .\\
o ; N ~

)-~-
Maximum 20 ppm. N H=-

Water (2.5.12)
Maximum 2.0 per cent, determined on 0.300 g. 0
U~
-
-tt H" NH, H
N
s CH3
CH. 3

Sulfated ash (2.4.14)

Maximum 0.5 per cent, determined on 1.0 g.
E. (2R)-2-[[[(2S,5R,6R)-6-[[(2R)-2-amino-2-
ASSAY phenylacetyl] amino]-3,3-dimethyl-7-oxo-4-thia-1-
Liquid chromatography (2.2.29) as described in the test for azabicyclo [3.2.0]hept-2-yl] carbonyl]amino]-2-phenylacetic
related substances with the following modifications. acid (ampicillinyl-D-phenylglycine), .

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1-178 Ampicillin Sodium 2020

'
en
~.

~ I °
H, NH

'
2
H
~
HNX--CH3
C0 2H

1*)<CH3
N--.../- S

F. (2RS,4S)-2-[[ [(2R)-2-amino-2-phenylacetyl]
amino]methyl]-5,5-dimethyl-l ,3-thiazolidine-4-carboxylic
acid (penilloic acids of ampicillin),

HN~
~.~H r-
I""'.
M.co-oligomers of ampicillin and of penicilloic acids of
~NH ampicillin.
UH~ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur

G. (3R,6R)-3,6-diphenylpiperazine-2,5-dione,

Ampicillin Sodium
(Ph. Bur. monograph 0578)

o ~. C02Na
\-~~CH,
en.
H-. NH'H
H. 3-phenylpyrazin-2-ol,
N--t-t-S CH3

~
I 0
H H

371.4 69-52-3

Action and use

Penicillin antibacterial.
Preparation
I. (2S,5R,6R)-6-[[(2R)-2-[[(2R)-2-amino-2-
Ampicillin Injection
phenylacetyl]amino]-2-phenylacetyl]amino]-3,3-dimethyl-
7-oxo-4-thia-l-azabicyclo[3.2.0]heptane-2-carboxylic acid PhEur _
(n-phenylglycylampicillin),
DEFINTI10N
Sodium (2S,5R,6R)-6-[[(2R)-2-amino-2-phenylacetyl]
amino]-3,3-dimethyl-7-oxo-4-thia-l-azabicyclo[3.2.0]
heptane-2-carboxylate.
Semi-synthetic product derived from a fermentation product.
Content
91.0 per cent to 102.0 per cent (anhydrous substance).
J. (2S,5R,6R)-6-[ (2,2-dimethylpropanoyl) amino]-3,3-
diulethyl-7-oxo-4-thia-l-azabicyclo[3.2.0]heptane-2- CHARACTERS
carboxylic acid, Appearance
White or almost white powder, hygroscopic.
Solubility
Freely soluble in water, sparingly soluble in acetone,
practically insoluble in fatty oils and in liquid paraffin.
IDENTIFICATION
Firstidentification: A, D.
Second identification: B, C, D.
K. (2R)-2- [(2,2-dimethylpropanoyl)amino]-2-phenylacetic A. Infrared absorption spectrophotometry (2.2.24).
acid,
Preparation Dissolve 0.250 gin 5 mL of water R, add
0.5 mL ofdzlute acetic acid R, swirl and allow to stand for
10 min in iced water. Filter the crystals through a small
sintered-glass filter (40) (2.1.2), applying suction, wash with
2-3 mL of a mixture of 1 volume of water Rand 9 volumes
of acetone R, then dry in an oven at 60°C for 30 min.
L. '(2R)-2-amino-2-phenylacetic acid (n-phenylglycine), Comparison ampicillin trihydrate CRS.
B. Thin-layer chromatography (2.2.27).
Testsolution Dissolve 25mg of the substance to be
examined in 10 mL of sodium hydrogen carbonate solution R.

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2020 Ampicillin Sodium 1-179

Reference solution (a) Dissolve 25 mg of ampicillin To 5.0 mL of this solution add 5.0 mL of reference
trihydrate CRS in 10 mL of sodium hydrogen carbonate solution (a).
solution R. Reference solution (c) Dilute 1.0 mL of reference solution (a)
Reference solution (b) Dissolve 25 mg of amoxicillin to 20.0 mL with mobile phase A.
trihydrate CRS and 25 mg of ampicillin trihydrate CRS in Reference solution (d) To 0.20 g of the substance to be
10 mL of sodium hydrogen carbonate solution R. examined add 1.0 mL of waterR. Heat the solution at 60°C
Plate TLC silanised silica gelplate R. for 1 h. Dilute 0.5 mL of this solution to 50.0 mL with
Mobile phase Mix 10 volumes of acetone Rand 90 volumes mobile phase A.
of a 154 gIL solution of ammonium acetate R previously Column:
adjusted to pH 5.0 with glacial acetic acidR. - size: 1= 0.25 m, 0 =
4.6 mm;
Application 1 J.LL. - stationary phase: octadecylsilyl silica gelfor chromatography R
(5 urn).
Development Over a path of 15 cm.
Mobile phase:
Drying In air.
- mobile phaseA: mix 0.5 mL of dilute acetic acid R, 50 mL
Detection Expose to iodine vapour until the spots appear of 0.2 M potassium dihydrogen phosphateR and 50 mL of
and examine in daylight. acetonitrile R, then dilute to 1000 mL with waterR;
System suitability Reference solution (b): - mobile phase B: mix 0.5 mL of dilute acetic acid R, 50 mL
- the chromatogram shows 2 clearly separated spots. of 0.2 M potassium dihydrogen phosphate Rand 400 mL of
Results The principal spot in the chromatogram obtained acetonimleR, then dilute to 1000 mL with waterR;
with the test solution is similar in position, colour and size to
the principal spot in the chromatogram obtained with Time Mobile phase A Mobile phase B
reference solution (a). (min) (per cent VIJ') (per cent VIJ')

C. Place about 2 mg in a test-tube about 150 mm long and 0- tR 85 15

about 15 mm in diameter. Moisten with 0.05 mL of water R tR - (tR + 30) 85 - 0 15 - 100

and add 2 mL of sulfuric acid-forrnaldehyde reagent R. Mix the (tR + 30) - (tR + 45) 0 100

contents of the tube by swirling; the solution is practically (tR + 45) - (tR + 60) 85 15

colourless. Place the test-tube in a water-bath for 1 min; tR =retention time of ampicillin determined with reference solution (c)
a dark yellow colour develops.
D. It gives reaction (a) of sodium (2.3.1). If the mobile phase composition has been adjusted to achieve
the required resolution, the adjusted composition will apply
TESTS at time zero in the gradient and in the assay.
Appearance of solution Flow rate 1.0 mlJmin.
Solutions A and B are not more opalescent than reference
suspension Il (2.2.1) and the absorbance (2.2.25) of Detection Spectrophotometer at 254 nm.
solution B at 430 nm is not greater than 0.15. Injection 50J.LLof reference solutions (b) and (c) with
Place 1.0g in a conical flask and add slowly and with isocratic elution at the initial mobile phase composition and
continuous swirling 10 mL of 1 M hydrochloric acid 50 ilL of test solution (b) and reference solution (d)
(solution A). Separately dissolve 1.0 g in waterR and dilute according to the elution gradient described under Mobile
to 10.0 mL with the same solvent (solution B). Examine phase; inject mobile phase A as a blank according to the
immediately after dissolution. elution gradient described under Mobile phase.
Identification of peaks Use the chromatogram obtained with
pH (2.2.3)
reference solution (d) to identify the peaks due to ampicillin
8.0 to 10.0.
and ampicillin dimer.
Dissolve 2.0 g in carbon dioxide-free waterR and dilute to
Relative retention With reference to ampicillin: ampicillin
20 mL with the same solvent. Measure 10 min after
dimer = about 2.8.
dissolution.
System suitabz1ity Reference solution (b):
Specific optical rotation (2.2. 7)
- resolution: minimum 3.0 between the peaks due to
+ 258 to + 287 (anhydrous substance).
ampicillin and cefradin; if necessary adjust the ratio A:B
Dissolve 62.5 mg in a 4 gIL solution of potassium hydrogen of the mobile phase.
phthalate R and dilute to 25.0 mL with the same solvent.
Limits:
Related substances - ampicillin dimer: not more than 4.5 times the area of the
Liquid chromatography (2.2.29). principal peak in the chromatogram obtained with
Test solution (a) Dissolve 31.0 mg of the substance to be reference solution (c) (4.5per cent); '"
examined in mobile phase A and dilute to 50.0 mL with - any other impurity: for each impurity, not more than twice
mobile phase A. the area of the principal peak in the chromatogram
Testsolution (b) Dissolve 31.0 mgofthe substance to be obtained with reference solution (c) (2 per cent).
examined in mobile phase A and dilute to 10.0 mL with N,N-Dimethylaniline (2.4.26, Method B)
mobile phase A. Prepare immediately before use. Maximum 20 ppm.
Reference solution (a) Dissolve 27.0 mg of anhydrous 2-Ethylhexanoic acid (2.4.28)
ampicillin CRS in mobile phase A and dilute to 50.0 mL with Maximum 0.8 per cent m/m.
mobile phase A.
Methylene chloride
Reference solution (b) Dissolve 2.0 mg of cefradine CRS in Gas chromatography (2.2.28).
mobile phase A and dilute to 50 mL with mobile phase A.

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1-180 Ampicillin Sodium 2020

Internalstandard solution Dissolve 1.0 mL of ethylene

chloride R in water R and dilute to SOO.O mL with the same
solvent.
Test solution (a) Dissolve 1.0 g of the substance to be
examined in water R and dilute to 10.0 mL with the same
solvent.
B. (2S,5R,6R)-6-[[(2S)-2-amino-2;.phenylacetyl]amino]-3,3-
Test solution (b) Dissolve 1.0 g of the substance to be dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-
examined in water R, add 1.0 mL of the internal standard carboxylic acid (t-ampicillin),
solution and dilute to 10.0 mL with water R.
Reference solution Dissolve 1.0 mLof methylene chloride R in
waterR and dilute to SOO.O mL with the same solvent.
To 1.0 mL of this solution add 1.0 mL of the internal
standard solution and dilute to 10.0 mL with water R.
Column:
- material: glass;
- size: 1 = 1.5 m, (2) = 4 mm;
- stationary phase: diatomaceous earth for gas C. (4S)-2-(3,6-dioxo-S-phenylpiperazin-2-yl)-S,S-
chromatography R impregnated with 10 per cent mlm of dimethylthiazolidine-4-carboxylic acid (diketopiperazines
macrogol 1000 R. of ampicilliri),
Carnergas nitrogen for chromatography R.
Flow rate 40 mUmin.
Temperature:
- column: 60 DC;
- injection port: 100 DC;
- detector: ISO DC.
Detection Flame ionisation. D. (4S)-2-[[[(2R)-2-amino-2-phenylacetyl]
Calculate the content of methylene chloride taking its density amino] carboxymethyl]-S, S-dimethyhhiazolidine-4-
at 20 DC to be 1.325 g/mL. carboxylic acid (penicilloic acids of ampicillin),
Limit:
- methylene chloride: maximum 0.2 per cent m/m.
Water (2.5.12)
Maximum 2.0 per cent, determined on 0.300 g.
Bacterial endotoxins (2.6.14)
Less than O.lS IV/mg, if intended for use in the manufacture
of parenteral preparations without a further appropriate
procedure for the removal of bacterial endotoxins. E. (2R)-2-[[[(2S,5R,6R)-6-[[(2R)-2-amino-2-phenylacetyl]
ASSAY amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicydo[3.2.0]hept-
Liquid chromatography (2.2.29) as described in the test for 2-yl]carbonyl] amino ]-2-phenylacetic acid (ampicillinyl-D-
related substances with the following modifications. phenylglycine),
Mobilephase Initial composition of the mixture of mobile
phases A and B, adjusted where applicable.
Injection Test solution (a) and reference solution (a).
System suitabt7ity Reference solution (a):
- repeatability: maximum relative standard deviation of
1.0 per cent after 6 injections.
Calculate the percentage content of ampicillin sodium by F. (2RS,4S)-2-[[[(2R)-2-amino-2-phenylacetyl]
multiplying the percentage content of ampicillin by 1.063. amino]methyl] -S,5-dimethylthiazolidine-4-carboxylic acid
(penilloic acids of ampicillin),
STORAGE
In an airtight container. Ifthe substance is sterile, store in a
sterile, airtight, tamper-proof container.
HN~
r- r-
o+r
IMPURITIES
NH

G . (3R,6R)- 3,6-diphenylpiperazine-2,S-dione,

A. (2S,5R,6R)-6-amino-3,3-dimethyl-7-oxo-4-thia-l-
azabicyclo [3.2.0] heptane-2-carboxylic acid
(6-aminopenicillanic acid),

H. 3-phenylpyrazin-2-01,

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2020 Ampicillin Trihydrate 1-181

H
\ NH20 H
Ampicillin Trihydrate
o:i 1+-<-·
,

~ I H\ NH H
O' CO~
CH3
(Ph. Bur. monograph 0168)

(('r
,

~
I 0
N- -
H H
.
S'><CH3

1. (2S,5R,6R)-6- [[(2R)-2-[[(2R)-2-amino-2-
phenylacetyl]amino]-2-phenylacetyl]amino]-3,3-dimethyl-
7-oxo-4-thial-azabicyclo [3.2.0]heptane-2-carboxylic acid
(n-phenylglycylampicillin),
7177-48-2

Action and use

Penicillin antibacterial.
Preparations
Ampicillin Capsules
Ampicillin Oral Suspension
J. (2S,5R,6R)-6-[ (2,2-dimethylpropanoyl)amino]-3,3- Co-fluampicil Capsules
dimethyl-7-oxo-4-thia-I-azabicyclo [3.2.0]heptane-2-
Co-fluampicil Oral Suspension
carboxylic acid,
PhEur _

DEFINITION
(2S,5R,6R)-6-[[(2R)-2-Amino-2-phenylacetyl]amino]-3,3-
dimethyl-7-oxo-4-thia-I-azabicyclo[3.2.0]heptane-2-
carboxylic acid trihydrate.
Semi-synthetic product derived from a fermentation product.
Content
K. (2R)-2-[ (2,2-dimethylpropanoyl)amino ]-2-phenylacetic 96.0 per cent to 102.0 per cent (anhydrous substance).
acid,
CHARACTERS
Appearance
White or almost white, crystalline powder.
Solubility
Slightly soluble in water, practically insoluble in ethanol
(96 per cent) and in fatty oils. It dissolves in dilute solutions
L. (2R)-2-amino-2-phenylacetic acid (n-phenylglycine), of acids and of alkali hydroxides.
IDENTIFICATION
Firstidentification: A, D.
Second identification: B, C, D.
A. Infrared absorption spectrophotometry (2.2.24).
Comparison ampicillin trihydrate CRS.
B. Thin-layer chromatography (2.2.27).
Testsolution Dissolve 25 mg of the substance to be
examined in 10 mL of sodium hydrogen carbonate solution R.
Reference solution (a) Dissolve 25 mg of ampicillin
trihydrate CRS in 10 mL of sodium hydrogen carbonate
solution R.
Reference solution (b) Dissolve 25 mg of amoxicillin
M. co-oligomers of ampicillin and of penicilloic acids of trihydrate CRS and 25 mg of ampicillin trihydrate CRS in
ampicillin,
10 mL of sodium hydrogen carbonate solution R.
Plate TLC silanised silica gelplate R.
Mobile phase Mix 10 volumes of acetone R and 90 volumes
of a 154 gIL solution of ammonium acetate R previously
adjusted to pH 5.0 with glacialacetic acid R.
Application 1~.
Development Over. a path of 15 cm.
Drying In air.
Detection Expose to iodine vapour until the spots appear
N. oligomers of penicilloic acids of ampicillin.
and examine in daylight.
_ _ _ _ _ _ _ _ _ _ _ _ _ _--:- PhEur
System suitabz7ity Reference solution (b):
- the chromatogram shows 2 clearly separated spots.

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1-182 Ampicillin Trihydrate 2020

Results The principal spot in the chromatogram obtained If the mobile phase composition has been adjusted to achieve
with the test solution is similar in position, colour and size to the required resolution, the adjusted composition will apply
the principal spot in the chromatogram obtained with at time zero in the gradient and in the assay.
reference solution (a). Flow rate 1.0 mIJmin.
C. Place about 2 mg in a test-tube about 150 rom long and Detection Spectrophotometer at 254 nm.
about 15 rom in diameter. Moisten with 0.05 mL of waterR
Injection 50 ~ of reference solutions (b) and (c) with
and add 2 mL of sulfuric acid-formaldehyde reagent R. Mix the
isocratic elution at the initial mobile phase composition and
contents of the tube by swirling; the solution is practically
50 JlL of test solution (b) according to the elution gradient
colourless. Place the test-tube in a water-bath for 1 min;
described under Mobile phase; inject mobile phase A as a
a dark yellow colour develops.
blank according to the elution gradient described under
D. Water (see Tests). Mobile phase.
TESTS System suitabz1ity Reference solution (b):
Appearance of solution - resolution: minimum 3.0 between the peaks due to
The solutions are not more opalescent than reference ampicillin and cefradin; if necessary, adjust the ratio A:B
suspension II (2.2.1). of the mobile phase.
Dissolve 1.0 g in 10 mL of 1 M hydrochloric acid. Separately Limit:
dissolve 1.0 g in 10 mL of dilute ammonia R2. Examine - any impurity: for each impurity, not more than the area of
immediately after dissolution. the principal peak in the chromatogram obtained with
pH (2.2.3) reference solution (c) (1.0 per cent).
3.5 to 5.5. N,N-Dimethylaniline (2.4.26, Method B)
Dissolve 0.1 g in carbon dioxide-free water R and dilute to Maximum 20 ppm.
40 mL with the same solvent. Water (2.5~12)
Specific optical rotation (2.2.7) 12.0 per cent to 15.0 per cent, determined on 0.100 g.
+ 280 to +,305 (anhydrous substance). Sulfated ash (2.4.14)
Dissolve 62.5 mg in water R and dilute to 25.0 mL with the Maximum 0.5 per cent, determined on 1.0 g.
same solvent. ASSAY
Related substances Liquid chromatography (2.2.29) as described in the test for
Liquid chromatography (2.2.29). related substances with the following modifications.
Test solution (a) Dissolve 31.0 mg of the substance to be Mobile phase Initial composition of the mixture of mobile
examined in mobile phase A and dilute to 50.0 mL with phases A and B, adjusted where applicable.
mobile phase A. Injection Test solution (a) and reference solution (a).
Test solution (b) Dissolve 31.0 mg of the substance to be System suitability Reference solution (a):
examined in mobile phase A and dilute to 10.0 mL with - repeatability: maximum relative standard deviation of
mobile phase A. Prepare immediately before use! 1.0 per cent after 6 injections.
Reference solution (a) Dissolve 27.0 mg of anhydrous Calculate the percentage content of ampicillin from the
ampicillin CRS in mobile phase A and dilute to 50.0 mL with declared content of anhydrous ampicillin CRS.
mobile phase A.
STORAGE
Reference solution (b) Dissolve 2 mg of cefradine CRS in In an airtight container.
mobile phase A and dilute to 50 mL with mobile phase A.
To 5 mL of this solution, add 5 mL of reference solution (a). IMPURITIES
Reference solution (c) Dilute 1.0 mL of reference solution (a)
to 20.0 mL with mobile phase A.
Column:
=
- size: I 0.25 m, 0 = 4.6 mm;
- stationary phase: octadecylsilyl silica gelfor chromatography R
(5 JlID).
Mobile phase: A. (2S,5R,6R)-6-amino-3,3-dimethyl-7-oxo-4-thia-l-
- mobile phaseA: mix 0.5 mL of dilute acetic acid R, 50 mL azabicyclo [3.2.Ojheptane-z-carboxylic acid
of 0.2 M potassium dihydrogen phosphate Rand 50 mL of (6-aminopenicillanic acid),
acetonitrile R, then dilute to 1000 mL with water R;
° .
H

.
- mobile phase B: mix 0.5 mL of dilute acetic acid R, 50 mL C0 2H
of 0.2 M potassium dihydrogen phosphate Rand 400 mL of H..NH'H 't-~5CH,
ro
~
acetonitrile R, then dilute to 1000 mL with waterR;
I --t-i-s
. .N. H H CH
3

Time Mobile phase A Mobile phase B .~ . °

(min) (per cent VIV) (per cent VIV)
0- tR 85 15 B. (2S,5R,6R)-6-[[ (2S)-2-amino-2-phenylacetyl] amino] -3,3-
te - (tR + 30) 85 ~ 0 15 ~ ioo dimethyl-7-oxo-4-thia-I-azabicyclo[3.2.0]heptane-2-
(tR + 30) - (tR + 45) o 100 carboxylic acid (t-ampicillin),
(tR + 45) - (tR + 60) 85 15
te = retention time of ampicillin determined with reference solution (c)

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2020 Ampicillin Trihydrate 1-183

7-oxo-4-thia-l-azabicyc1o[3.2.0]heptane-2-carboxylic acid
(n-phenylglycylampicillin),

C. (4S)-2-(3,6-dioxo-5-phenylpiperazin-2-yl)-5,5-dimethyl-
1,3-thiazolidine-4-carboxylic acid (diketopiperazines of J. (2S,5R,6R)-6-[(2,2-dimethylpropanoyl) amino]-3,3-
ampicillin), dimethyl-7-oxo-4-thia-l-azabicyclo [3.2.0]heptane-Z-
carboxylic acid,

D. (4S)-2- [[[(2R)-2-amino-2-phenylacetyl] amino]

'carboxymethyl] -5,5-dimethyl-l ,3-thiazolidine-4-earboxylic
K. (ZR)-Z-[ (Z,Z-dimethylpropanoyl)amino]-2-phenylacetic
acid (penicilloic acids of ampicillin),
acid,

L. (2R)-Z-amino-Z-phenylacetic acid (n-phenylglycine),

E. (2R)-2-[[[(ZS,5R,6R)-6-[[(ZR)-Z-amino-Z-
phenylacetyl] amino]-3,3-dimethyl-7-oxo-4-thia-l-
azabicyclo [3.2. O]hept-2-yl] carbonyl] amino] ';'Z-phenylacetic
acid (ampicillinyl-D-phenylg1ycine),

F. (2RS,4S)-2-[ [[ (2R)-Z-amino-Z-phenylacetyl]
amino] methyl] -5 ,5-dimethyl-l,3-thiazolidine-4-carboxylic M.co-oligomers of ampicillin and of penicilloic acids of
acid (penilloic acids of ampicillin), ampicillin,

,~ H ('l
cftrNHHNy./

N. (3S)-6-[[(ZR)-2-amino-2-phenylacetyl] amino]-Z,2-
G. (3R,6R)-3,6-diphenylpiperazine-2,5-dione, dimethyl-7-oxo-2,3,4,7-tetrahydro-l,4-thiazepine-3-
carboxylic acid.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur

}f.3-phenylpyrazin-2-o~

I. (2S,5R,6R)-6-[[(ZR)-2-[[(2R)-Z-amino-2-
phenylacetyl] amino]-2-phenylacetyl] amino]-3,3-dimethyl-

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 1-184 Amylmetacresol 2020

- size: 1= 30 m, 0 = 0.25 mID;

Amylmetacresol - stationary phase: macrogol 20 000 R (film thickness
0.5 um),
(Ph. Bur. monograph 2405)
Carrier gas heliumfor chromatography R.
Linear velocity 33 cm/s.
Split ratio 1:30.
Temperature:

178.3 1300-94-3 Time Temperature

(min) eC)
Action and use Column o- 17.5 100 -> 240
Antiseptic. 17.5 - 32.5 240
PhEur _ Injection port 250
Detector 250
DEFINITION
5-Methyl-2-pentylphenol. Detection Flame ionisation.
Content Injection 1.0 IlL of test solution (a) and reference
98.0 per cent to 102.0 per cent. solutions (a), (b) and (d).
CHARACTERS Identification of impurities Use the chromatogram supplied
Appearance with amylmetacresol for peak identification CRS and the
->, Clear or almost clear liquid, or solid crystalline mass, chromatogram obtained with reference solution (b) to
colourless or slightly yellow when freshly prepared. identify the peaks due to impurities A, G and K.
The substance changes colour during storage by darkening Relativeretention With reference to amylmetacresol
and/or discolouration to dark yellow, brownish-yellow or (retention time = about 16 min): impurity G
pink. (diastereoisomer 1) = about 0.51; impurity G
Solubility (diastereoisomer 2) = about 0.53; impurity D = about 0.77;
Practically insoluble in water, very soluble in acetone and in impurity B = about 0.78; impurity K = about 0.95; ,
ethanol (96 per cent). =
impurity A about 0.99.
It solidifies at about 22°C. System suitability Reference solution (a):
- resolution: minimum 1.5 between the peaks due to
IDENTIFICATION impurities D and B.
Infrared absorption spectrophotometry (2.2.24).
Limits:
Preparation Film between 2 plates of potassium bromide R. - impurityA: maximum 0.6 per cent;
Comparison amylmetacresol CRS. - impurities G (sum of the 2 diastereoisomers), K: for each
TESTS impurity, maximum 0.15 per cent;
Related substances - unspecified impurities: for each impurity, maximum
Gas chromatography (2.2.28): use the normalisation 0.10 per cent;
procedure. - total: maximum 1.0 per cent;
- disregard limit: the area of the peak due to amylmetacresol
Internal standard solution Dissolve 0.100 g of
in the chromatogram obtained with reference solution (d)
butylhydroxytoluene R in 2-propanol R and dilute to .10.0 mL
(0.05 per cent).
with the same solvent.
Sulfated ash (2.4.14)
Test solution (a) Dissolve 0.1000 g of the substance to be
Maximum 0.1 per cent, determined on 1.0 g.
examined in 2-propanol R and dilute to 10.0 mL with the
same solvent. ASSAY
Test solution (b) To 2.0 mL of test solution (a) add 2.0 mL Gas chromatography (2.2.28) as described in the test for
of the internal standard solution and dilute to 10.0 mL with related substances with the following modification.
2-propanol R. Injection 1.0 IlL of test solution (b) and reference
Reference solution (a) Dissolve 10 mg of m-cresol R solution (c).
(impurity B) and 10 mg of p-cresol R (impurity D) in Calculate the percentage content of C 12H1SO from the
2-propanol R and dilute to 100.0 mL with the same solvent. declared content of amylmetacresol CRS.
Reference solution (b) Dissolve the contents of a vial of STORAGE
amylmetacresol for peak identification CRS (containing In an airtight, non-metallic container, protected from light.
impurities A, G and K) in 1.0 mL of 2-propanol R.
IMPURITIES
Reference solution (c) Dissolve 0.1000 g of
Specified impurities A, G, K.
amylmetacresolCRS in 2-propanol R and dilute to 10.0 mL
with the same solvent. To 2.0 mLof this solution add Other detectable impurities (thefollowing substances would, if
2.0 mL of the intemalstandard solution and dilute to present at a sufficient level, be detected by oneor otherof the tests
10.0 mL with 2-propanol R. in the monograph. They arelimited by the general acceptance
criterion for other/unspecified impurities and/orby the general
Reference solution (d) Dilute 1.0 mL of test solution (a) to
100.0 mL with 2-propanol R. Dilute 1.0 mL of this solution
monograph Substances for pharmaceutical use (2034). It is
therefore not necessary to identify these impurities for
to 20.0 mL with 2-propanol R.
demonstration of compliance. See also 5.10. Control of impurities
Column: in substances for pharmaceutical use) B, C, D, B, F, H, I, J.
- material: fused silica;

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2020 Anastrozole 1-185

Anastrozole
(Ph. Eur. monograph 2406)
A. 4-methyl- 2-pentylphenol,

B. 3-methylphenol (m-cresol),

293.4 120511-73-1

Action and use

Aromotase inhibitor; treatment of breast carcinoma.
C. 5-methyl-2- [(2RS)-2-methylbutyl] phenol, Preparation
Anastrozole Tablets
PhEur _

DEFINITION
2,2'-[5-(IH-l,2,4-Triazol-l-ylmethyl)benzene-l,3-diyl]bis(2-
D. :4-methylphenol (p-cresol), methylpropanenitrile) .
Content
98.0 per cent to 102.0 per cent (anhydrous substance).
CHARACTERS
Appearance
White or almost white powder.
E. 1-(2-hydroxy-4-methylphenyl)pentan-l-one, Solubility
Very slightly soluble in water, freely soluble in anhydrous
ethanol, practically insoluble in cyclohexane.
It shows polymorphism (5.9).
IDENflFICATION
Infrared absorption spectrophotometry (2.2.24).
F. 1-(2-hydroxy-5-methylphenyl)pentan-l-one, Comparison anastrozole CRS.
If the spectra obtained in the solid state show differences,
dissolve the substance to be examined and the reference
substance separately in acetone R, evaporate to dryness and
record new spectra using the residues.
TESTS
G. 5-methyl-2~pentylcyc1ohexanone, Related substances
Liquid chromatography (2.2.29).
Solvent mixture acetonitrile R1, waterfor chromatography R
(50:50 VIV).
Testsolution (a) Dissolve 25 mg of the substance to be
H. ethyl pentanoate, examined in the solvent mixture and dilute to 100.0 mL with
the solvent mixture.
Testsolution (b) Dissolve 25.0 mg of the substance to be
examined in the solvent mixture and dilute to 200.0 mL with
the solvent mixture.
1. 3-methylphenyl pentanoate, Reference solution (a) Dilute 1.0 mL of test solution (a) to
100.0 mL with the solvent mixture. Dilute 1.0 mL of this
solution to 10.0 mL with the solvent mixture.
Reference solution (b) Dissolve 2~5 mg of anastrozole
impurity E CRS in 20.0 mL of the solvent mixture. Dilute
1.0 mL of the solution to 50.0 mL with test solution (a).
J. 4-methylphenyl pentanoate, Reference solution (c) Dissolve 25.0 mg of anastrozole CRS in
K. unknown structure. the solvent mixture and dilute to 200.0 mL with the solvent
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur mixture.
Column:
- size: 1= 0.15 m, 0 = 4.6 mm;

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1-186 Anastrozole 2020

- stationary phase: end-capped ethylene-bridged polar-embedded

octadecylsilyl silica gelfor chromatography (hybrid material) R
(3.5 urn).
Mobile phase: F'N,
- mobile phase A: phosphoric acid R, waterfor N
chromatography R (0.1:100 V/V); "=N
- mobile phase B: phosphoric acid R, acetonitrile Rl
(0.1:100 V/V);

Time Mobile phase A Mobile phase B B. (2RS)-2,3-bis [3-(l-cyano-1-methylethyl)-5-(lH-1,2,4-

(min) (per cent VIV) (per cent VIV) triazol-1-ylmethyl)phenyl] -2-methylpropanenitrile,
0-2 95 5
2 - 54 95 --> 35 5 --> 65

Flow rate 1.0 mUmin.

Detection Spectrophotometer at 215 nm.
Injection 20 ilL of test solution (a) and reference
solutions (a) and (b).
Identification of impurities Use the chromatogram obtained C. 2,2'-[5-(bromomethyl)benzene-1 ,3-diyl] bis(2~
with reference solution (b) to identify the peak due to methylpropanenitrlle),
impurity E.
Relative retention With reference to anastrozole (retention
time = about 29 min): impurity E = about 1.05.
System suitabilz"ty Reference solution (b):
- resolution: minimum 3.5 between the peaks due to
anastrozole and impurity E.
Calculation ofpercentage contents:
- for each impurity, use the concentration of anastrozole in
D. 2,2'- [5-(dibromomethyl)benzene-I,3-diyl] bis(2-
reference solution (a).
methylpropanenitrile),
Limits:
- unspecified impurities: for each impurity, ~aximum
0.10 per cent;
- total: maximum 0.2 per cent;
- reporting threshold: 0.05 per cent.
Water (2.5.32)
Maximum 0.3 per cent, determined on 50.0 mg.
Sulfated ash (2.4.14)
Maximum 0.1 per cent, determined on 1.0 g. E. 2,2'-[5-(hydroxymethyl)benzene-1,3-diyl]bis(2-
methylpropanenitrile),
ASSAY
Liquid chromatography (2.2.29) as described in the test for
related substances with the following modification.
Injection Test solution (b) and reference solution (c).
Calculate the percentage content of C17H19Ns taking into
account the assigned content of anastrozole CRS. F. 4-methylbenzenesulfonic acid,

IMPURITIES
Other detectable impurities (the following substances would, if·
presentat a sufficientlevel, be detected by one or otherof the tests
in the monograph. They are limited by the generalacceptance
criterion for other/unspecified impurities and/or by the general
monograph Substances for pharmaceutical use (2034). It is
therefore not necessary to identify these impurities for
demonstration of compliance. See also 5.10. Control of impurities G. 2,2'-[5-(4H-1,2,4.:..triazol-4-ylmethyl)benzene-1,3-diyl]bis
in substances for pharmaceutical use) A, B, C, D, E, F, G, (2-methylpropanenitrile),
H,1.
CN

{J~.
: I .e~,H, and enantiomer

H,eX.·
H CN

A. 2-[3- [(lRS)-1.;.cyanoethyl]-5-(lH-1,2,4-triazol-1-ylmethyl) H. 2,2'-(5-methylbenzene-1,3-diyl)bis{2-

phenyl] -2-methylpropanenitrile, methylpropanenitrile),

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2020 Animal Epithelia and Outgrowths for Allergen Products 1-187

eN studies, in order to assess this aspect along with the source

Y
CH3
material characteristics upon storage.
CI ~I CH3
Control methods and acceptance criteria relating to identity
and purity of the animal epithelia and outgrowths are
H3C established. The acceptance criteria must ensure the
H CN
3C consistency of the animal epithelia and outgrowths source
material from a qualitative and quantitative point of view.
I. 2,2'-[5-(chloromethyl)benzene-l,3-diyl] bis(2- The animal epithelia and outgrowths source material is
methylpropanenitrile) . stored under controlled conditions justified by stability data.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _---'- PhEur The collection and production, as well as the handling of the
source material, are such that consistent composition is
ensured from batch to batch.
ANIMAL EPITHELIA AND OUTGROWTHS FOR
Animal Epithelia and Outgrowths ****
**
ALLERGEN PRODUCTS REFERENCE BATCH
* An appropriate reference batch is established for each animal
for Allergen Products ***** epithelia and outgrowths source material. The nature of the
(Ph. Bur. monograph 2621) reference batch depends on the testing approach to verify
PhEur _ batch-to-batch consistency and to establish acceptable
quality. The reference batch may be, for example, an internal
DEFINITION reference preparation (if available), a source material extract
Animal epithelia and outgrowths for allergen products consist or a sample of a production batch. Its characterisation must
of hair, epithelium fragments, dander, feathers and other be described. The extent of characterisation of the reference
structures that grow from the epidermis of mammals or batch depends on the nature ofthe animal epithelia and
birds. outgrowths source material, knowledge of the allergenic
Animal epithelia and outgrowths may contain proteins components and availability of suitable reagents.
deposited from the saliva and/or secretions from the The reference batch is stored under controlled conditions
sebaceous glands of the animal. They may be further ensuring its stability,
processed (e.g. cut or washed) using qualified methods or are BATCH-TO-BATCH CONSISTENCY
unprocessed. To establish batch-to-batch consistency, one or more of the
PRODUCTION following tests are performed on each batch. The choice of
Animal epithelia and outgrowths for allergen products are tests must be justified.
obtained from healthy animals selected to avoid possible Total protein (2.5.33)
transmissible agents of disease. The exact species and/or Protein profile
variety of animal is stated. Typical production steps, Determined by using suitable electrophoresis methods
including animal management, source material collection and (2.2.31, 2.2.54).
purification, are specified. The origin, quality, and
Allergen profile
traceability of the source material must be demonstrated.
Relevant allergenic components are identified by means of
It is expected that, where. applicable, the animal care and suitable techniques using allergen-specific antibodies.
husbandry follows the principles described for the protection
of vertebrate animals used for experimental and other
Major allergen content
scientific purposes. A responsible veterinarian or another Determined by using suitable immunochemical methods
competent person confirms the identity of the species and (2.7.1) such as enzyme-linked immunosorbent assay
that the animals are healthy. It is verified that the skin is (EUSA).
visibly clean and intact before harvest and that the animals Total allergenic activity
have not been recently treated with preparations for Determined by testing inhibition of the binding capacity of
cutaneous application, such as antiparasitic drugs. specific immunoglobulin E antibodies or by a suitable
The collection ofanimal epithelia and outgrowths must be equivalent in vitro method.
performed without injuring the skin of the animal. CHARACTERS
Confirmation that measures are in place to prevent cross- Animal epithelia and outgrowths for allergen products are
contamination by animal epithelia and outgrowths from other supplied as coloured powders or other materials such as
animals is provided, including during animal management, feathers, dander or hairs.
collection and processing. Methods involving the. grinding of
whole skin and/or pelts must not be used.
IDENTIFICATION
The identity of animal epithelia and outgrowths is confirmed
Where major changes to the productionof the animal
by their' relevant macroscopic and microscopic characteristics
epithelia and outgrowths take place (e.g. when a new process
in comparison to those ofa reference batch Or reference
or supplier is introduced), such changes are qualified.
documents. Identity may also be confirmed using other
Microbial contamination of the animal epithelia and methods such as EUSA or by genetic identification, if
outgrowths may be unavoidable and should be monitored on performed by generally accepted methods.
a representative number of batches of source material
according to a justified sampling plan and each time a new TESTS
supplier and/or a new process for the source material Foreign matter
production is introduced; if a determination of microbial Foreign matter is defined as vermin (e.g. mites and fleas),
contamination is not applicable, this must be justified. dirt, and foreign animal epithelia and outgrowths. Foreign
Microbial contamination values and potential increases in matter is determined by appropriate tests (e.g. microscopic
microbial contamination are monitored during stability examination, EUSA), visual inspection and/or tactile

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1-188 Antazoline Hydrochloride 2020

inspection. Foreign matter is below a predefined and justified D. It gives reaction (a) of chlorides (2.3.1).
limit. TESTS
Water (2.5.12 or 2.5.32) or loss on drying (2.2.32) Solution S
The water content of dried material is determined; Dissolve 2.0 g in carbon dioxide-free water R prepared from
specification limits must be supported by batch analysis and distilled waterR, heating at 60°C if necessary. Allow to cool
stability data. and dilute to 100 mL with the same solvent.
STORAGE Appearance of solution
The source materials are stored under controlled conditions Solution S is clear (2.2.1) and not more intensely coloured
justified by stability data. than reference solution Y7 (2.2.2" Method II).
LABELLING Acidity or alkalinity
The label states: To 10 mL of solution S add 0.2 mL of methyl red solution R.
- the species of the source animal; Not more than 0.1 mL of O.OlM hydrochloric acid or 0.01 M
- the nature of the animal epithelia and outgrowths. sodium hydroxide is required to change the colour of the
_ _ _--,- PhEur indicator.
Related substances
Examine by thin-layer chromatography (2.2.27), using silica
gel GF254 R as the coating substance. Heat the plate at
Antazoline Hydrochloride 110 °C for 15 min before using.
Test solution (a) Dissolve 0.10 g of the substance to be
(Ph. Bur. monograph 0972) examined in methanolR and dilute to 5 mL with the same
solvent.
Test solution (b) Dilute 1 mL of test solution (a) to 5 mL
with methanolR.
, Hel Reference solution (a) Dilute 0.5 mL of test solution (a) to
100 mL with methanolR.
Reference solution (b) Dissolve 20 mg of antazoline
hydrochloride CRS in methanolR and dilute to 5 mL with the
same solvent.
301.8 2508-72;.7
Reference solution (c) Dissolve 20 mg of xylometazoline
Action and use hydrochloride CRS in 1 mL of test solution (a) and dilute to
Histamine HI receptor antagonist; antihistamine. 5 mL with methanolR.
Apply to the plate 5 J.!L of each solution. Develop over a
PhEur --------
path of 15.cm using a mixture of 5 volumes of
DEFINITION diethylamine R, 10 volumes of methanol Rand 85 volumes of
Antazoline hydrochloride contains not less than 99.0 per cent ethyl acetate R. Dry the plate in a current of warm air for
and not more than the equivalent of 101.0 per cent of 15 min. Examine in ultraviolet light at 254 nm. The test is
N-benzyl-N- [(4,5-dihydro-lH-imidazol-2-yl)methyl] aniline not valid unless the chromatogram obtained. with reference
hydrochloride, calculated with reference to the dried solution (c) shows two dearly separated principal spots;
substance. Spray with a mixture of equal volumes of a 200 gIL solution
of ferric chloride R and a 5 gIL solution of potassium
CHARACTERS ferricyanide R. Examine immediately in daylight. Any spot in
A white or almost white, crystalline powder, sparingly soluble the chromatogram obtained with test solution (a), apart from
in water, soluble in alcohol, slightly soluble in methylene the principal spot, is not more intense. than the spot in the
chloride. chromatogram obtained with reference solution (a)
It melts at about 240°C, with decomposition. (0.5 per cent).
IDENTIFICATION Loss on drying (2.2.32)
First identification: A" D. Not more than 0.5 per cent, determined on 1.000 gby
Second identification: B" C, D. drying in an oven at 105°C for 3 h.
A; Examine by infrared absorption spectrophotometry Sulfated ash (2.4.14)
(2.2.24), comparing with the spectrum obtained with Not more than 0.1 per cent, determined on the residue
antazoline hydrochloride CRS. Examine the substances as discs obtained in the test for loss on drying.
prepared using potassium chloride R. ASSAY
B. Examine the chromatograms obtained in the test for Dissolve 0.250 gin 100 mL of alcohol R. Add 0.1 mL of
related substances in daylight after spraying. The principal phenolphthalein solution R1. Titrate with 0.1. M alcoholic
spot in the chromatogram obtained with test solution (b) is potassium hydroxide.
similar in position, colour and size to the principal spot in 1 mL of 0.1 M alcoholic potassiumhydroxide is equivalent to
the chromatogram obtained with reference solution (b). 30.18 mg of C17HzoC1N3.
C. To 5 mL of solution S (see Tests) add, drop by drop,
dilute sodium hydroxide solution R until an alkaline reaction is
produced. Filter. The precipitate, washed with two
quantities, each of 10 mL, of water R and dried in a
desiccator under reduced pressure, melts (2.2.14) at 119°C
to 123 °C.

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2020 Apomorphine Hydrochloride Hemihydrate 1-189

IMPURITIES precipitate is formed. The precipitate slowly becomes

greenish. Add 0.25 mL of 0.05 M iodine and shake.
The precipitate becomes greyish-green. Collect the
precipitate. The precipitate dissolves in methylene chloride R
giving a violet-blue solution and in ethanol (96 per cent) R
giving a blue solution.
D. To 2 mL of solution S (see Tests) add 0.1 mL of nitric
acid R.· Mix and filter. The filtrate gives reaction (a) of
A. N-(2-aminoethyl)-2-(benzylphenylamino)acetamide. chlorides (2.3.1).
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur TESTS
Solution S
Dissolve 0.25 g without heating in carbon dioxide-free water R
and dilute to 25 mL with the same solvent.
Apomorphine Hydrochloride Appearance of solution
Solution S is clear (2.2.1) and not more intensely coloured
Hemihydrate than reference solution BYs or GYs (2.2.2J Method II).
(Ph. Bur. monograph 0136) pH (2.2.3)
4.0 to 5.0 for solution S.
''6c5)H
HO ""'./
~ ~
Specific optical rotation (2.2.7)
-52 to -48 (dried substance).
~I Dissolve 0.25.g in a 2.06 gIL solution of hydrochloric acid R
N
H I
and dilute to 25.0 mL with the same acid solution.
CH3
Related substances
312.8 41372-20-7 Liquid chromatography (2.2.29).
Test solution Dissolve 50.0 mg of the substance to be
Action and use examined in a 1 per cent V/V solution of glacial aceticacid R
Dopamine receptor agonist; treatment of Parkinson's disease. and dilute to 20.0 mL with the same solution.
Preparation Reference solution (a) Dilute 1.0 mL of the test solution to
Apomorphine Hydrochloride for Homoeopathic Preparations 100.0' mL with a 1 per cent V/V solution of glacial acetic
PhEur ~ __,.----- acid R. Dilute 1.0 mL of this solution to 10.0 mL with a
1 per cent V/V solution of glacial acetic acid R.
DEFINITION Reference solution (b) Dissolve 12.5 mg of apomorphine
(6aR)-6-Methyl-5, 6,6a, 7-tetrahydro-4H-dibenzo [de,g] impurity B CRS in a 1 per cent V/V solution of glacial acetic
quinoline-l 0, ll-diol hydrochloride hemihydrate. acid R and dilute to 10.0 mL with the same solution.
Content Reference solution (c) Dilute 2.0 mL of reference solution (b)
98.5 per cent to 101.5 per cent (dried substance). to 10.0 mL with a 1 per cent V/V solution of glacial acetic
CHARACTERS acid R. Dilute 2.0 mL of this solution to 100.0 mL with a
Appearance 1 per cent V/V solution of glacial acetic acid R.
White or slightly yellowish-brown or green-tinged greyish, Reference solution (d) Dissolve 25 mg of boldineR in a
crystalline powder or crystals; on exposure to air and light, 1 per cent V/V solution of glacial acetic acid R and dilute to
the green tinge becomes more pronounced. 10.0 mL with the same solution. To 1 mL of this solution
add 1 mL of the test solution and dilute to 10.0 mL with a
Solubility
1 per cent V/V solution of glacial acetic acid R.
Sparingly soluble in water and in ethanol (96 per cent),
practically insoluble in toluene. Column:
- size: 1= 0.15 m, 0 = 4.6 mm;
IDENTIFICATION - stationaryphase: end-capped octadecylsilyl silica gel for
First identification: B J D. chromatography R (5 urn);
Second identification: A, C, D. - temperature: 35 0 C.
A. Ultraviolet and visible absorption spectrophotometry Mobile phase:
(2.2.25). - mobile phaseA: 1.1 gIL solution of sodium
Test solution Dissolve 10.0 mg in a 10.3 gIL solution of octanesulfonate R, adjusted to pH 2.2 with a
hydrochloric acid R and dilute to 100.0 mL with the same acid 50 per cent m/m solution of phosphoric acid R;
solution. Dilute 10.0 mL of the solution to 100.0 mL with a - mobile phase B: acetonitrile R;
10.3 gIL solution of hydrochloric acid R.
Spectral range 230-350 nm Time Mobile phase A Mobile phase B
(min) (per cent VIP) (per cent VIP)
Absorption maximum At 273 nm.
0-2 85 15
Shoulder At 300-310 nm. 2 - 32 85 -> 68 15 -> 32
Specific absorbance at the absorption maximum 530 to 570. 32 - 37 68 32
B. Infrared absorption spectrophotometry (2.2.24).
Comparison apomorphine hydrochloride hemihydrate CRS. Flow rate 1.5 mUmin.
C. To 5 mL of solution S (see Tests) add a few millilitres of Detection Spectrophotometer at 280 nm.
sodium hydrogen carbonate solution R until a permanent, white

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1-190 Aprepitant 2020

Injection 10 ut,
Identification of impurities Use the chromatogram obtained
with reference solution (b) to identify the peak due to
impurity B.
Relative retention With reference to apomorphine (retention
time = about 18 min): impurity B =about 0.4;
boldine = about 0.9.
System suitability Reference solution (d):
- resolution: minimum 2.5 between the peaks due to boldine
C. (6aR)-9-[7,8-didehydro-4,5Cl-epoxy-3-hydroxy-17-
and apomorphine.
methylmorphinan-6Cl-yl]-6-methyl-5,6,6a,7-tetrahydro-4H-
Limits: dibenzo [de,g] quinoline-1 0, ll-diol (morphine-apomorphine
- impurity B: not more than 0.75 times the area of the dimer).
corresponding peak in the chromatogram obtained with _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
reference solution (c) (0.15 per cent);
- unspecified impurities: for each impurity, not more than the
area ~f the principal peak in the chromatogram obtained
with reference solution (a) (0.10 per cent);
Aprepitant ***
- total: maximum 0.5 per cent;
- disregard limit: 0.5 times the area of the principal peak in
** **
the chromatogram obtained with reference solution (a) (Ph. Bur. monograph 2757) *****
(0.05 per cent).
Loss on drying (2.2.32)
2.5 per cent to 4.2 per cent, determined on 1.000 g by
drying in an oven at 105°C for 2 h.
Sulfated ash (2.4.14)
Maximum 0.1 per cent, determined on 1.0 g.
ASSAY
Dissolve 0.250 g in a mixture of 5.0 ml, of 0.01 M
hydrochloric acid and 50 rnL of ethanol (96 per cent) R. Carry
out a potentiometric titration (2.2.20), using 0.1 M sodium 534.4 170729-80-3
hydroxide. Read the volume added between the first 2 points
of inflexion. Action and use
Neurokinin-1 (NKI) receptor antagonist; prevention of
1 rnL of 0.1 M sodium hydroxide is equivalent to 30.38 mg of
nausea and vomiting associated with emetogenic
C 17H1SClNOZ' chemotherapy.
STORAGE Preparation
In an airtight container, protected from light. Aprepitant Capsules
IMPURITIES PhEur _
Specifiedimpurities B.
Other detectable impurities (the following substances would, if DEFINITION
presentat a sufficient level, be detected by one or otherof the tests 5-[[(2R,3S)-2-[(lR)-1-[3,5-Bis(trifluoromethyl)
in the monograph. They are limited by the general acceptance phenyl] ethoxy] -3-(4-fluorophenyl)morpholin-4-yl]methyl}-
criterion for other/unspecified impurities and/or by the general 1,2-dihydro-3H-1 ,2,4-triazol-3-one.
monograph Substances for pharmaceutical use (2034). It is Content
therefore not necessary to identify these impurities for 98.0 per cent to 102.0 per cent (anhydrous substance).
demonstration of compliance. See also 5.10. Control of impurities
CHARACTERS
in substances for pharmaceutical use) A, C.
Appearance
White or almost white powder.
Solubility
Very slightly soluble in water, sparingly soluble in anhydrous
ethanol, practically insoluble in heptane.
It shows polymorphism (5.9).

A. (6aR)-1 0-methoxy-6-methyl-5,6,6a,7-tetrahydro-4H- IDENTIFICATION

dibenzo [de,g] quinolin-I l-ol (apocodeine), A. Specific optical rotation (see Tests).
/CH 3
B. Infrared absorption spectrophotometry (2.2.24).
H N Comparison aprepitant CRS.

~.
If the spectra obtained in the solid state show differences,
dissolve the substance to be examined and the reference
substance separately in anhydrous ethanol R, evaporate to
HO 0" H6H
H dryness on a water-bath and record new spectra using the
residues.
B. 7,S-d.idehydro-4,5Cl-epoxy-17-methylmorphinan-3,6Cl-diol
(morphine),

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2020 Aprepitant 1-191

TESTS Sulfated ash (2.4.14)

Specific optical rotation (2.2.7) Maximum 0.1 per cent, determined on 1.0 g in a platinum
+ 66.0 to + 70.0 (anhydrous substance), measured at 25°C. crucible.
Dissolve 0.250 g in methanolR and dilute to 25.0 mL with ASSAY
the same solvent. Liquid chromatography (2.2.29) as described in the test for
Related substances related substances with the following modification.
Liquid chromatography (2.2.29): use the normalisation Injection Test solution and reference solution (a).
procedure. Calculate the percentage content of C23H21F7N403 taking
Solvent mixture acetonitrile R1, waterfor chromatography R into account the assigned content of aprepitani CRS.
(50:50 V/V).
IMPURITIES
Test solution Dissolve 40.0 mg of the substance to be Specified impurities A.
examined in the solvent mixture and dilute to 50.0 mL with
the solvent mixture. Other detectable impurities (the following substances would, if
present at a sufficient level, be detected by one or otherof the tests
Reference solution (a) Dissolve 40.0 mg of aprepitantCRS in in the monograph. They are limitedby the general acceptance
the. solvent mixture and dilute to 50.0 mL with the solvent criterion for other/unspecified impurities and/or by the general
mixture. monograph Substances for pharmaceutical use (2034). It is
Reference solution (b) Dilute 1.0 mL of the test solution to therefore not necessary to identify these impurities for
100.0 mf.with the solvent mixture. Dilute 1.0 mL ofthis demonstration of compliance. See also 5.10. Control of impurities
solution to 20.0 mL with the solvent mixture. in substances for pharmaceutical use) B, C.
Reference solution (c) Dissolve 4 mg of aprepitant for system
suitability CRS (containing impurity A) in 5.0 mL of the
solvent mixture.
Column:
- size: 1= 0.25 m, 0 = 4.6 mm;
- stationaryphase: end-capped octadecylsilyl silicagelfor
chromatography compatible with 100 per cent aqueous mobile
phasesR (5 urn);
- temperature: 35°C.
A. 5-[[(2R,3S)-2-[(lR)-1-[3,5-bis(trifluoromethyl)
Mobile phase:
phenyl] ethoxy]-3-phenylmorpholin-4-yl]methyl]-1 ,2-
- mobile phase A: 0.1 per cent V/V solution of phosphoric
dihydro- 3H-l,2,4-triazol-3-one,
acid R;
- mobile phase B: acetonitrile R1;

Time Mobile phase A Mobile phase B

(min) (per cent VIV) (per cent Viii')
0-2 65 35
2 - 22 65 ..... 20 35 ..... 80
22 - 32 20 80

Flow rate 1.5 mUmin. B. 5-[[(2R,3S)-2-[(lR)-1-[3,5-bis(trifluoromethyl)

Detection Spectrophotometer at 220nm. phenyl] ethoxy]-3-(4'-fiuorobiphenyl-3-yl)morpholin-4-
Injection 20 J.1L of the test solution and reference yl]methyl] -1,2-dihydro-3H-l,2,4-triazol-3-one,
solutions (b) and (c).
Identification of impurities Use the chromatogram supplied
with aprepitantfor system suitabz7ity CRS and the
chromatogram obtained with reference solution (c) to identify
the peak due to impurity A.
Relative retention With reference to aprepitant (retention
time = about 15 min): impurity A = about 0.97.
System suitability Reference solution (c):
- resolution: minimum 1.5 between the peaks due to
impurity A and aprepitant.
Limits:
- impurity A: maximum 0.15 per cent;
- unspecified impurities: for each impurity, maximum C. 5-[[(2R,3S)-2-[(lR)-1-[3,5-bis(trifluoromethyl)
0.10 per cent; phenyl] ethoxy]-3-(4'-fiuorobiphenyl-4-yl)morpholin-4-
- total: maximum 0.2 per cent; yl]methyl] - i ,2-dihydro-3H-l ,2,4-triazol-3-one.
- reporting threshold: 0.05 per cent (reference solution (b)). _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _----'- PhEur

Water (2.5.32)
Maximum 0.2 per cent, determined on 0.200 g by direct
sample introduction.

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1-192 Aprotinin 2020

Test solution Dilute 1 mL of solution S to 50 mL with buffer

Aprotinin solution pH 7.2 R.
(Ph. Eur. monograph 0580) Trypsin solution - Dissolve 10 mg of trypsin BRP in 0.002 M
hydrochloric acid and dilute to 100 mL with the same acid.
H-Arg-Pro-Asp-Phe-Cys-Leu-Glu-Pro-Pro-Tyr-
10
Casein solution Dissolve 0.2 g of casein R in buffersolution
Thr - Gly - Pro- Cys - Lys - Ala - Arg -lie -lie - Arg- pH 7.2 R and dilute to 100 mL with the same buffer
20
~-P~-~-A~-~-~-~-~-~-C%­
solution.
30
Precipitating solution glacial acetic acid R, waterR, anhydrous
Gin- Thr - Phe- Val- Tyr - Gly - Gly - Cys- Arg- Ala-
40 ethanolR (1:49:50 VIVIV).
Lys - Arg- Asn- Asn -Phe -Lys-Ser - Ala-Glu - Asp-
50 Mix 1 mL of the test solution with 1 mL of the trypsin
Cys- Met- Arg- Thr - Cys - Gly - Gly -:- Ala- OH solution. Allow to stand for 10 min and add 1 mL of the
58
casein solution. Incubate at 35 "C for 30 min. Cool in iced
water and add 0.5 mL of the precipitating solution. Shake
6511 9087-70-1 and allow to stand at room temperature for 15 min.
The solution is cloudy. Carry out a blank test under the
Action and use same conditions using buffer solution pH 7.2 R instead of the
, Antifibrinolytic.
test solution. The solution is not cloudy.
PhEur _ TESTS
DEFINITION Solution S
Aprotinin is a polypeptide consisting of a chain of 58 amino Prepare a solution of the substance to be examined
acids. It inhibits stoichiometrically the activity of several containing 15 Ph. Eur. U.lmL, calculated from the activity
proteolytic enzymes such as chymotrypsin, kallikrein, plasmin stated on the label.
and trypsin. It contains not less than 3.0 Ph. Eur. U. Appearance of solution
of aprotinin activity per milligram, calculated with reference Solution S is clear (2.2.1).
to the dried substance. Absorbance (2.2.25)
PRODUCTION Maximum 0.80 by measuring at the absorption maximum at
The animals' from which aprotinin is derived must fulfil the 277 run.
requirements for the health of animals suitable for human Prepare a solution of the substance to be examined
consumption. containing 3.0 Ph. Eur. U.lmL.
The method of manufacture is validated to demonstrate that Des-Ala-aprotinin and des-Ala-des-Gly-aprotlnin
the product, if tested, would comply with the following test. Capillary zone electrophoresis (2.2.47): use the normalisation
Histamine (2.6.10) procedure.
Maximum 0.2 !lg of histamine base per 3 Ph. Eur. U. Test solution Prepare a solution of the substance to be
examined in water R containing not less than
CHARACTERS
1 Ph. Eur. U.lmL.
Appearance
Almost white hygroscopic powder. Reference solution Dilute aprotinin solution BRP in waterR to
obtain the same concentration as the test solution.
Solubility
Soluble in water and in isotonic solutions, practically Capillary:
insoluble in organic solvents. - material: uncoated fused silica;
- size: effective length = 45-60 em, 0 =·75 urn.
IDENTIFICATIO.N Temperature 25 DC.
A. Thin-layer chromatography {2.2.27).
CZE buffer Dissolve 8.21 g of potassium dihydrogen
Test solution Solution S (see Tests). phosphate R in 400 mL of waterR, adjust to pH 3.0 with
Reference solution Dilute aprotinin solution BRP in water R to phosphoric acid R, dilute to 500.0 mL with waterR and filter
obtain a concentration of 15 Ph. Eur. U.lmL. through a membrane filter (nominal pore size 0.45 urn).
Plate TLC silica gel G plate R. Detection Spectrophotometer at 214 run.
Mobile phase water R, glacial acetic acid R (80:100 VIV) Between-run rinsing Rinse the capillary for at least 1 min
containing 100 gIL of sodium acetate R. with 0.1 M sodium hydroxide filtered through a membrane
Application 10!lL. filter (nominal pore size 0.45 urn) and for 2 min with the
Development Over a path of 12 em. CZE buffer.
Drying In air. Injection . Under pressure or vacuum (for example, 3 s at a
differential pressure of 3.5 kPa).
Detection Spray with a solution of 0.1 g of ninhydrin R in a
mixture of 6 ml.of a 10 gIL solution of cupric chloride R, Migration Apply a field strength of 0.2 kVlcm, using the
21 mL of glacial acetic acid Rand 70 mL of anhydrous CZE buffer as the electrolyte in both buffer reservoirs.
ethanolR. Dry the plate at 60 "C. Run time 30 min.
Results The principal spot in the chromatogram obtained Identification of impurities Use the e1ectropherogram supplied
with the test solution is similar in position, colour and size to with aprotinin solution BRP and the e1ectropherogram
the principal spot in the chromatogram obtained with the obtained with the reference solution to identify the peaks due
reference solution. to impurities A and B.
B. Determine the ability of the substance to be examined to Relative migration With reference to aprotinin (migration
~bit trypsin activity using the method described below. time = about 22 min): impurity A = about 0.98;
impurity B = about 0.99.

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2020 Aprotinin 1-193

System suitability Reference solution after at least Reference solution Treat the substance to be examined to
6 injections: obtain about 2 per cent aprotinin oligomers. For example,
- migration time: aprotinin = 19.0 min to 25.0 min; heat freeze-dried aprotinin at about 110 "C for about 4 h.
- resolution: minimum 0.8 between the peaks due to Then dissolve in water R to obtain a concentration of about
impurities A and B; minimum 0.5 between the peaks due 5 Ph. Eur. U.lmL.
to impurity Band aprotinin; Column 3 columns coupled in series:
- peak distribution: the electropherogram obtained is - size: l = 0.30 m, 0 =7.8 mm;
qualitatively and quantitatively similar to the - stationaryphase: hydrophilic silica gelfor chromatography R
electropherogram supplied with aprotinin solution BRP; of a grade suitable for fractionation of globular proteins in
- height of the principal peak: at least 1000 times the height the relative molecular mass range of 20 000 to
of the baseline noise. If necessary, adjust the sample load 10000 000 (8 urn),
to give peaks of sufficient height.
Mobile phase acetonitrile R, glacial acetic acid R, waterfor
Limits: chromatography R (2:2:6 VIVIV); filter and degas.
- impurity A: maximum 8.0 per cent; Flow rate 1.0 mUmin.
-'-- impurity B: maximum 7.5 per cent.
Detection Spectrophotometer at 277 nm.
Pyroglutamyl-aprotinin and related compounds
Injection 100 ~.
Liquid chromatography (2.2.29): use the normalisation
procedure. Run time 40 min.
Test solutlon' Prepare a solution of the substance to be Relative retention With reference to aprotinin monomer
examined ,in mobile phase A, containing about (retention time = 24.5 min to 25.5 min): aprotinin
5 Ph. Eut;'U./mL. =
dimer about 0.9.
Reference solution 'Dissolve the contents of a vial of aprotinin System suitability Reference. solution:
for systemsttitabilitjJ CRS in 2.0 mL of mobile phase A. - resolution: minimum 1.3 between the peaks due to
aprotinin dimer and monomer;
Column:
- symmetryfactor. maximum 2.5 for the peak due to
- size: l = 0.075 m, 0= 7.5 mm;
aprotinin monomer.
- stationaryphase: strong cation-exchange silica gelfor
chromatography R (10 urn); Limit:
- temperature: 40 DC. - total: maximum 1.0 per cent.
Mobile phase: Loss on drying (2.2.32)
- mobile phaseA: dissolve 3.52 g of potassium dihydrogen Maximum 6.0 per cent, determined on 0.100 g by drying in
phosphate Rand 7.26 g of disodium hydrogen phosphate vacuo.
dihydrate R in 1000 mL of waterfor chromatography R; Bacterial endotoxins (2.6.14)
filter and degas; Less than 0.14 ill per European Pharmacopoeia Unit of
- mobile phase B: dissolve 3.52 g of potassium dihydrogen aprotinin, if intended for use in the manufacture of
phosphate R, 7.26 g of disodium hydrogen phosphate parenteral preparations without a further appropriate
dihydrate Rand 66.07 g of ammonium sulfate R in procedure for the removal of bacterial endotoxins.
1000 niL of waterfor chromatography R; filter and degas;
ASSAY
The activity of aprotinin is determined by measuring its
TiIne Mobile phase A Mobile phase B
(min) (per cent VIP) (per cent VIJI) inhibitory action on a solution of trypsin of known activity.
0-21 92-> 64 8 -> 36
The inhibiting activity of the aprotinin is calculated from the
64 -> 0 36 -> 100
difference between the initial activity and the residual activity
21 - 30
of the trypsin.
The inhibiting activity of aprotinin is expressed in European
Flow rate 1.0 mUmin.
Pharmacopoeia Units. 1 Ph. Eur. U. inhibits 50 per cent of
Detection Spectrophotometer at 210 nm. the enzymatic activity of 2 microkatals of trypsin.
Injection 40 j.lL. Use a reaction vessel with a capacity of about 30 mL,
Relative retention With reference to aprotinin (retention provided with:
time = 17.0 min to 20.0 min): impurity C = about 0.9. - a device that will maintain a temperature of 25 ± 0.1 DC;
System suitability Reference solution: - a stirring device, such as a magnetic stirrer;
- resolution: minimum 1.5 between the peaks due to - a lid with 5 holes for accommodating the electrodes, the
impurity C and aprotinin; tip of a burette, a tube for the admission of nitrogen and
- symmetryfactor: maximum 1.3 for the peak due to the introduction of the reagents.
aprotinin. An automatic or manual titration apparatus may be used.
Limits: In the latter case the burette is graduated in 0.05 mL and the
- impurity C: maximum 1.0 per cent; pl-l-meter is provided with a wide reading scale and glass-
- any otherimpurity: maximum 0.5 per cent; silver-silver chloride or other suitable electrodes.
- sum of impurities other than C: maximum 1.0 per cent. Test solution Prepare a solution of the substance to be .
Aprotinin oligomers examined in 0.0015 M borate buffersolution pH 8.0 R
Size-exclusion chromatography (2.2.30): use the expected to contain 1.67 Ph. Eur. U.lmL (about 0.6 mg
normalisation procedure. (m mg) per millilitre).
Test solution Prepare a solution of the substance to be Trypsin solution Prepare a solution of trypsin BRP containing
examined in waterR containing about 5 Ph. Eur. U./mL. about 0.8 microkatals per millilitre (about 1 mg/mL), using
O. 001 M hydrochloric acid as the solvent. Use a freshly
prepared solution and keep in iced water.

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1-194 Aprotinin 2020

Glp - Arg - Pro- Asp- Phe- Cys- Leu- Glu- Pro - Pro - Tyr-
Trypsin and aprotinin solution To 4.0 mL of the trypsin 1 11
solution add 1.0 mL of the test solution. Dilute immediately Thr - Gly- Pro - Cys- Lys - Ala - Arg-lie -lie - Arg-
21
to 40.0 mL with 0.0015 M borate buffer solution pH 8.0 R. Tyr- Phs - Tyr- Asn- Ala-Lys - Ala - Gly-Leu -Cys-
Allow to stand at room temperature for 10 min and then 31
Gin- Thr - Phe- Val- Tyr - Gly - Gly - Cys- Arg - Ala -
keep in iced water. Use within 6 h of preparation. 41
~-~-A~-A~-P~-~-~-~-~-~p­
Dilute trypsin solution Dilute 0.5 mL of the trypsin solution 51
to 10.0 mL with 0.0015 M borate buffer solution pH 8.0 R. C%-~-~-~-C%-~-~-~-OO
59
Allow to stand at room temperature for 10 min and then
keep in iced water.
Maintain an atmosphere of nitrogen in the reaction flask and C. (5-oxoprolyl)aprotinin (pyroglutamylaprotinin).
stir continuously; introduce 9.0 mL of 0.0015 M borate buffer _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur

solution pH 8.0 Rand 1.0 mL of a freshly prepared 6.9 gIL

solution of benzoylarginine ethyl ester hydrochloride R. Adjust to
pH 8.0 with 0.1 M sodium hydroxide. When the temperature
has reached equilibrium at 25 ± 0.1 DC, add 1.0 mL of the Aprotinin Concentrated Solution
trypsin and aprotinin solution and start a timer. Maintain at
pH 8.0 by the addition of 0.1 M sodium hydroxide and note (Ph. Bur. monograph 0579)
the volume added every 30 s. Continue the reaction for
6 min. Determine the number of millilitres of 0.1 M sodium H - Arg- Pro- Asp- Phe- Cys- Leu- Glu- Pro- Pro- Tyr-
LO
hydroxide used per second (nl mL). Carry out, under the Thr - Gly - Pro- Cys- Lys- Ala - Arg-lie -lie - Arg-
20
same conditions, a titration using 1.0 mL of the dilute
~-P~-~-A~-~-~-~-~-~-C%-
trypsin solution. Determine the number of mil1i1itres of 30

0.1 M sodium hydroxide used per second (nz mL). Gin ~ Thr - Phe- Val- Tyr - Gly - Gly - Cys- Arg- Ala-
40
Calculate the aprotinin activity in European Pharmacopoeia Lys - Arg- Asn- Asn- Phe- Lys- Ser - Ala - Glu- Asp-
50
Units per milligram using the following expression: Cys - Met- Arg- Thr - Cys- Gly - Gly - Ala - OH
58

4000( 2nz - nd
m 6511

The estimated activity is not less than 90 per cent and not ; Action and use
more than 110 per cent of the activity stated on the label. Antifibrinolytic.
STORAGE PhEur _
In an airtight, tamper-proof container, protected from light.
DEFINITION
LABELLING Aprotinin concentrated solution is a solution of aprotinin, a
The label states: polypeptide consisting of a chain of 58 amino acids, which
- the number of European Pharmacopoeia Units of inhibits stoichiometrically the activity of several proteolytic
aprotinin activity per milligram; enzymes such as chymotrypsin, kallikrein, plasmin and
- where applicable, that the substance is suitable for use in trypsin. It contains not less than 15.0 Ph. Eur. U.
the manufacture of parenteral preparations. of aprotinin activity per millilitre.
IMPURITIES PRODUCTION
The animals from which aprotinin is derived must fulfil the
H - Arg - Pro- Asp - Phe - Cys - Leu- Glu- Pro - Pro- Tyr-
I w requirements for the health of animals suitable for human
~-~-~-C~-~-~-~-~-~-~- consumption.
20
~-P~-~-~n-~-~-~-~-~-C%­ The method of manufacture is validated to demonstrate that
30
~-~-P~-~-~-~-~-~-~-~-
the product, if tested, would comply with the following test.
40
~-~-A~-~n-~e-~-S~-~-~-A~­
Histamine (2.6.10)
50 Maximum 0.2 ug of histamine base per 3 Ph. Eur. U.
Cys - Met- Arg - Thr - Cys - Gly - OH
56 CHARACTERS
Appearance
A. aprotinin-(l-56)-peptide, Clear, colourless liquid.

H- Arg- Pro- Asp - Phe -Cys - Leu- Glu-Pro -Pro - Tyr-

IDENTIFICATION
I w A. Thin-layer chromatography (2.2.27).
Thr - Gly- Pro - Cys - Lys - Ala - Arg -lie -lie - Arg-
20 Test solution Solution S (see Tests).
~-P~-~-A~-~-~-~-~-~-~-
30 Reference solution Dilute aprotinin solution BRP in water R to
~-~-P~-~-~-~-~-C%-~-~- obtain a concentration of 15 Ph. Eur. U.lmL.
40
Lys - Arg- Asn - Asn - Phe - Lys- Ser - Ala - Glu- Asp- Plate TLC silica gel G plate R.
50
Cys - Met- Arg - Thr - Cys - Gly - Gly - OH Mobile phase water R, glacial acetic acid R (80:100 V/V)
57
containing 100 gIL of sodium acetate R.
Application 10 jlL.
B. aprotinin-(l-57)-peptide, Development Over a path of 12 em.
Drying In air.

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2020 Aprotinin 1-195

Detection Spray with a solution of 0.1 g of ninhydrin R in a Migration Apply a field strength of 0.2 kV/cm, using the
mixture of 6 mL of a 10 gIL solution of cupric chloride R, CZE buffer as the electrolyte in both buffer reservoirs.
21 mL of glacial acetic acid Rand 70 mL of anhydrous Run time 30 min.
ethanol R. Dry the plate at 60°C. Identification of impurities Use the electropherogram supplied
Results The principal spot in the chromatogram obtained with aprotinin solution BRP and the electropherogram
with the test solution is similar in position, colour and size to obtained with the reference solution to identify the peaks due
the principal spot in the chromatogram obtained with the to impurities A and B.
reference solution. Relative migration With reference to aprotinin (migration
B. Determine the ability of the preparation to be examined to time = about 22 min): impurity A = about 0.98;
inhibit trypsin activity using the method described below. impurity B = about 0.99.
Test solution 'Dilute 1 mL of solution S to 50 mL with buffer System suitabz1ity Reference solution after at least 6
solutionpH 7.2 R. injections:
Trypsin solution Dissolve 10 mg of trypsin BRP in 0.002 M - migration time: aprotinin = 19.0 min to 25.0 min;
hydrochloric acid and dilute to 100 mL with the same acid. - resolution: minimum 0.8 between the peaks due to
Casein solution Dissolve 0.2 g of casein R in buffersolution impurities A and B; minimum 0.5 between the peaks due
pH 7.2 R and dilute 1:0 100 mL with the same buffer to impurity Band aprotinin;
solution. - peak distribution: the electropherogram obtained is
Precipitating. solution glacialacetic acid R, water R, anhydrous qualitatively and quantitatively similar to the
electropherogram supplied with aprotinin solution BRP;
ethanol R (1::49:50 VIVIV).
- height of the principal peak: at least 1000 times the height
Mix 1 mLofthe test solution with 1 mL of the trypsin of the baseline noise. If necessary, adjust the sample load
solution. Allow to stand for 10 min and add 1 mL of the to give peaks of a sufficient height.
casein solution. Incubate at 35°C for 30 min. Cool in iced
waterand add 0.5 mL of the precipitating solution. Shake Limits:
. - impurity A: maximum 8.0 per cent;
and allow to stand at room temperature for 15 min.
- impurity B: maximum 7.5 per cent.
The solution is cloudy. Carry out a blank test under the
same conditions using buffersolution pH 7.2 R instead of the Pyroglutamyl-aprotinin and related compounds
test solution. The solution is not cloudy. Liquid chromatography (2.2.29): use the normalisation
procedure.
TESTS
Testsolution Dilute the preparation to be examined in
Solution S
Prepare a solution containing 15 Ph. Eur. U .ImL, if mobile phase A to a concentration of about
necessary by dilution, on the basis of the activity stated on 5 Ph. Eur. U.lmL.
the label. Reference solution Dissolve the contents of a vial of aprotinin
for systemsuitabilityCRS in 2.0 mL of mobile phase A.
Appearance of solution
Solution S is clear (2.2.1). Column:
- size: 1 = 0.075 m, 0 = 7.5 mm;
Absorbance (2.2.25) - stationaryphase: strong cation-exchange silica gelfor
Maximum 0.80 by measuring at the absorption maximum at chromatography R (10 urn);
277 om. - temperature: 40 "C.
Prepare a solution containing 3.0 Ph. Eur. U.lmL. Mobile phase:
Des-Ala-aprotinin and des-Ala-des-Gly-aprotinin - mobile phase A: dissolve 3.52 g of potassium dihydrogen
Capillary zone electrophoresis (2.2.47): use the normalisation phosphate Rand 7.26 g of disodium hydrogen phosphate
procedure. dihydrate R in 1000 mL of waterfor chromatography R;
Test solution Dilute the preparation 10 be examined in filter and degas;
water R to obtain a concentration of not less than 1 Ph Eur. - mobile phase B: dissolve 3.52 g of potassium dihydrogen
U.lmL. phosphate R, 7.26 g of disodium hydrogen phosphate
Reference solution Dilute aprotinin solution BRP in water R to dihydrate Rand 66.07 g of ammonium sulfate R in
obtain the same concentration as the test solution. 1000 mL of waterfor chromatography R; filter and degas;
Capillary:
Time Mobile phase A Mobile phase B
- material: uncoated fused silica; (min) (per cent VIV) (per cent VIV)
- size: effective length = 45-60 em, 0 = 75 urn. 0-21 92 -.64 8 -. 36
Temperature 25 -c. 21 - 30 64 -. 0 36 -. 100
CZE buffer Dissolve 8.21 g of potassium dihydrogen
phosphate R in 400 mL of water R, adjust to pH 3.0 with Flow rate 1.0 mIJmin.
phosphoric acid R, dilute to 500.0 mL with waterR and filter
Detection Spectrophotometer at 210 nm.
through a membrane filter (nominal pore size 0.45 urn).
Injection 40 ilL.
Detection Spectrophotometer at 214 nm.
Relative retention With reference to aprotinin (retention
Between-run rinsing Rinse the capillary for at least 1 min
time = 17.0 min to 20.0 min): impurity C = about 0.9.
with 0.1 M sodium hydroxide filtered through a membrane
filter (nominal pore size 0.45 urn) and for 2 min with the System suitability Reference solution:
CZE buffer. - resolution: minimum 1.5 between the peaks due to
impurity C and aprotinin;
Injection Under pressure or vacuum (for example, 3 s at a
- symmetryfactor: maximum 1.3 for the peak due to
differential pressure of 3.5 kPa).
aprotinin.

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1-196 Aprotinin 2020

Limits: - a lid with 5 holes for accommodating the electrodes, -the

- impurity C: maximum 1.0 per cent; tip of a burette, a tube for the admission of nitrogen and
- any otherimpurity: maximum 0.5 per cent; the introduction of the reagents.
- sum of impurities other than C: maximum 1.0 per cent. An automatic or manual titration apparatus may be used.
Aprotinin oligomers In the latter case the burette is graduated in 0.05 mL and the
Size-exclusion chromatography (2.2.30): use the pH-meter is provided with a wide reading scale and glass-
normalisation procedure. silver-silver chloride or other suitable electrodes.
Test solution Dilute the preparation to be examined in Test solution With O.OOlSM borate buffersolution pH 8.0 R
water R to obtain a concentration of about 5 Ph. Eur. U.lmL. prepare an appropriate dilution (D) of the aprotinin
Reference solution Treat the substance to be examined to concentrated solution expected, on the basis of the stated
obtain about 2 per cent aprotinin oligomers. For example, potency, to contain 1.67 Ph. Eur. U.lmL.
heat freeze-dried aprotinin at about 110°C for about 4 h. Trypsin solution Prepare a solution of trypsin BRP containing
Then dissolve in water R to obtain a concentration of about about 0.8 microkatals per millilitre (about 1 mglmL), using
5 Ph. Eur. U.lmL. 0.001 M hydrochloric acid as the solvent. Use a freshly
Column 3 columns coupled in series: prepared solution and keep in iced water.
~ size: 1= 0.30 m, 0 = 7.8 mm; Trypsin and aprotinin solution To 4.0 mL of the trypsin
- stationaryphase: hydrophilic silica gelfor chromatography R solution add 1.0 mL of the test solution. Dilute immediately
of a grade suitable for fractionation of globular proteins in to 40.0 mL with 0.0015 M borate buffersolution pH 8.0 R.
the relative molecular mass range of 20 000 to Allow to stand at room temperature for 10 min and then
10000000 (8 urn). keep in iced water. Use within 6 h of preparation;
Mobile phase acetonitrile R, glacial acetic acid R, waterfor Dilute trypsin solution Dilute 0.5 mL of the trypsin solution
chromatography R (2:2:6 VIVIV); filter and degas. to 10.0 mL with 0.0015 M borate buffersolution pH 8.0 R.
Flow rate 1.0 mlJmin. Allow to stand at room temperature for 10 min and then
keep in iced water.
Detection Spectrophotometer at 277 nm.
Maintain an atmosphere of nitrogen in the reaction flask and
Injection 100 ~L.
stir continuously; introduce 9.0 mt of 0.0015 M borate buffer
Run time 40 min. solution pH 8.0 Rand 1.0 mL of a freshly prepared 6.9 g/L
Relative retention With reference to aprotinin monomer solution of benzoylarginine ethyl esterhydrochloride R. Adjust to
(retention time = 24.5 min to 25.5 min): aprotinin pH 8.0 with 0.1 M sodium hydroxide. When the temperature
dimer = about 0.9. has reached equilibrium at 25 ± 0.1 "C, add 1.0 mL of the
System suitability Reference solution: trypsin and aprotinin solution and start a timer. Maintain at
- resolution: minimum 1.3 between the peaks due to pH 8.0 by the addition of 0.1 M sodium hydroxide and note
aprotinin dimer and monomer; the volume added every 30 s. Continue the reaction for
- symmetryfactor. maximum 2.5 for the peak due to 6 min. Determine the number of millilitres of 0.1 M sodium
aprotinin monomer. hydroxide used per second (n! mL). Carry out, under the
Limit: same conditions, a titration using 1.0 mL of the dilute
- total: maximum 1.0 per cent. trypsin solution. Determine the number of millilitres of
0.1 M sodium hydroxide used per second (nz mL).
Specific activity of the dry residue
Minimum 3.0 Ph. Eur. U. of aprotinin activity per milligram Calculate the aprotinin activity in European Pharmacopoeia
of dry residue. Units per millilitre using the following expression:
Evaporate 25.0 mL to dryness ina water-bath, dry the 4000(2n2 - nl) x D
residue at 110°C for 15 hand weigh. From the mass of the
residue and the activity determined as described below, D dilution factor of the aprotinin concentrated solution to be
calculate the number of EUropean Pharmacopoeia Units per examined.in order to obtain a solution containing
milligram of dry residue. 1.67 Ph. Eur. U.lmL.

Bacterial endotoxins (2.6.14) The estimated activity is not less than 90 per cent and not
Less than 0.14 ill per European Pharmacopoeia Unit of more than 110 per cent of the activity stated on the label.
aprotinin, if intended for use in the manufacture. of
parenteral preparations without a further appropriate STORAGE
procedure for the removal of bacterial endotoxins. In an airtight, tamper-proof container, protected from light.
ASSAY LABELLING
The activity of aprotinin is determined by measuring its The labelstates:
inhibitory action on a solution of trypsin of known activity. - the number of European Pharmacopoeia Units of
The inhibiting activity of the aprotinin is calculated from the aprotinin activity per millilitre;
difference between the initial activity and the residual activity - where applicable, that the substance is suitable for use in
of the trypsin. the manufacture of parenteral preparations.
The inhibiting activity of aprotinin is expressed in European
Pharmacopoeia Units. 1 Ph. Eur. U. inhibits 50 per cent of
the enzymatic activity of 2 microkatals of trypsin.
Use a reaction vessel with a capacity of about 30 mL,
provided with:
- a device that will maintain a temperature of 25 ± 0.1 °C;
- a stirring device, such as a magnetic stirrer;

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2020 Arachis Oil 1-197

IMPURITIES Results The chromatogram obtained is similar to the

corresponding chromatogram shown in Figure 2.3.2.-1.
H- Arg - Pro - Asp - Phe- Cys- Leu- Glu- Pro - Pro- Tyr-
1 . 10 B. Composition of fatty acids (see Tests).
Thr - Gly - Pro - Cys- Lys- Ala - Arg -lie -lie - Arg-
20 TESTS
~-P~-~-~-~-~-~-~-~-C~- Acid value (2.5.1)
30
Gin- Thr - Phe - Val- Tyr - Gly - Gly - Cys - Arg - Ala - Maximum 0.5, determined on 10.0 g.
40
~-~~A~-~n-P~-~-~-~-~-A~­ Peroxide value (2.5.5, Method A)
50 Maximum 5.0.
Cys - Met - Arg - Thr - Cys - Gly - OH
56 Unsaponifiable matter (2.5.7)
Maximum 1.0 per cent, determined on 5.0 g.
A. aprotinin-(1-56)-peptide, Alkaline impurities (2.4.19)
It complies with the test.
H- Arg- Pro - Asp - Phe- C~ - Leu- Glu ~ Pro - Pro - Tyr-
1 10 Composition-of fatty acids' (2.4.22, Method A)
Thr - Gly ~ Pro - Cys- Lys- Ala - Arg -lie -lie - Arg-
20 Use the mixture of calibrating substances in Table 2.4.22.-3.
Tyr - Phe - Tyr - Asn- Ala - Lys- Ala - Gly - Leu- Cys- Composition of thefatty-acidfraction of the oil:
30
Gin- Thr - Phe - Val- Tyr - Gly - Gly - Cys - Arg - Ala - - saturatedfatty acids of chain length less than C16 : maximum
40
~-~-~n-A~-P~-~-~-~-~-~p­
0.4 per cent;
50 - palmitic add: 5.0 per cent to 14.0 per cent;
Cys- Met - Arg - Thr - Cys- Gly - Gly - OH ~ stearic acid: 1.3 per cent to 6.5 per cent;
57
- oleic acid: 35.0 percent to 76.0 per cent;
- linoleic acid: 8.0 per cent to 43.0 per cent;
B. aprotiIJ.in-(1-57)-peptide,
- linolenic acid: maximum 0.6 per cent;
~-~-~-~p-~-C~-~-~-~-~-~-
- arachidic acid: 0.5 per cent to 3.0 per cent;
I 11 - eicosenoic acid: 0.5 percent to 3.0 per cent;
Thr - Gly - Pro - Cys- Lys - Ala - Arg -lie -lie - Arg- - behenic acid: 1.0 per cent to 5.0 per cent;
21
~-P~-~-~-~-~-~-~-~-C~- - erucic acid: maximum 0.5 per cent;
, 31
Gin- Thr - Phe - Val- Tyr - Gly - Gly - Cys - Arg - Ala -
- lignoceric acid: 0.5 per cent to 3.0 per cent.
41
Water (2.5.32)
Lys- Arg- Asn- Asn-Phe-Lys-Ser- Ala- Glu- Asp-
51 Maximum 0.1 per cent, determined on 1.00 g.
Cys - Met - Arg - Thr - Cys - Gly - Gly7 Ala - OH
59 STORAGE
In a well-filled container, protected from light.
C. (5-oxoprolyl)aprotinin (pyroglutamylaprotiIJ.in). _ _ _ _ _ _ _ _ _ _--:- PhEur
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur

Hydrogenated Arachis Oil

Arachis Oil Hydrogenated Peanut Oil
Peanut Oil (ph. Bur. monograph 1171)
(RefinedArachis oa, Ph. Bur. monograph 0263) PhEur _

Preparation DEFINITION
Arachis Oil Enema .Oil obtained by refining, bleaching, hydrogenating and
PhEur _ deodorising oil obtained from the shelled seeds of Arachis
DEFINITION hypogaea L. Each type of hydrogenated arachis oil is
characterised by its nominal drop point.
The refined fatty oil obtained from the shelled seeds of
Arachis hypogaea L. A suitable antioxidant may be added. CHARACTERS
CHARACTERS Appearance
White or faintly yellowish, soft mass which melts to a clear,
Appearance
pale yellow liquid when heated.
Clear, yellowish, viscous liquid.
Solubility
Solubility
Practically insoluble in water, freely soluble in methylene
Very slightly soluble in ethanol (96 per cent), miscible with
chloride and in light petroleum (bp: 65-70 °C), very slightly
light petroleum.
soluble in ethanol (96 per cent).
Relative density
About 0.915. IDENTIFICATION
First identification: A~ C.
It solidifies at about 2°C.
Second identification: A, B.
IDENTIFICATION
A. Drop point (see Tests).
First identification: B.
B. Identification of fatty oils by thin-layer chromatography
Secondidentification: A. (2.3.2).
A. Identification of fatty oils by thin-layer chromatography
(2.3.2).

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 1-198 Arginine 2020

Results The chromatogram obtained is similar to the Source Nickel hollow-cathode lamp.
corresponding chromatogram shown in Figure 2.3.2.-1. Wavelength 232 nm.
C. Composition of fatty acids (see Tests). Atomisation deoice Graphite furnace.
TESTS Carrier gas argon R.
Drop point (2.2.17) STORAGE
32°C to 43 DC, and within 3 °C of the nominal value.
Protected from light.
Acid value (2.5.1)
LABELLING
Maximum 0.5.
The label states the nominal drop point.
Dissolve 10.0 gin 50 mL of the prescribed solvent by _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
heating on a water-bath.
Peroxide value (2.5.5, Method A)
Maximum 5.0.
Dissolve 5.0 gin 30 mL of the prescribed solvent by heating
on a water-bath.
Arginine
, Unsaponifiable matter (2.5.7) (Ph. Bur. monograph 0806)
Maximum 1.0 per cent.
Alkaline impurities (2.4.19)
It complies with the test.
Composition of fatty acids (2.4.22, Method A)
Use the mixture of calibrating substances in Table 2.4.22.-3.
Column: 174.2 74-79-3
- material: fused silica;
=
- size: l = 25 m, 0 0.25 mrn; Action and use
- stationary phase: poly(cyanopropyl)szloxane R (film thickness Amino acid; nutrient.
0.2 urn). PhEur -,-- _
Carrier gas helium for chromatography R.
DEFINITION
Flow rate 0.7 mlJmin. (2S)-2-Amino-5-guanidinopentanoic acid.
Split ratio 1:100. Product of fermentation or of protein hydrolysis.
Temperature: Content
- column: 180°C for 20 min;
98.5 per cent to 101.0 per cent (dried substance).
- injection port and detector: 250 DC.
Detection Flame ionisation. CHARACTERS
Appearance
Composition of thefatty-acid fraction of the oil:
White or almost white, crystalline powder or colourless
- saturated fatty acids of chain length less than C14: maximum
crystals, hygroscopic.
0.5 per cent;
- myristic acid: maximum 0.5 per cent; Solubility
- palmitic acid: 7.0 per cent to 16.0 per cent; Freely soluble in water, very slightly soluble in ethanol
- stearic acid: 3.0 per cent to 19.0 per cent; (96 per cent).
- oleic acid and isomers: 54.0 per cent to 78.0 per cent; IDENTIFICATION
- linoleic acid and isomers: maximum 10.0 pet cent; First identification: A, C.
- arachidic acid: 1.0 per cent to 3.0 per cent;
Second identification: A, B, D, E.
- eicosenoic acids: maximum 2.1 per cent;
- behenic acid: 1.0 per cent to 5.0 per cent; A. Specific optical rotation (see Tests).
- erucic acid and isomers: maximum 0.5 per cent; B. Solution S (see Tests) is strongly alkaline (2.2.4).
.~ lignoceric acid: 0.5 per cent to 3.0 per cent. C. Infrared absorption spectrophotometry (2.2.24).
Nickel Comparison arginine CRS.
Maximum 1 ppm. If the spectra obtained show differences, dry the substance to
Atomic absorption spectrometry (2.2.23, Method II). be examined and the reference substance in an oven at
Testsolution Into a platinum or silica crucible previously 105°C and record new spectra.
tared after ignition introduce 5.0 g. Cautiously heat and D. Thin-layer chromatography (2.2.27).
introduce into the substance a wick formed from twisted Testsolution Dissolve 10 mg of the substance to be
ashless filter paper. Ignite the wick. When the substance has examined in a 10.3 gIL solution of hydrochloric acid Rand
ignited stop heating. After combustion, ignite in a muffle dilute to 50 mL with the same solution.
furnace at about 600 ± 50°C. Continue ignition until white Reference solution Dissolve. 10 mg of arginine CRS in a
ash is obtained. After cooling, take up the residue with 10.3 gIL solution of hydrochloric acid R and dilute to 50 mL
2 quantities, each of 2 mL, of dilute hydrochloric acid Rand with the same solution.
transfer into a 25 mL graduated flask. Add 0.3 mL of nitric
acid R and dilute to 25.0 mL with waterR. ' Plate TLC silica gelplate R.
Reference solutions Prepare 3 reference solutions by adding Mobzle phase concentrated ammonia R, 2-propanol R
1.0mL, 2.0 mL and 4.0 mL of nickelstandard solution (30:70 VIV).
(O:2ppm Ni) R to 2.0 mL of the test solution and diluting to Application 5 ilL.
10.0 mL with waterR. Development Over 2/3 of the plate.

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2020 Arginine 1-199

Drying At 105 "C until the ammonia disappears completely. wavelengths, use the result obtained at 570 run for
Detection Spray with ninhydrin solution R and heat at 105 "C quantification.
for 15 min. Limits:
Results The principal spot in the chromatogram obtained - any ninhydrin-positive substance: for each impurity,
with the test solution is similar in position, colour and size to maximum 0.2 per cent;
the principal spot in the chromatogram obtained with the - total: maximum 0.5 per cent;
reference solution. - reporting threshold: 0.05 per cent.
E. Dissolve about 25 mg in 2 mL of water R. Add 1 mL of The thresholds indicated under Related substances
a-naphthol solution Rand 2 mL of a mixture of equal volumes (Table 2034.-1) in the general monograph Substances for
of strong sodium hypochlorite solution R and waterR. A red phamzaceutical use (2034) do not apply.
colour develops. Chlorides (2.4.4)
TESTS Maximum 200 ppm.
Solution S To 5 mL of solution S add 0.5 mL of dilute nitric acid Rand
Dissolve 2.5 g in distilled waterR and dilute to 50 mL with dilute to 15 mL with water R.
the same solvent. Sulfates (2.4.13)
Appearance of solution Maximum 300 ppm.
Solution S is clear (2.2.1) and not more intensely coloured To 10 mL of solution S, add 1.7 mLof dilute hydrochloric
than reference solution BY6 (2.2.2~ Method II). acid R and dilute to 15 mL with distilled water R.
Specific optical rotation (2.2.7) Ammonium
+ 255,to + 28.5 (dried substance). Amino acid analysis (2.2.56) as described in the test for
Dissolve 2.00 g in hydrochloric acid R1 and dilute to 25.0 mL ninhydrin-positive substances with the following
with the same acid. modifications.
Ninhydrin-positive substances Injection 'Test solution, reference solution (c) and blank
Amino acid analysis (2.2.56). For analysis, use Method 1. solution.
The concentrations of the test solution and the reference Limit:
solutions may be adapted according to the sensitivity of the - ammonium at 570 nm: not more than the area of the
equipment used. The concentrations of all solutions are corresponding peak in the chromatogram obtained with
adjusted so that the system suitability requirements described reference solution (c) (0.02 per cent), taking into account
in general-chapter 2.2.46 are fulfilled, keeping theratios of the peak due to ammonium in the chromatogram
concentrations between all solutions as described. obtained with the blank solution.
SolutionA water R or a sample preparation buffer suitable Iron (2.4.9)
for the apparatus used. Maximum 10 ppm.
Test solution Dissolve 30.0 mg of the substance to be In a separating funnel, dissolve 1.0 g in 10 mL of dilute
examined in solution A and dilute to 50.0 mL with hydrochloric acid R. Shake with 3 quantities, each of 10 mL,
solution A. of methyl isobutyl ketone R1, shaking for 3 min each time,
To the combined organic layers add 10 mL of water Rand
Reference solution (a) Dilute 1.0 mL of the test solution to
shake for 3 min. Use the aqueous layer.
100.0 mL with solution A. Dilute 2.0 mL of this solution to
10.0 mL with solution A. Loss on drying (2.2.32)
Reference solution (b) Dissolve 30.0 mg oiproline R in Maximum 0.5 per cent, determined on 1.000 g by drying in
solution A and dilute to 100.0 mL with solution A. Dilute an oven at 105 "C.
1.0 mL of the solution to 250.0 ml..with solution A. Sulfated ash (2.4.14)
Reference solution (c) Dilute 6.0 mL of ammonium standard Maximum 0.1 per cent, determined on 1.0 g.
solution (l00 ppm NH4J R to 50.0 mL with solution A. Dilute ASSAY
1.0 mL of this solution to 100.0 mL with solution A. Dissolve 0.150 gin 50 mL of waterR. Titrate with 0.1 M
Reference solution (d) Dissolve 30 mg of isoleucine Rand hydrochloric acid, determining the end-point potentiometrically
30 mg of leucine R in solution A and dilute to 50.0 mL with (2.2.20).
solution A. Dilute 1.0 mL of the solution to 200.0 mL with 1 mL of 0.1 M hydrochloric acidis equivalent to 1?.42 mg of
solution A. e6Hl~402'
Blank solution Solution A. STORAGE
Inject suitable, equal amounts of the test, blank and reference In an airtight container, protected from light.
solutions into the amino acid analyser. Run a program
suitable for the determination of physiological amino acids. IMPURITIES
Other detectable impurities (the following substances would, if
System suitability Reference solution (d):
presentat a sufficient leoel, be detected by one or otherof the tests
- resolution: minimum 1.5 between the peaks due to
in the monograph. They are limitedby the general acceptance
isoleucine and leucine.
criterion for other/unspecified impurities. It is therefore not
Calculation of percentage contents: necessary to identify these impurities for demonstration of
- for any ninhydrin-positive substance detected at 570 nm, compliance. See also5.10. Control of impurities in substances for
use the concentration of arginine in reference solution (a); phamzaceutical use) A, B, C.
- for any ninhydrin-positive substance detected at 440 nm,
use the concentration of proline in reference solution (b);
if a peak is above the reporting threshold at both

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2020 Arginine Hydrochloride 1-201

TESTS
Arginine Hydrochloride Solution S
(Ph. Bur. monograph 0805) Dissolve 25.g hl. distilled water R and dilute to 50 mL with
the same solvent.
Appearance of solution
Solution S is clear (2.2.1) and not more intensely coloured
than reference solution BY6 (2.2.2, Method II).
Specific optical rotation (2.2.7)
210.7 1119-34-2 + 21.0 to + 23.5 (dried substance).
Dissolve 2.00 g in hydrochloric acid R1 and dilute to 25.0 mL
Action and use with the same acid.
Amino acid; nutrient.
Ninhydrin-positive substances
Preparations Amino acid analysis (2.2.56). For analysis, use Method 1.
Arginine Hydrochloride Infusion The concentrations of the test solution and the reference
Arginine Hydrochloride Oral Suspension solutions may be adapted according to the sensitivity of the
Arginine Hydrochloride Sterile Concentrate equipment used. The concentrations of all solutions are
adjusted so that the system suitability requirements described
PhEur _
in general chapter 2.2.46 are fulfilled, keeping the ratios of
DEFINITION concentrations between all solutions as described.
(2S)-2-Amino-5-guanidinopentanoic acid hydrochloride. Solution A waterR or a sample preparation buffer suitable
Product of fermentation or of protein hydrolysis. for the apparatus used.
Content Test solution Dissolve 30.0 mg of the substance to be
98.5 per cent to 101.0 per cent (dried substance). examined in solution A and dilute to 50.0 mL with
solution A.
CHARACTERS
Reference solution (a) Dilute 1.0 mL of the test solution to
Appearance
100.0 mL with solution A. Dilute 2.0 mL of this solution to
White or. almost white, crystalline powder or colourless
10.0 mL with solution A.
crystals.
Reference solution (b) Dissolve 30.0 mg of proline R in
Solubility solution A and dilute to 100.0 mL with solution A. Dilute
Freely soluble in water, very slightly soluble in ethanol 1.0 mL of the solution to 250.0 mL with solution A.
(96 per cent).
Reference solution (c) Dilute 6.0 mL of ammonium standard
IDENTIFICATION solution (100 ppm NH.J R to 50.0 mL with solution A. Dilute
First identification: A, B, E. 1.0 mL of this solution to 100.0 mL with solution A.
Second identification: A, C, D, B. Reference solution (d) Dissolve 30 mg of isoleucine R and
A. Specific optical rotation (see Tests). 30 .mg of leucine R in solution A and dilute to 50.0 mL with
B. Infrared absorption spectrophotometry (2.2.24). solution A. Dilute 1.0 mL of the solution to 200.0 mL with
solution A.
Comparison argininehydrochloride CRS.
Blank solution Solution A.
C. Thin-layer chromatography (2.2.27).
Inject suitable, equal amounts of the test, blank and reference
Test solution Dissolve 10 mg of the substance to be
solutions into the amino acid analyser. Run a program
examined in water R and dilute to 50 mL with the same
suitable for the determination of physiological amino acids.
solvent.
System suitability Reference solution (d):
Reference solution Dissolve 10 mg of arginine
- resolution: minimum 1.5 between the peaks due to
hydrochloride CRS in waterR and dilute to 50 mL with the
isoleucine and leucine.
same solvent.
Calculation of percentage contents:
Plate TLC silica gel plate R.
- for any ninhydrin-positive substance detected at 570 nm,
Mobile phase concentrated ammonia R, 2-propanol R use the concentration of arginine in reference solution (a);
(30:70 VIV). - for any ninhydrin-positive substance detected at 440 nm,
Application 5 .~L. use the concentration of proline in reference solution (b);
Development Over 2/3 of the plate. if a peak is above the reporting threshold at both
Drying At 105°C until the ammonia disappears completely. wavelengths, use the result obtained at 570 nm for
quantification.
Detection Spray with ninhydrin solution R and heat at 105°C
for 15 min. Limits:
- any ninhydrin-positive substance: for each impurity,
Results The principal spot in the chromatogram obtained
maximum 0.2 per cent;
with the test solution is similar in position, colour and size to
- total: maximum 0.5 per cent;
the principal spot in the chromatogram obtained with the
- reporting threshold: 0.05 per cent.
reference solution.
The thresholds indicated under Related substances
D. Dissolve about 25 mg in 2 mL of water R. Add 1 mL of
(Table 2034.-1) in the general monograph Substances for
a-naphthol solution Rand 2 mL of a mixture of equal volumes
-pharmaceutical use (2034) do not apply.
of strong sodium hypochlorite solution R and water R. A red
colour develops. Sulfates (2.4.13)
Maximum 300 ppm.
E. It gives reaction (a) of chlorides (2.3.1).

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2020 Aripiprazole 1-203

Flow rate 70 mIJmin. Solubility

Temperature: Practically insoluble in water, soluble in methylene chloride,
- column: 80°C; very slightysoluble in ethanol (96 per cent).
- detector: 40 "C. It shows polymorphism (5.9).
Detection Discharge ionisation. IDENTIFICATION
Injection 1 mL. Infrared absorption spectrophotometry (2.2.24).
Sample rate 100 mlJmin. Comparison aripiprazole CRS.
Relative retention With reference to impurity C (retention If the spectra obtained in the solid state show differences,
=
time = about 4.7 min): impurity A aboutOi-l; dissolve the substance to be examined and the reference
impurity B = about 0.7. substance separately in methylene chloride R, evaporate to
System suitability Reference gas: dryness and record new spectra using the residues.
- resolution: minimum 3.0 between the peaks due to TESTS
impurities A and B and minimum 2.0 between the peaks Appearance of solution
due to impurities Band C. If intended for use in the manufacture of parenteral
Limits: preparations, the solution is clear (2.2.1) and not more
- impurity A: not more than the area of the corresponding intensely coloured than reference solution GY5 (2.2.2,
peak in the chromatogram obtained with the reference gas Method II).
(5;0 PPm. VIV); Dissolve 0.5 g in a mixture of 10 volumes of acetic acid Rand
- total: maximum 0.0040 per cent of the sum of the areas of 90 volumes of anhydrous ethanol R and dilute to 20 mL with
all the peaks (40.0 ppm VIV). the same mixture of solvents. Sonicate for about 15 min,
Water (2.5.28) shaking .occasionally, until dissolution is complete.
Maximum ro.O ppm VI17, determined using an electrolytic Related substances
hygrometer;' Liquid chromatography (2.2.29). Protect the solutions from
STORAGE light.
In gaseous or liquid state, in suitable containers, complying Solvent mixture acetic acidR,.methanolR, acetonitrile R,
with the legal regulations. waterR (1:10:30:60 VIVIVIV).
IMPURITIES Test solution Dissolve 50.0 mg of the substance to be
Specified impurities A, D. examined in the solvent mixture and dilute to 50.0 mL with
Other detectable impurities B, C. the solvent mixture. Dilute 5.0 mL of the solution to
50.0 mL with the solvent mixture.
A. oxygen,
Reference solution (a) Dilute 1.0 mL of the test solution to
B. nitrogen, 100.0 mL with the solvent mixture. Dilute 1.0 mL of this
C. methane, solution to 10.0 mL with the solvent mixture.
b. water. Reference solution (b) Dissolve 5 mg of the substance to be
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _-'-- PhEur examined and 5 mg of aripiprazole impurity F CRS in the
solvent mixture and dilute to 100 mL with the solvent
mixture. Dilute 1 mL of the solution to 50 mL with the
solvent mixture.
Aripiprazole Reference solution (c) Dissolve 50.0 mg of aripiprazole CRS
in the solvent mixture and dilute to 50.0 mL with the solvent
(Ph. Bur. monograph 2617) mixture. Dilute 5.0 mL of the solution to 50.0 mL with the
solvent mixture.
H
Column:
rN~°'Y'(NyO
=
- size: 1= 0.10 m, (2) 4.6 nun;
('I(~ VV - stationary phase: end-capped octadecylsilyl silica gelfor

yCI chromatography R (311m).

Mobile phase:
CI
- mobile phaseA: acetonitrile R, 0.05 per cent VIV solution of
trifluoroacetic acid R (10:90 VIV);
448.4 129722-12-9 - mobile phase B: 0.05 per cent VIV solution of trifluoroacetic
acid R, acetonitrile R (10:90 VIV);
Action and use
Dopamine D z receptor antagonist; neuroleptic.
Time Mobile phase A Mobile phase B
PhEur _ (min) (per cent VIV) (per cent VIV)
0-2 80 20
DEFINITION 2 - 10 80 --+ 65 20 --+ 35
7-[4- [4-(2,3-Dichlorophenyl)piperazin-l-yl]butoxy]-3,4- 10 - 20 65 --+ 10 35 --+ 90
dihydroquinolin-2 (1H)-one. 20 - 25 10 90
Content
98.0 per cent to 102.0 per cent (dried substance). Flow rate 1.2 mlJmin.
CHARACTERS Detection Spectrophotometer at 254 nm.
Appearance
White or almost white crystals or crystalline powder.

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 1-204 Aripiprazole 2020

Injection 20 ul, of the test solution and reference

r NH

QC.I .
solutions (a) and (b). N~
Relative retention With reference to aripiprazole (retention
time = about 11 min): impurity F = about 1.1. ~ CI

System suitability Reference solution (b): CI

- resolution: minimum 2.0 between the peaks due to
aripiprazole and impurity F. B. 1-(2,3-dichlorophenyl)piperazine,
Calculation of percentage contents:
H
- for each impurity, use the concentration of aripiprazole in
rN~°Y'yNyO
reference solution (a).
, N-J . VV
Limits:
- unspecified impurities: for each impurity, maximum
0.10 per cent;
(X
~ CI

- total: maximum 0.2 per cent;

- reporting threshold: 0.05 per cent. C. 7- [4-[4-(2-chlorophenyl)piperazin-1-yl] butoxy]-3,4-
dihydroquinolin-2(1H)-one,
Loss on drying (2.2.32)
- Maximum 0.5 per cent, determined on 1.000 g by drying in H
an oven at 105°C for 3 h. rN~°Y'yNyO
Sulfated ash (2.4.14) (l(N~ VV
Maximum 0.1 per cent, determined on 1.0 g.
Bacterial endotoxins (2.6.14)
Less than 5 IV/mg, if intended for use in the manufacture of
Y CI
parenteral preparations without a further appropriate
procedure for the removal of bacterial endotoxins. D. 7-[4-[4-(3-chlorophenyl)piperazin-1-yl]butoxy]-3,4-
Dissolve 1.0 mg of the substance to be examined in 20 mL dihydroquinolin-2(1H)-one,
of a 5.17 gIL solution of hydrochloric acid R. H
ASSAY - I-< N ~O'(().N
I .0
QC.I .
liquid chromatography (2.2.29) as described in the test for N~ ~ #
related substances with the following modifications.
Injection Test solution and reference solution (c). ~ CI

System suitability Reference solution (c): CI

- symmetryfactor. maximum 2.0.
E. 7- [4-[4-(2,3-dichlorophenyl)piperazin-1-yl]
Calculate the percentage content of C23H27ChN302 taking
butoxy] quinolin-2 (lH)-one,
into account the assigned content of aripiprazole CRS.
STORAGE o H
Protected from light. If the substance is sterile, store in a r~~°Y'yNyO
sterile, airtight, tamper-proof container.
('yN-J VV
LABELLING
The label states, where applicable, that the substance is yCI
suitable for use in the manufacture of parenteral CI
preparations.
F. 7- [4-[4-(2,3-dichlorophenyl)-1-oxidopiperazin-1-yl]
IMPURITIES butoxy]-3,4-dihydroquinolin-2(1H)-one,
Other detectable impurities (the following substances would, zf

(rNl
presentat a sufficient level, be detected by one or otherof the tests
rN~
in the monograph. They are limitedby the general acceptance
criterion for other/unspecified impurities and/or by the general
monograph Substances for pharmaceutical use (2034). It is
therefore not necessary to identify these impurities for
demonstration of compliance. See also 5.10. Control of impurities
in substances for pharmaceutical use) A, B, C, D, E, F, G.
0'9)1
#

HN
CI~
~ N~ - ~
~N:QyQC~.

.
I -<"
CI CH3
"" I
CI
CI «~
~
.
NH
I. 0

H o 0
HOY'!(.
",,' N"y0
.0v G. 7,7'-[ethane-1,1-diylbis[(2,3-dichlorobenzene-4,1-diyl)
piperazine-4, 1-diylbutane-4, l-diyloxy]]bis [3,4-
dihydroquinolin-2(1H)-one].
A.7-hydroxy-3,4-dihydroquinolin-2(1H)-one,
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur

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2020 Articaine Hydrochloride 1-205

TESTS
Articaine Hydrochloride Solution S
(Ph. Eur. monograph 1688) Dissolve 0.50 g in water R and dilute to 10 mL with the
same solvent.
Appearance of solution
Solution S is clear (2.2.1) and not more intensely coloured
than reference solution BY6 (2.2.2, Method 1).
pH (2.2.3)
4.2 to 5.2.
Dissolve 0.20 g in carbon dioxide-free waterR and dilute to
320.8 23964-57-0 20.0 mL with the same solvent.
Action and use Related substances
Local anaesthetic. Liquid chromatography (2.2.29).
Test solution Dissolve 10.0 mg of the substance to be
PhEur _
examined in the mobile phase and dilute to 10.0 mL with
DEFINITION the mobile phase.
Methyl 4-methyl-3- [[(2RS)-2-(propylamino) Reference solution (a) Dilute 1.0 mL of the test solution to
propanoyl] amino] thiophene-2-carboxylate hydrochloride. 100.0 mL with the mobile phase. Dilute 1.0 mL of this
Content solution to 10.0 mL with the mobile phase.
98.5 percent to 101.0 per cent (dried substance). Reference solution (b) Dissolve 5.0 mg of articaine
impurity A CRS and 2.5 mg of articaine impurity E CRS in
CHARACTERS
the mobile phase and dilute to 50.0 mL with the mobile
Appearance
phase. Dilute 1.0 mL of the solution to 50.0 mL with the
White· or almost white, crystalline powder.
mobile phase.
Solubility Column:
Freely soluble in water and in. ethanol (96 per cent). - size: I = 0.25 m, (2) = 4.6 mm;
IDENTIFICATION - stationary phase: spherical end-capped octadecylsilyl silica gel
First identification: B, D. for chromatography R (5 11m);
Second identification: A, C, D. - temperature: 45°C.
A. Dissolve 50.0 mg in a 1 gIL solution of hydrochloric acid R Mobile phase Mix 25 volumes of acetonitrile Rand
and dilute to 100.0 mL with the same acid. Dilute 5.0 mL of 75 volumes of a solution prepared as follows: dissolve 2.02 g
the solution to 100.0 mL with a 1 gIL solution of hydrochloric of sodium heptanesulfonate Rand 4.08 g of potassium
acid R. Examined between 200 nm and 350 nm (2.2.25), the dihydrogen phosphate R in water R and dilute to 1000 mL with
solution shows an absorption maximum at 272 nm. the same solvent. Adjust to pH 2.0 with phosphoric acid R.
The specific absorbance at the maximum is 290 to 320. Flow rate 1 mL/min.
B. Infrared absorption spectrophotometry (2.2.24). Detection Spectrophotometer at 276 nm.
Preparation Place dropwise 20 ilL of the test solution on Injection 10 ilL.
300 mg discs. Run time 5 times the retention time of articaine.
Test solution Dissolve 0.1 gin 5 mL of water R, add 3 mL Relative retention With reference to articaine (retention
of a saturated solution of sodium hydrogen carbonate Rand time = about 9 min): impurity A = about 0.8;
shake twice with 2 mL of methylene chloride R. Combine the impurity E = about 0.86.
methylene chloride layers, dilute to 5.0 mL with methylene System suitability Reference solution (b):
chloride R and dry over anhydrous sodium sulfateR. - resolution: minimum 1.2 between the peaks due to
Comparison articaine hydrochloride CRS. impurities A and E.
C. Thin-layer chromatography (2.2.27). Limits:
Test solution Dissolve 20 mg of the substance to be - impurityA: not more than the area of the corresponding
examined in 5 rnL of ethanol (96 per cent) R. peak in the chromatogram obtained with reference
Reference solution Dissolve 20 mg of articaine solution (b) (0.2 per cent);
hydrochloride CRS in 5 mL of ethanol (96 per cent) R. - unspecified impurities: for each impurity, not more than the
area of the principal peak in the chromatogram obtained
Plate TLC silica gel F254 plateR.
with reference solution (a) (0.10 per cent);
Mobilephase triethylamine R, ethyl acetate R, heptane R - sum of impurites otherthan A: not more than 5 times the
(10:35:65 V/V/V). area of the principal peak in the chromatogram obtained
Application 5 ilL. with reference solution (a) (0.5 per cent);
Development Over a path of 15 em. - disregard limit: 0.5 times the area of the principal peak in
Drying In air. the chromatogram obtained with reference solution (a)
(0.05 per cent).
Detection Examine in ultraviolet light at 254 nm.
Loss on drying (2.2.32)
Results The principal spot in the chromatogram obtained
Maximum 0.5 per cent, determined on 1.000 g by drying in
with the test solution is similar in position and size to the
an oven at 105°C for 5 h.
principal spot in the chromatogram obtained with the
reference solution. Sulfated ash. (2.4.14)
D. It gives reaction (a) of chlorides (2.3.1). Maximum 0.1 per cent, determined on 1.0 g.

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1-206 Ascorbic Acid 2020

ASSAY
Dissolve 0.250 g in a mixture of 5.0 mL of 0.01 M
hydrochloric acid and 50 mL of ethanol (96 per cent) R. Carry
out a potentiometric titration (2.2.20) using 0.1 M sodium
hydroxide. Read the volume added between the 2 points of
inflexion.
F. 4-methyl.;N-propyl-3-[[(2RS)-2-(propylamino)propanoyl]
1 mL of 0.1 M sodium hydroxide is equivalent to 32.08 mg of amino]thiophene-2-carboxamide (articaine acid
C13H21ClNz03S. propionamide),
STORAGE
Protected from light.
IMPURITIES
Specified impurities A.
Other detectable impurities (the following substances would, if
present at a sufficient level, be detected by one or other of the tests
in the monograph. They are limited by the general acceptance G. methyl 3-[[(2RS)-2-(butylamino)propanoyl]amino]-4-
criterionfor other/unspecified impurities and/or by the general methylthiophene-2-carboxylate (butylarticaine),
monograph Substances for pharmaceutical use (2034). It is
therefore not necessary to identify these impurities for
demonstration of compliance. See also 5.10. Control of impurities
in substances for pharmaceutical use) B, C, D, E, F, G, H,
I, J.

H. methyl 3- [[(2RS)-2-( dipropylamino)propanoyl] amino]-4-

methylthiophene-2-carboxylate (dipropylarticaine),

A. methyl 4-methyl-3-[[2-(propylamino)acetyl]amino]
thiophene-2-carboxylate (acetamidoarticaine),

I. methyl 3-amino-4-methylthiophene-2-carboxylate
(3-aminoarticaine),

B. 4-methyl- 3-[[(2RS)-2-(propylamino)propanoyl] amino]

thiophene-2-carboxylic acid (articaine acid),

J. methyI3-[[(2RS)-2-bromopropanoyl]amino]-4-
methylthiophene-2-carboxylate (bromo compound).
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur

C. I-methylethyl 4-methyl-3-[[(2RS)-2-(propylamino)
propanoyl] amino] thiophene-2-carboxylate (articaine
isopropyl ester), Ascorbic Acid
(Ph. Bur. monograph 0253)

HO
~;~,
~o
D. methyl 3-[[(2RS)-2-(ethylamino)propanoyl]amino]-4- HO OH
methylthiophene-z-carboxylate (ethylarticaine),
o . 176.1 50-81-7
3
/ 0 & ~_ H .CH3CH
H3 C . ~ X Action and use
s ~ "-~ 3
CH
Vitamin C.
---== 0 Preparations
CH3 and enantiomer
Ascorbic Acid Injection
Ascorbic Acid Tablets
E. methyl 4-methyl- 3- [[(2RS)-2- [(l-methylethyl)amino]
propanoyl] amino]thiophene-2-carboxylate Ascorbic Acid Chewable Tablets
(isopropylarticaine), Paediatric Vitamins A, C and D Oral Drops

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2020 Ascorbic Acid 1-207

Potassium Ascorbate Eye Drops Allow the solutions to stand for 1 h. Any opalescence in the
Vitamins Band e Injection test solution is not more intense than that in the reference
solution.
When Vitamin e is prescribed or demanded, Ascorbic Acid
shall be dispensed or supplied. Related substances
Liquid chromatography (2.2.29). Prepare the solutions
PhEur ....:.- _
immediately before use.
DEFINITION Phosphate buffer solution Dissolve 6.8 g of potassium
(5~)-5-[(I~-1,2-Dihydroxyerllyl]-3,4-dihydroxyfUran-2(5li)­ dihydrogen phosphate R in uiater for chromatography Rand
one. dilute to about 175 mL with the same solvent. Filter through
a membrane filter (nominal pore size 0.45 um) and dilute to
Content
1000 mL with water for chromatography R.
99.0 per cent to 100.5 per cent.
Testsolution Dissolve 0.500 g of the substance to be
CHARACTERS examined in the mobile phase and dilute to 10.0mL with
Appearance the mobile phase.
White or almost white, crystalline powder or colourless
Reference solution (a) Dissolve 10.0 mg of ascorbic acid
crystals, becoming discoloured on exposure to air and
impurity C C~S in the mobile phase and dilute to 5.0 mL
moisture.
with the mobile phase.
Solubility Reference solution (b) Dissolve 5.0 mg of ascorbic acid
Freely soluble .in water, sparingly soluble in ethanol
impurity D C~S and 5.0 mg of ascorbic acid C~S in the
(96 per cent).
mobile phase, add 2.5 mL of reference solution (a) and
mp dilute to 100.0 mL with the mobile phase.
About 190 "C, with decomposition. Reference solution (c) Dilute 1 mL of the test solution to
IDENfIFICATION 200 mL with the mobile phase. Mix 1 mL of this solution
Firstidentification: B, C. and 1 mL of reference solution (a).
Second identification: A, C, D. Column:
A. Ultraviolet and visible 'absorption spectrophotometry - size: 1 = 0.25 m, (2) = 4.6 mm;
(2.2.25). - stationary phase: aminopropylsilyl silica gelfor
chromatography ~ (5 JlIl1);
Testsolution Dissolve 0.10 g in water R and dilute
- temperature: 45 "C.
immediately to 100 . 0 mL with the same solvent. Add 1.0 mL
of the solution "to 10 mL of a 10.3 gIL solution of hydrochloric Mobile phase Phosphate buffer solution, acetonitrile ~1
acidR and dilute to 100.0 mL with uuuer R. (25:75 VIV).
Absorption" maximum At 243 nm, determined immediately Flow rate 1.0 mLImin.
after dissolution. Detection Spectrophotometer at 210 nm.
Specific absorbance at the absorption maximum 545 to 585. injection 20 JlL of the test solution and reference
B. Infrared absorption spectrophotometry (2.2.24). solutions (b) and (c).'
Comparison ascorbic acid C~S. Run time 2.5 times the retention time of ascorbic acid.
e. pH (2.2.3): 2.1 to 2.6 for solution S (see Tests). Identification of impurities Use the chromatogram obtained
with reference solution (b) to identify the peaks due to
D. To 1 mL of solution S add 0.2 mL of dilute nitric add R
impurities e and D.
and 0;2 mL of silver nitrate solution ~2. A grey precipitate is
formed. . Relatiueretention With reference to ascorbic acid (retention
time = about 11 min): impurity D = about 0.4;
TESTS 'impurity e = about 1.7. .
Solution S Systemsuitability:
Dissolve 1.0 g in carbon 'dioxide-free waterR and dilute to
- resolution: minimum 3.0 between the peaks due to
20 mL with the same solvent.
ascorbic acid and impurity e in the chromatogram
Appearance of solution obtained with reference solution (c);
Solution S is clear (2.2.1) and not more intensely coloured - signal-to-noise ratio: minimum 20 for the peak due to
than reference solution BY7 (2.2.2, Method II). impurity C in the chromatogram obtained with reference
Specific optical rotation (2.2.7) solution (b).
+ 20.5 to + 21.5. Limits:
Dissolve 2.50 g in water R and dilute to 25.0 mL with the - impurities C, D: for each impurity, not more than
same solvent. 1.5 times the area of the corresponding peak in the
chromatogram obtained with reference solution (b)
Impurity E
(0.15 per cent);
Maximum 0.2 per cent.
- unspecified impurities: for each impurity, not more than the
Testsolution Dissolve 0.25 gin 5 mL of toater R, Neutralise area of the peak due to ascorbic acid in the chromatogram
using dilute sodium hydroxide solution R, then add 1 mL of obtained with reference solution (b) (0.10 per cent);
dilute acetic acidR and 0.5 mLof calcium chloride solution R. - sum of impurities otherthan C and D: not more than twice
Reference solution Dissolve 70 mg of oxalic acidR (dihydrate the area of the peak due to ascorbic acid in the
of impurity E) in waterR and dilute to 500 mL with the chromatogram obtained with reference solution (b)
same solvent; to 5mL of the solution add 1 mL of dilute (0.2 per cent);
acetic acid R and 0.5 ml.of calcium chloride solution R.

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 1-208 Ascorbyl Palmitate 2020

~
- disregard limit: 0.5 times the area of the peak due to . o~ OCH3
ascorbic acid in the chromatogram obtained with H ..
reference solution (b) (0.05 per cent). HO- . .( OH 0

Copper HO
Maximum 5 ppm.
Atomic absorption spectrometry (2.2.23, Method 1). D. methyl L-xylo-hex-2-ulosonate (methyl L-sorbosonate),
Test solution Dissolve 2.0 g in 0.1 M nitricacid and dilute to
25.0 mL with the same acid.
Reference solutions Prepare the reference solutions (0.2 ppm,
0.4 ppm and 0.6 ppm) using copper standardsolution (10 ppm E. oxalic acid,
Cu) R, diluting with 0.1 M nitricacid.
Source Copper hollow-cathode lamp.
Wavelength 324.S nm. HO
~;O,
~O
Atomisation device Air-acetylene flame.
HO OH
Adjust the zero of the apparatus using 0.1 M nitric acid.
. Iron F. (5R)-5-[(lR)-1,2-dihydroxyethyl]-3,4-dihydroxyfuran-2
Maximum 2 ppm. (5H)-one,
Atomic absorption spectrometry (2.2.23, Method 1).
H
Test solution Dissolve 5.0 gin 0.1 M nitricacid and dilute to
25.0 mL with the same acid. H~C~O
Reference solutions- Prepare the reference solutions (0.2 ppm,
0.4 ppm and 0.6 ppm) using iron standardsolution (20 ppm HO OH
Fe) R, diluting with 0.1 M nitric acid.
Source Iron hollow-cathode lamp. G. (R)-[(2R)-3,4-dihydroxy-5-oxo-2,5-dihydrofuran-2-yl]
Wavelength 24S.3 nm. hydroxyacetic acid,
Atomisation device Air-acetylene flame.

~
O H
Adjust the zero of the apparatus using 0.1 M nitricacid. .OH
Sulfated ash (2.4.14) H,eo .•. ~ 0
Maximum 0.1 per cent, determined on 1.0 g.
ASSAY HO OH
Dissolve 0.150 g in a mixture of 10 mL of dilute sulfuric
acid R and SO mL of carbon dioxide-free waterR. Add 1 mL of Ii. methyl (R)-[(2R)-3,4-dihydroxy-5-oxo-2,5-dihydrofUran-
starch solution R. Titrate with 0.05 M iodine until a' persistent 2-yl]hydroxyacetate.
violet-blue colour is obtained. _ _ _ _ _ _ _ _ _ _ _ _...:....-_~-----PhEur
1 mL of 0.05 M iodine is equivalent to S.Sfmg of C 6Hs0 6 •
STORAGE
In a non-metallic container, protected from light.
IMPURITIES
AscQrbyl Palmitate
Specified impurities C, D, B. (Ph. Bur. monograph 0807)
Other detectable impurities (the following substances would, if

~
present at a sufficientlevel, be detected by one or otherof the tests
in the monograph. They are limited by the general acceptance o
criterion for other/unspecified impurities and/or by the general
3C
H t1~4
r 1 "0l ¢.....
= . HH
'. °
monograph Substances for pharmaceutical use (2034). It is
o
therefore not necessary to identify these impurities for HO OH
demonstration of compliance. See also 5.10. Control of impurities
in substances for pharmaceutical use) A, F, G, H.
414.5 137-66-6
(('OyCHO Action and use
U Excipient.

A. furan-2-carbaldehyde, PhEur _ _~----------------_

DEFINITION
~X1:2H (2~-2-[(2R)-3,4-Dihydroxy-5-oxo-2,5-dihydro£Uran-2-yl]-2­
hydroxyethyl hexadecanoate.
HOJt{\OH 0
Content
HO
98.0 per cent to 100.5 per cent (dried substance).

C. L-xylo-hex-2-:-ulosonic acid (t-sorboscnic acid), CHARACTERS

Appearance
White or yellowish-white powder.

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2020 Asparagine 1-209

Solubility CHARACTERS
Practically insoluble in water, freely soluble in ethanol Appearance
(96 per cent) and in methanol, practically insoluble in White or almost white, crystalline powder or colourless
methylene chloride and in fatty oils. crystals.
IDENTIFICATION Solubility
A. Specific optical rotation (see Tests). Slightly soluble in water, practically insoluble in ethanol
B. Infrared absorption spectrophotometry (2.2.24). (96 per cent) and in methylene chloride.
Comparison ascorbyl palmitate CRS. IDENTIFICATION
C. Dissolve about 10 mg in 5 mL of methanol R. First identification: A, B, D.
The solution decolourises dichlorophenolindophenol standard Second identification: A, C, D.
solution R. A. Specific optical rotation (see Tests).
TESTS B. Infrared absorption spectrophotometry (2.2.24).
Solution S Comparison asparagine monohydrate CRS.
Dissolve 2.50 g in methanol R and dilute to 25.0 mL with the C. Thin-layer chromatography (2.2.27).
same solvent.
Test solution Dissolve 10 mg of the substance to be
Appearance of solution examined in water R and dilute to 10 mL with the same
Solution S is dear (2.2.1) and not more intensely coloured solvent.
than reference solution BY4 (2.2.2, Method 1).
Reference solution Dissolve 10 mg of asparagine
Specific optical rotation (2.2.7) monohydrate CRS in water R and dilute to 10 mL with the
+ 21 to + 24 (dried substance), determined on solution S. same solvent.
Related substances Plate TLC silica gel plate R.
The thresholds indicated under Related substances Mobile phase glacial acetic-acid R, water R, butanol R
(Table 2034.-1) in the general monograph Substances for (25:25:50 V/V/V).
pharmaceutical use (2034) do not apply.
Application 5 J1l...
Loss on drying (2.2.32) Development Over 2/3 of the plate.
Maximum 1.0 per cent, determined on 1.000 g by drying in
Drying At 110°C for 15 min.
vacuo at 60°C for 5 h.
Detection Spray with ninhydrin solution R and heat at 105°C
Sulfated ash (2.4.14)
for 10 min.
Maximum 0.1 per cent, determined on 1.0 g.
Results The principal spot in the chromatogram obtained
ASSAY with the test solution is similar in position, colour and size to
Dissolve 0.200 gin 50 mL of ethanol (96 per cent) R. the 'principal spot in the chromatogram obtained with the
Add 30 mL of water R and titrate with 0.05 M iodine until a reference solution. .
yellow colour is obtained.
D. Loss on drying (see Tests).
1 mL of 0.05M iodine is equivalent to 20.73 mg of
TESTS
C22H3S07'
Solution S
STORAGE Dissolve with heating 2.0 g in carbon dioxide-free water Rand
In an airtight container, protected from light. dilute to 100 mL with the same solvent.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
Appearance of solution
Solution S is clear (2.2.1) and colourless (2.2.2, Method II).
pH (2.2.3)
4.0 to 6.0 for solution S.
Asparagine Monohydrate Specific optical rotation (2.2.7)
(Ph. Bur. monograph 2086) + 33.7 to + 36.0 (dried substance).
Dissolve 2.50 g in a 309.0 gIL solution of hydrochloric acid R
and dilute to 25.0 mL with the same acid.
Related substances
Liquid chromatography (2.2.29). Prepare the solutions
immediately beforeuse.
150.1 5794-13-8
Test solution Dissolve 0.100 g of the substance to be
Action and use examined in water R and dilute to 10.0 mL with the same
Amino acid. solvent.
Reference solution (a) Dilute 1.0 mL of the test solution to
PhEur ----------- 100.0 mL with water R.
DEFINITION Reference solution (b) Dilute 1.0 mL of reference solution (a)
(2S)-2,4-Diamino-4-oxobutanoic acid monohydrate. to 10.0 mL with water R.
Content Reference solution (c) Dissolve 5.0 mg of aspartic .acid R
99.0 per cent to 101.0 per cent (dried substance). (impurity A) in water R and dilute to 10.0 mL with the same
solvent. Dilute 1.0 mL of the solution to 10.0 mL with
water R.

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1-210 Asparagine 2020

Reference solution (d) Dissolve 3.0 mg of asparagine organic phases with 10 mL of waterR for 3 min.
impurity C CRS in 40 mL of water R using sanification and The aqueous phase complies with the limit test for iron.
dilute to 50.0 mL with the same solvent. Dilute 1.0 mL of Loss on drying (2.2.32)
the solution to 10.0 mL with waterR. 10.5 per cent to 12.5 percent, determined on 1.000 g by
Reference solution (e) Mix 5.0 mL of reference solution (c) drying in an oven at 130 "C for 3 h.
with 2.5 mL of reference solution (a) and dilute to 10.0 mL Sulfated ash (2.4.14)
with water R. Maximum 0.1 per cent, determined on 1.0 g.
Column:
- size: 1= 0.25 m, 0 = 4.6 mm; ASSAY
- stationary phase: end-cappedoctadecylsilyl silica gelfor Dissolve 0.110 g in 5 mL of anhydrous formic acid R.
chromatography R (5 urn); Add 50 mL of anhydrous acetic acid R. Titrate with 0.1 M
- temperature: 25 "C. perchloric acid, determining. the end-point potentiometrically
(2.2.20).
Mobilephase Dissolve 13.6 g of potassium dihydrogen
phosphate Rand 2.16 g of sodium octanesulfonate R in about 1 mL of 0.1 M perchloric acid is equivalent to 13.21 mg
900 mL of waterfor chromatography R. Adjust to pH 2.2 with of C4HgN203 •
phosphoric acid R and dilute to 1000 mL with waterfor IMPURITIES
chromatography R. Add 5 mL of acetonitrile Rl. Specified impurities A, C.
Flow rate 0.7 mUmin. Other detectable impurities (the following substances would, if
Detection Spectrophotometer at 210 urn. present at a sufficientlevel, be detected by one or otherof the tests
Injection 20 IlL. in the monograph. They are limitedby the general acceptance
criterion for other/unspecified impurities and/orby the general
Run time Twice the retention time of asparagine.
monograph Substances for pharmaceutical use (2034). It is
Identification of impurities Use the chromatogram obtained therefore not necessary to identify these impurities for
with reference solution (c) to identify the peak due to demonstration of compliance. See also 5.10. Control of impurities
impurity A; use the chromatogram obtained with reference in substances for pharmaceutical use) B, D, E, F, G, H.
solution (d) to identify the peak due to impurity C.
Relative retentz"onWith reference to asparagine (retention
time = about 6.6 min): impurity C = about 0.6;
=
impurity A about 1.2.
System suitability Reference solution (e):
A. (2S)-2-aminobutanedioic acid (aspartic acid),
- resolution: minimum 5.0 between the peaks due to
asparagine and impurity A. .
Calculation of percentage contents:
- for impurity A, use the concentration of impurity A in
reference solution (c);
- for impurity C, use the concentration of impurity C in B. (2S)-2-aminopentanedioic acid (glutamic acid),
reference solution (d);
- for impurities other thanA and C, use the concentration
of asparagine monohydrate in reference solution (b).
Limits:
- impurity A: maximum 0.5 per cent;
- impurity C: maximum 0.1 per cent;
- unspecified impurities: for each impurity, maximum
0.05 per cent; C. 2,2'-[(28,58)-3,6-dioxopiperazine-2,5-diyl]diacetamide,
- total: maximum 0.8 per cent;
- reporting threshold: 0.03 per cent.
Chlorides (2.4.4)
Maximum 200 ppm. D. (2E)-but-2-enedioic acid (fumaric acid),
Dilute 12.5 mL of solution S to 15 mL with water R.
Sulfates (2.4.13)
Maximum 200 ppm.
To 0.75 g add 2.5 mL of dilute hydrochloric acid R and dilute
to 15 mL with distilled water R. Examine after 30 min.
Ammonium (2.4.1, Method B) E. (2S)-2,5-diamino-5-oxopentanoic acid (glutamine),
Maximum 0.1 per cent, determined on 10 mg.
Prepare the standard using 0.1 mL of ammonium standard
solution (100 ppm NH-J R.
Iron (2.4.9)
Maximum 10 ppm.
Dissolve 1.0 g in dilute hydrochloric acid R and dilute to F. (2S)-2-[[(2S)-2,4-diamino-4-oxobutanoyl]amino]
10 mL with the same acid Shake 3 times with 10 mL of butanedioic acid (asparaginylaspartic acid),
methyl isobutyl ketone Rl for 3 min. Wash the combined

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2020 Aspartame 1-211

COzH
B. Infrared absorption spectrophotometry (2.2.24),

o~:H' Preparation Discs.

Comparison aspartame CRS.
o H NH C. Thin-layer chromatography (2.2.27).
HZN~COZH Test solution Dissolve 15 mg of the substance to be
examined in 2.5 mL of water R and dilute to 10 mL with
G. (2S)-4-amino-2-[[(2S)-2-amino-3-carboxypropanoyl] acetic acid R.
amino] -4-oxobutanoic acid (c-aspartylasparagine), Reference solution Dissolve 15 mg of aspartame CRS in
2.5 mL of water R and dilute to 10 mL with acetic acid R.
o H NHz
Plate TLC silica gel G plate R.
H,N~~I.P~HZH Mobile phase water R, anhydrous formic acid R, methanol R,
o yo methylene chloride R (2:4:30:64 VIVIVIV).
NHz
Application20 llL.
Development Over a path of 15 em.
H. (2S)-4-amino-2-[[(2S)-2,4-diamino-4-oxobutanoyl] Drying In air.
amino]-4-oxobutanoic acid (asparaginylasparagine). Detection Spray with ninhydrin solution R and heat at
_ _-"-_-----"-'-- -'-- PhEur 100-105 DC for 15 min.
Results The spot in the chromatogram obtained with the
test solution is similar in position, colour and size to the spot
in the chromatogram obtained with the reference solution.
Aspartame D. Dissolve about 20 mg in 5 ml, of methanol R and add
1 mL of alkaline hydroxylamine solution Rl, Heat on a water-
(Ph. Bur. monograph 0973) bath for 15 min. Allow to cool and adjust to about pH 2
with dilute hydrochloric acid R. Add 0.1 mL oi ferric chloride
solution Rl, A brownish-red colour is produced.
TESTS
Solution S
Dissolve 0.8 g in carbon dioxide-free water R and dilute to
100 mL with the same solvent.
Appearance of solution
Solution S is clear (2.2.1) and not more intensely coloured
294.3 22839-47-0 than reference solution GY6 (2.2.2, Method!!).
Conductivity (2.2.38)
Action and use Maximum 30 J-lS·cm- 1 •
Sweetening agent.
Dissolve 0.80 g in carbon dioxide-free water R prepared from
PhEur ------------- distilled water R and dilute to 100.0 mL with the same
solvent. Measure the conductivity of the solution (C I ) and
DEFINITION
that of the water used for preparing the solution (C z).
(3S)-3-~o-4-[[(2S)-I-methoxy-l-oxo-3-phenylpropan-2­
The readings must be stable within 1 per cent over a period"
yl]amino]-4-oxobutanoic acid (methyl o-t-aspartyl-t-
of 30 s.
phenylalaninate) .
Calculate the conductivity of the solution of the substance 'to
Content be examined using the following expression:
98.0 per cent to 102.0 per cent (dried substance).
CHARACTERS
Appearance
White or almost white, slightly hygroscopic, crystalline Specific optical rotation (2.2.7)
powder. + 14.5 to + 16.5 (dried substance).
Dissolve 2.00 g in a 690 gIL solution of anhydrous formic
Solubility
acid R and dilute to 50.0 mL with the same solution.
Sparingly soluble or slightly soluble in water and in ethanol
Measure within 30 min of preparation.
(96 per cent), practically insoluble in hexane and in
methylene chloride. Related substances
Liquid chromatography (2.2.29).
IDENTIFICATION
Test solution Dissolve 0;60 gof the substance to be
First identification: B.
examined in a mixture of 1.5 volumes of glacial acetic acid R
Second identification: A, C, D. and 98.5 volumes of water R and dilute to 100.0 mL with
A. Ultraviolet and visible absorption spectrophotometry the same mixture of solvents.
(2.2.25). Reference solution (a) Dissolve 4.5 mg of aspartame
Test solution Dissolve 0.1 g in ethanol (96 per cent) Rand impurity A CRS in a mixture of 1.5 'volumes of glacial acetic
dilute to 100 mL with the same solvent. acid Rand 98.5 volumes of water R and dilute to 50.0 mL
Spectral range 230-300 run. with the same mixture of solvents.
Absorption maxima At 247 nm, 252 nm, 258 nm and Reference solution (b) Dissolve 30.0 mg of phenylalanine R
264 nm. (impurity C) in a mixture of 15 volumes of glacial acetic

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 1-212 .Aspartic Acid 2020

acid Rand 85 volumes of waterR and dilute to 100.0 mL

with the same mixture of solvents. Dilute 1.0 mL of this
solution to 10.0 mL with water R.
Reference solution (c) Dilute 5.0 mL of the test solution to
10.0 mL with water R. Dilute 3.0 mL of this solution to
100.0 mL with water R.
A. 2-[(2S,5S}.. 5-benzyl-3,6-dio~opiperazin-2-yl]acetic acid,
Reference solution (d) Dissolve 30.0 mg of l--aspartyi-t»
phenylalanine R (impurity B) in a: mixture of 15 volumes of
glacialacetic acid Rand 85 volumes of water R and dilute to
100.0 mL with the same mixture of solvents. Dilute 1.0 mL
of the solution to 10.0 mL with water R. Mix 1.0 mL of this
solution with 1.0 mL of reference solution (b).
Column
- size: I = 0.25 m, 0 = 4.0 mm;
- stationaryphase: octadecylsilyl silica gelfor chromatography R
(5-10 urn). B. (3S)-3-amino-4-[[(1S)-1-carboxy-2-phenylethyl]amino]-4-
, Mobile phase Mix 10 volumes of acetonitrile Rand oxobutanoic acid (e-t-aspartyl-t-phenylalanine),
90 volumes of a 6.8 gIL solution of potassium dihydrogen
phosphate R previously adjusted to pH 3.7 with phosphoric
acid R.
Flow rate 1 mUmin.
Detection Spectrophotometer at 220 nm. C. (2S)-2-amino-3-phenylpropanoic acid (t-phenylalanine).
Injection 20 /.lL. _ _ _ _---,- PhEur
Run time Twice the retention time of aspartame.
System suitability Reference solution (d):
- resolution: minimum 3.5 between the peaks due to
impurities Band C. Aspartic Acid
Limits:
- impurity A: not more than the area of the principal peak (ph. Bur. monograph 0797)
in the chromatogram obtained with reference solution (a)
(1.5 per cent);
- impurity C: not more than the area of the principal peak
in the chromatogram obtained with reference solution (b)
(0.5 per cent); 133.1
- sum of impurities otherthan A and C: not more than the
area of the principal peak in the chromatogram obtained Action and use
with reference solution (c) (1.5 per cent); Amino acid.
- disregard limit: disregard any peak due to the solvent.
PhEur _
Loss on drying (2.2.32)
Maximum 4.5 per cent, determined on 1.000 g by drying in DEFINITION
an oven at 105 °C. (2S)-2-Aminobutanedioic acid (t-aspartic acid).
Sulfated ash (2.4.14) Content
Maximum 0.2 per cent, determined on 1.0 g. 98.5 per cent to 101.5 per cent (dried substance).
ASSAY CHARACTERS
Dissolve 0.250g in 1.5 mL of anhydrous formic acid Rand Appearance
60 mL of anhydrous acetic acid R. Titrate immediately with White or almost white, crystalline powder or colourless
0.1 M perchloric acid, determining the end-poult crystals.
potentiometrically (2.2.20). Solubility
1 mL of 0.1 M perchloric acid is equivalent to 29.43 mg Slightly soluble in water, practically insoluble in ethanol
of C14HlSN20S' (96 per cent). It dissolves in dilute mineral acids and in
dilute solutions of alkali hydroxides.
STORAGE
In an airtight container. IDENTIFICATION
First identification [carry out either tests A J C or tests CJ D.]
IMPURITIES
Specifiedimpurities A J C. Secondidentification: A.; BJ B.
Other detectable impurities (the following substances would, if A. Specific optical rotation (2.2.7): + 24.0 to + 26.0 (dried
presentat a sufficient level, be detected by one or otherof the tests substance) .
in 'the monograph. They arelimited by the general acceptance Dissolve 2.00 g in hydrochloric acid Rl and dilute to 25.0 mL
criterion for other/unspecified impurities and/or by the general with the-same acid.
monograph Substances for pharmaceutical use (2034). It is B. A suspension of 1 g in 10 mL of waterR is strongly acid
therefore not necessary to identify these impurities for (2.2.4).
demonstration of compliance. See also 5.10. Control of impurities C. Infrared absorption spectrophotometry (2.2.24).
in substances for pharmaceutical use) B.
Comparison aspartic acid CRS.

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2020 Aspartic Acid 1-213

D. Enantiomeric purity (see Tests). Other dicarboxylic acids

E. Thin-layer chromatography (2.2.27). Liquid chromatography (2.2.29).
Test solution Dissolve 10 mg of the substance to be Testsolution· Dissolve 0.500 g of the substance to be
examined in 2 mL of dilute ammonia R1 and dilute to 50 mL examined in 2.0 mL of a 618 gIL solution of hydrochloric
with waterR. acid R and dilute to 10.0 mL with water R.
Reference solution Dissolve 10 mg of aspartic acid CRS in Reference solution (a) Dissolve 20.0 mg of malic acidR
2 mL of dilute ammonia R1 and dilute to 50 mL with (impurity A) in waterR and dilute to 20.0 mL with the same
water R. solvent.
Plate TLC silica gelplate R. Reference solution (b) Dissolve 10.0 mg of maleic acidR
Mobilephase glacial acetic acid R, waterR, butanolR (impurity H) in waterR and dilute to 10.0 mL with the same
(20:20:60 VIVIV). solvent. Dilute 1.0 mL of the solution to 10.0 mL with
waterR.
Application 5 ilL.
Reference solution (c) Dilute 1.0 mL of reference solution (b)
Development Over 2/3 of the plate. to 10.0 mL with reference solution (a).
Drying In air. Reference solution (d) Dilute 1.0 mL of reference solution (a)
Detection Spray with ninhydrin solution R and heat at 105 DC to 10.0 mL with water R.
for 15 min. Reference solution (e) Dissolve 10.0 mg of fumaric acidR
Results The principal spot in the chromatogram obtained (impurity B)· in waterR and dilute to 10.0 mL with the same
with the test solution is similar in position, colour and size to solvent. Dilute 1.0 mL of the solution to 100.0 mL with
the principal spot in the chromatogram obtained with the waterR.
reference solution. Column:
TESTS - size: I = 0.30 m,0 = 7.8 mm;
Appearance of solution - stationary phase: cation-exchange resin R (9 urn);
The solution is clear (2.2.1) and not more intensely coloured - temperature: 30 DC.
than reference solution BY6 (2.2.2, MethodII). Mobile phase 0.39 gIL solution of sulfuric acid R.
Dissolve 0.5 g in a 103 gIL solution of hydrochloric acid Rand Flow rate 0.6 mUmin.
dilute to 10 mL with the same acid. Detection Spectrophotometer at 214 nm.
Enantiomeric purity Injection 10 IlL.
Liquid chromatography (2.2.29).
Run time 4 times the retention time of impurity H.
Test solution Dissolve 0.100 g of the substance to be
Identification of impurities Use the chromatogram obtained
examined in waterR and dilute to 100.0 mL with the same
with reference solution (c) to identify the peaks due to
solvent.
impurities A and H; use the chromatogram obtained with
Reference solution (a) Dissolve 0.100 g of ti-aspartic acid R reference solution (e) to identify the peak due to impurity B.
(impurity 1) in waterR and dilute to 100.0 mL with the same
Relativeretention With reference to impurity H (retention
solvent.
time = about 7.5 min): impurity A = about 1.2;
Reference solution (b) Dissolve 0.100 g of the substance to impurity B = about 2.0.
be examined in 90 mL of water R, add 0.3 mL of reference
System suitability Reference solution (c):
solution (a) and dilute to 100.0 mL with waterR.
- resolution: minimum 1.5.between the peaks due to
Reference solution (c) Dilute 0.3 mL of reference solution (a) impurities H and A.
to 100.0 mL with water R. Calculation of percentage contents:
Column: - for impurity A, use the concentration of impurity A in
- size: 1= 0.15 m, (2) = 4.6 mm; reference solution (d);
- stationary phase: L-penicillamine coated silica gelfor chiral - for impurities Band H, use the concentration of
separations R (5 11m); impurity H in reference solution (b);
- temperature: 30 DC. - for impurities other than Band H, use the concentration
Mobilephase .2-propanol R, 0.5 gIL solution of copper sulfate of impurity A in reference solution (d).
pentahydrate R (5:95 VIV). Limits:
Flow rate 1.0 mL/min. - impurity A: maximum 0.2 per cent;
Detection Spectrophotometer at 230 nm. - impurity B: maximum 0.10 per cent;
Injection 20 ilL. - impurity H: maximum 0.10 per cent;
- unspecified impurities: for each impurity, maximum
Relative retention With reference to aspartic acid (retention 0.10 per cent;
time = about 12 min): impurity I = about 0.85. - total: maximum 0.3 per cent;
System suitabz1ity Reference solution (b): - reporting threshold: 0.05 per cent.
- resolution: minimum 2.0 between the peaks due to
Ninhydrin-positive substances
impurity I and aspartic acid.
Amino acid analysis (2.2.56). For analysis, use Method 1.
Calculation of percentage content:
The concentrations of the test and reference solutions may
- for impurity I, use the concentration of impurity I in
be adapted according to the sensitivity of the equipment
reference solution (c).
used. The concentrations of all solutions are adjusted so that
Limit: the system suitability requirements described in general
- impurityI: maximum 0.3 per cent. chapter 2.2.46 are fulfilled, keeping the ratios of
concentrations between all solutions as described.

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 1-214 Aspartic Acid 2020

Solution A dilute hydrochloric acid Rl or a sample preparation Injection Test solution, reference solution (c) and blank
buffer suitable for the apparatus used. solution.
Test solution Dissolve 30.0 mg of the substance to be Limit:
examined in solution A and dilute to 50.0 mL with - ammonium at 570 nm:nbt more than the area of the
solution A. corresponding peak in the chromatogram obtained with
Reference solution (a) Dilute 1.0 mL of the test solution to reference solution (c) (0.02 per cent), taking into account
100.0 mL with solution A. Dilute 2.0 mL of this solution to the peak due to ammonium in the chromatogram
10.0 mL with solution A. obtained with the blank solution.
Reference solution (b) Dissolve 30.0 mg of proline R in Iron (2.4.9)
solution A and dilute to 100.0 mL with solution A. Dilute Maximum 10 ppm.
1.0 mL of this solution to 250.0 mL with solution A. In a separating funnel, dissolve 1.0 g in 10 mL of dilute
Reference solution (c) Dilute 6.0 mL of ammonium standard hydrochloric acid R. Shake with 3 quantities, each of 10 mL,
solution (100 ppm NH-J R to 50.0 mL with solution A. Dilute of methyl isobutyl ketone R1, shaking for 3 min each time.
1.0 mL of this solution to 100.0 mL with solution A. To the combined organic layers add 10 mL of water Rand
Reference solution (d) Dissolve 30 mg of isoleucine Rand shake for 3 min. Use the aqueous layer.
30 mg of leucine R in solution A and dilute to 50.0 mL with Loss on drying (2.2.32)
solution A. Dilute 1.0 mL of the solution to 200.0 mL with Maximum 0.5 per cent, determined on 1.000 g by drying in
solution A. an oven at 105°C.
Reference solution (e) Dissolve 30.0 mg of alanine R Sulfated ash (2.4.14)
(impurity D), 60.0 mg of asparagine R (impurity G) and Maximum 0.1 per cent, determined on 1.0 g.
30.0 mg of glutamic acid R (impurity C) in solution A and
ASSAY
dilute to 100.0 mL with solution A. Dilute 1.0 mL of the
Dissolve 0.100 gin 50 mL of carbon dioxide-free waterR, with
solution to 250.0 mL with solution A.
slight heating if necessary. Cool and titrate with O.lM sodium
Blank solution Solution A. hydroxide determining the end-point potentiometrically
Inject suitable, equal amounts of the test solution, blank (2.2.20).
solution and reference solutions (a), (b), (d) and (e) into the 1 mL of 0.1 M sodiumhydroxide is equivalent to 13.31 mg of
amino acid analyser. Run a program suitable for the C4H7N04 •
determination of physiological amino acids.
STORAGE
System suitability Reference solution (d):
-resolution: minimum 1.5 between the peaks due to Protected from light.
isoleucine and leucine. ." IMPURITIES
Calculation of percentage contents: Specified impurities A, B, C, D, H, G, 1.
- for impurities C, D and G, use the concentration of each Other detectable impurities (the following substances would, if
impurity in reference solution (e); present at a sufficient level, be detected by one or otherof the tests
- for any ninhydrin-positive substance detected at 570 nm, in the monograph. They are limited by the general acceptance
use the concentration of aspartic acid in reference criterion for other/unspecified impurities and/or by the general
solution (a); monograph Substances for pharmaceutical use (2034). It is
- for any ninhydrin-positive substance detected at 440 nm, therefore not necessary to identify these impurities for
use the concentration of proline in reference solution (b); demonstration of compliance. See also 5.10. Control of impurities
if a peak is above the reporting threshold at both in substances for pharmaceutical use) E, F.
wavelengths, use the result obtained at 570 nm for
quantification.
Limits:
- impurities C, D, G: for each impurity, maximum
0.2 per cent; A. (2RS)-2-hydroxYbutanedioic acid (malic acid), -
- any ninhydrin-positive substance: for each impurity,
maximum 0.10 per cent;
- total: maximum 1.0 per cent;
- reporting threshold: 0.05 per cent.
B. (2E)-but-2-enedioic acid (fumaric acid),
Chlorides (2.4.4)
Maximum 200 ppm.
Dissolve 0.25 gin 3 mL of dilute nitric acid R and dilute to
15 mL with water R. Add 1 mL of waterR instead of 1 mL
of dilute nitric acid R. C. (2S)-2-aminopentanedioic acid (glutamic acid),
Sulfates (2.4.13)
Maximum 300 ppm.
Dissolve 0.5 gin 4 mL of hydrochloric acid R and dilute to
15 mL with distilled water R. Carry out the evaluation of the
test after 30 min. D. (2S)-2-aminopropanoic acid (alanine),
Ammonium.
. Amino acid analysis (2.2.56) as described in the test for
ninhydrin-positive substances with the following modification.
E. butanedioic acid (succinic acid),

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2020 Aspirin 1-215

IDENTIFICATION
First identification: A, B.
Second identification: B, C, D.
A. Infrared absorption spectrophotometry (2.2.24).
F. (2S)-2,5-diamino-5-oxopentanoic acid (t-glutamine),
Comparison acetylsalicylic acid CRS.
B. To 0.2 g add 4 mL of dilute sodium hydroxide solution R
and boil for 3 min. Cool and add 5 mL of dilute sulfuric
acid R. A crystalline precipitate is formed. Filter, wash the
precipitate and dry at 100-105 °C. The melting point
G. (2S)-2,4-diamino-4-oxobutanoic acid (asparagine), (2.2.14) is 156°C to 161 °C.
C. In a test tube mix 0.1 g with 0.5 g of calcium hydroxide R.
Heat the mixture and expose to the fumes produced a piece
of filter paper impregnated with 0.05 mL of nitrobenzaldehyde
solution R. A greenish-blue or greenish-yellow colour develops
H. (2Z)-but-2-enedioic acid (maleic acid), on the paper. Moisten the paper with dilute hydrochloric
acid R. The colour becomes blue.
D. Dissolve with heating about 20 mg of the precipitate
obtained in identification test Bini 0 mL of water Rand
cool. The solution gives reaction (a) of salicylates (2.3.1).
1. (2R)"';2-amiIlobutanedioic acid (n-aspartic acid). TESTS
_ _ _-"- PhEur Appearance of solution
The solution is clear (2.2.1) and colourless (2.2. 2~
Method II).
Dissolve 1.0 g in 9 mL of ethanol (96 per cent) R.
Aspirin Related substances
Liquid chromatography (2.2.29). Prepare the solutions
(Acetylsalicylic Acid~ Ph. Bur. monograph 0309) immediately before use.
Test solution Dissolve 0.100 g of the substance to be
examined in acetonitrile for chromatography R and dilute to
10.0 mL with the same solvent.
Reference solution (a) Dissolve 50.0 mg of salicylic acid R
(impurity C) in the mobile phase and dilute to 50.0 mL with
the mobile phase. Dilute 1.0 mL of the solution to 100.0 mL
with the mobile phase.
180.2 50-78-2
Reference solution (b) Dissolve 10 mg of salicylic acid R
Action and use (impurity C) in the mobile phase and dilute to 10.0 mL with
Salicylate; non-selective cyclo-oxygenase inhibitor; the mobile phase. To 1.0 mL of the solution add 0.2 mL of
antipyretic; analgesic; anti-inflammatory. the test solution and dilute to 100.0 mL with the mobile
Preparations phase.
Aspirin Tablets Reference solution (c) Dissolve with the aid of ultrasound the
Aspirin Dispersible Tablets contents of a vial of acetylsalicylic acidfor peak
identification CRS (containing impurities A, B, D, E and F)
Aspirin Effervescent Soluble Tablets
in 1.0 mL of acetonitrile R.
Aspirin Gastro-resistant Tablets
Column:
Aspirin and Caffeine Tablets - size: I = 0.25 m, 0 = 4.6 mm;
Co-codaprin Tablets - stationary phase: octadecylsilyl silica gelfor chromatography R
Co-codaprin Dispersible Tablets (5 urn).
PhEur _ Mobilephase phosphoric acid R, acetonitrile for
chromatography R, waterR (2:400:600 V/V/V).
DEFINITION Flow rate 1 mLlmin.
2-(Acetyloxy)benzoic acid. Detection Spectrophotometer at 237 nm.
Content Injection 10 IlL.
99.5 per cent to 101.0 per cent (dried substance). Run time 7 times the retention time of acetylsalicylic acid.
CHARACTERS Identification of impurities Use the chromatogram obtained
Appearance with reference solution (a) to identify the peak due to
White or almost white, crystalline powder or colourless impurity C; use the chromatogram supplied with
crystals. acetylsalicylic acidfor peak identification CRS and the
Solubility chromatogram obtained with reference solution (c) to identify
Slightly soluble in water, freely soluble in ethanol the peaks due to impurities A, B, D, E and F.
(96 per cent). Relative retention .With reference to acetylsalicylic acid
mp (retention time = about 5 min): impurity A = about 0.7;
About 143°C (instantaneous method). impurity B = about 0.8; impurity C = about 1.3;

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1-216 Atazanavir Sulfate 2020

impurity D = about 2.3; impurity E = about 3.2;

impurity F = about 6.0.
System suitability Reference solution (b):
- resolution: minimum 6.0 between the peaks due to
acetylsalicylic acid and impurity C.
Limits:
- impurities A-, B-, C-, D-, B-, F: for each impurity, not more
E. 2-[(2-hydroxybenzoyl)oxy]benzoic acid (salsalate,
than 1.5 times the area of the principal peak in the
salicylsalicylic acid),
chromatogram obtained with reference solution (a)
(0.15 per cent);
- unspecified impurities: for each impurity, not more than
0.5 times the area of the principal peak in the
chromatogram obtained with reference solution (a)
(0.05 per cent);
- total: not more than 2.5 times the area of the principal
peak in the chromatogram obtained with reference
solution (a) (0.25 per cent);
- disregard limit: 0.3 times the area of the principal peak in
the chromatogram obtained with reference solution (a) F. 2-(acetyloxy)benzoic anhydride (acetylsalicylic anhydride).
(0.03 per cent). _----,- PhEur
Loss on drying (2.2.32)
Maximum 0.5 per cent, determined on 1.000 g by drying in
vacuo.
Sulfated ash (2.4.14) Atazanavir Sulfate
Maximum 0.1 per cent, determined on 1.0 g.
(ph. Bur. monograph 2898)
ASSAY
In a flask with a ground-glass stopper, dissolve 1.000 g in
10 mL of ethanol (96 per cent) R. Add 50.0 mL of 0.5 M
sodium hydroxide. Close the flask and allow to stand for 1 h.
Using 0.2 mL of phenolphthalein solution R as indicator, titrate
with 0.5 M hydrochloric acid. Carry out a blank titration.
1 mL of 0.5 M sodium hydroxide is equivalent to 45.04 mg of
C9H804 •
STORAGE
In an airtight container.
IMPURITIES
Specified impurities A-, B-, C-, D-, E-, F.
803 229975-97-7

Action and use

Antiviral (lllV).
PhEur _ _----,- ----,- _
A. 4-hydroxybenzoic acid,
DEFINITION
H02CYyC02H
Methyl [(5S, lOS, 11S, 14S)-II-benzjrl-5-tert-butyl-l O-hydroxy-
~OH 15, 15-dimethyl-3,6,13-trioxo-8-[[4-(pyridin-2-yl)phenyl]
methyl]-2-oxa-4,7,8, 12-tetraazahexadecan-14-yl] carbamate
B. 4-hydroxybenzene-l,3-dicarboxylic acid sulfate.
(4-hydroxyisophthalic acid), Content

~
'
OC C02H

OH
98.0 per cent to 102.0 per cent (anhydrous substance).
CHARACTERS
Appearance
White or pale yellow, slightly hygroscopic, crystalline powder
C. 2-hydroxybenzenecarboxylic acid (salicylic acid), that may contain agglomerates.
Solubility
C0 2H

OC
~.
I
oAo 0
CH3 Slightly soluble in water, freely soluble in ethanol
(96 per cent), practically insoluble in heptane.

oA() IDENTIFICATION
A. Specific optical rotation (see Tests).
B. Infrared absorption spectrophotometry (2;2.24).
Comparison atazanavir sulfate CRS.
D. 2-[[2-(acetyloxy)benzoyl]oxy]benzoic acid
(acetylsalicylsalicylic acid), C. It gives reaction (a) of sulfates (2.3.1).

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2020 Atazanavir Sulfate 1-217

TESTS System suitability Reference solution (c):

Specific optical rotation (2.2.7) - resolution: minimum 1.5 between the peaks due to
-44 to -40 (anhydrous substance), measured at 25 "C. impurity OF and atazanavir.
Dissolve 0.100 gin 8 mL of methanol R, using sonication if Calculation of percentage contents:
necessary, and dilute to 10.0 mL with the same solvent. -;- for each impurity, use the concentration of atazanavir
Related substances sulfate in reference solution (b).
Liquid chromatography (2.2.29). Limits:
Solvent mixture Mix equal volumes of acetonitrile Rl and a - unspecified impurities: for each impurity, maximum
freshly prepared 2.73 gIL solution of potassium dihydrogen 0.10 per cent;
phosphate R in waterfor chromatography R previously adjusted - total: maximum 0.5 per cent;
to pH 3.5 with dilute phosphoric add R. - reporting threshold: 0.05 per cent; disregard any peak with a
relative retention with reference to atazanavir of less than
Test solution (a) Dissolve 20.0 mg ofthe substance to be 0.2.
examined in 40 mL of the solvent mixture, sonicate for
3 min and dilute to 50.0 mL with the solvent mixture. Im.purity K
Liquid chromatography (2.2.29) as described in the test for
Test solution (b) Dissolve 50.0 mg of the substance to be
related substances with the following modifications.
examined in 40 mL of the solvent mixture, sonicate for
3 min and dilute to 50.0 mL with the solvent mixture. Mobilephase:
Reference solution (a) Dissolve 20.0 mg of atazanavir
Time Mobile phase A Mobile phase B
sulfate CRS in 40 rnl, of the solvent mixture, sonicate for (min) (per cent VIJI) (per cent VIJI)
3 min and dilute to 50.0 mL with the solvent mixture.
0-5 95 5
Reference solution (b) Dilute 1.0 mL oftest solution (a) to 5-8 95 ~ a 5 ->100
100.0 mL with the solvent mixture. Dilute 1.0 mL of this 8.,. 14 0 100
solution to 10.0 mL with the solvent mixture.
Reference solution (c) Dissolve 4 mg of atazanavirfor system Injection 20 J.LL of test solution (b) and reference
suitability CRS (containing impurity F) in 8 mL of the solvent solution (d).
mixture, sonicate for 3 min and dilute to 10 mL with the
Identification of impurities Use the chromatogram obtained
solvent mixture;
with reference solution (d) to identify the peak due to
Reference solution (d) Dissolve 2.0 mg of atazanavir impurity K.
impurity K CRS in 9 mL of the solvent mixture, sonicate for
Relativeretention With reference to atazanavir (retention
3 min and dilute to 10.0 mL with the solvent mixture. Dilute
5.0 mL of the solution to 100.0 mL with the solvent mixture.
time = about 10 min): impurity K about 0.4. =
Dilute 3.0 mL of this solution to 20.0 mL with the solvent Calculation of percentage content:
mixture. - for impurity K, use the concentration of impurity K in
reference solution (d).
Column:
- size: 1 = 0.15 m, 0 = 4.6 mrn; Limit:
- stationary phase: end-capped octadecylst'Zyl silica gelfor - impurityK: maximum 0.15 per cent.
chromatography R (3.0 1J1l1); Water (2.5.32)
- temperature: 25°C. Maximum 2.5 per cent, determined on 0.100 g by direct
Mobz1e phase: sample introduction.
- mobile phaseA: mix 25 volumes of acetonitrile R1 and Sulfated ash (2.4.14)
75 volumes of a freshly prepared 2.73 gIL solution of Maximum 0.2 per cent, determined on 1.0 g.
potassium dihydrogen phosphate R previously adjusted to
ASSAY
pH 3.5 with phosphoric acid R;
Liquid chromatography (2.2.29) as described in the test for
- mobile phaseB: mix 25 volumes of a freshly prepared
related substances with the following modifications.
2.73 gIL solution of potassium dihydrogen phosphate R
previously adjusted to pH 3.5 with phosphoric acid R, and Mobilephase Solvent mixture.
75 volumes of acetonitrile Rl; Injection Test solution (a) and reference solutions (a) and
(c).
Time Mobile phase A Mobile phase B Run time 1.6 times the retention time of atazanavir.
(min) (per cent VIP) (per cent VIJI)
Relativeretention With reference to atazanavir (retention
0-5 100 a time = about 9.5 min): impurity F = about 0.94.
5 - 45 100 ~ a a -> 100
System suitability Reference solution (c):
.:.- resolution: minimum 1.5 between the peaks due to
Flow rate 1.0 mIJrnin. impurity F and atazanavir.
Detection Spectrophotometer at 215 run. Calculate the percentage content of C38H5~6011S using the
Injection 10 ~ of test solution (a) and reference chromatogram obtained with reference solution (a) and
solutions (b) and (c). taking into account the assigned content of atazanavir
Identification of impurities Use the chromatogram supplied sulfate CRS.
with atazanavir for system suitability CRS and the
0

STORAGE
chromatogram obtained with reference solution (c) to identify
In an airtight container.
the peak due to impurity F.
Relative retention With reference to atazanavir (retention IMPURITIES
time = about 30 min): impurity F = about 0.99. Specified impurities K.

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1-218 Atazanavir Sulfate 2020

Other detectable impurities (the following substances would, if

present at a sufficientlevel, be detected by one or otherof the tests
in the monograph. They are limitedby the general acceptance
criterion for other/unspecified impurities and/or by the general
monograph Substances for pharmaceutical use (2034). It is
therefore not necessary to identify these impurities for
demonstration of compliance. See also 5.10. Control of impurities
in substances for pharmaceutical use) A, B, C, D, E, F, G, H,
I, J.

F. methyl [(5R, lOS, 11S, 14S)-II-benzyl-5-tert-butyl--I0-

hydroxy-IS, 15-dimethyl-3,6, 13-trioxo-8-[[4~(pyridin- 2-yl)
phenyl] methyl] -2-oxa-4,7,8, 12-tetraazahexadecan-I4-yl]
carbamate,
A. 4-(pyridin-2-yl)benzoic acid,

B. 4-(pyridin-2-yl)benzaldehyde,

G. methyl [(5S, lOS, lIS, 14R)-II..,benzyl-5-tert-butyl-I0-

hydroxy-IS, 15-dimethyl-3,6, 13-trioxo-8-[[4-(Pyridin-2-yl)
phenyl] methyl] -2-oxa-4,7,8,I2-tetraazahexadecan-14-yl]
carbamate,

C. methyl [(SS,IOS,IIS,14S)-II-benzyl-S-tert.:.butyl-IO-
hydroxy-15, 15-dimethyl-3,6,13-trioxo-2-oxa-4,7,8,12-
tetraazahexadecan-I4-yl] carbamate,

H. methyl [(SS, lOR, IIR, 14S)-II-benzyl-5-tert-butyl-10-

hydroxy-IS, 1S-dimethyl-3,6,13-trioxo-8-[[4-(Pyridin-2-yl)
phenyl] methyl] -2-oxa-4,7,8, 12-tetraazahexadecan-I4-yl]
carbamate,
D. (2S,3S)-3-amino-4-phenyl-I-[(E)-I-[[4-(pyridin-2-yl)
phenyl]methyl] -2-[[4-(pyridin-2-yl)phenyl] methylidene]
hydrazin-I-yl] butan-2-ol,

1. methyl [(2S)-I-[[(2S,3S)-3-hydroxy-l-phenyl-4-[(E)-I-
[[4-(pyridin-2-yl)phenyl]methyl]-2-[[4-(Pyridin-2-yl)
phenyl] methylidene]hydrazin-I-yl]butan-2-yl] amino] -3,3-
E. methyl [(SS,IOR,IIS,14S)-II-benzyl-S-tert-butyl-IO- dimethyl-I-oxobutan-2-yl] carbamate,
hydroxy-Ifi, 15-dimethyl-3,6, 13-trioxo-8-[[4-(pyridin-2-yl)
phenyl] methyl] -2-oxa-4,7,8,I2-tetraazahexadecan-I4-yl]
carbamate,

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2020 Atenolol 1-219

Test solution Dissolve 0.100 g in methanol R and dilute to

100.0 rnL with the same solvent. Dilute 10.0 mL of the
I~ solution to -100.0 mL with methanol R.
#
Spectral range 230-350 nrn.
H3C CH 3 0 Hq.. H H
Absorption maxima At 275 nrn and 282 nrn.
H3C
X 0 :Jl ~ '-N~N'v""O'/CH3
.... II /\ Absorbance ratio A27s/A282 = 1.15 to 1.20.
H o H3C CH3
C. Infrared absorption spectrophotometry (2.2.24).
Comparison atenolol CRS.
\ j)
D. Thin-layer chromatography (2.2.27).
Test solution Dissolve 10 mg of the substance to be
J. tert-butyl 2- [(2S,3S)-3-(tert-butoxyfonnamido)-2-hydroxy- examined in 1.0 mL of methanol R.
4-phenylbutyl] -2- [[4-(pyridin-2-yl)phenyl] methyl]
Reference solution Dissolve 10 mg of atenolol CRS in 1.0 mL
hydrazine-l-carboxylate,
of methanol R.
Plate TLC silanised silica gel F 254 plate R.
Mobile phase concentrated ammonia R1, methanol R
(1:99 VIV).
Application 10 ut,
Drying In air.
K. -(2S)-2-(methoxyfonnamido)-3,3-dirnethylbutanoic acid. Detection Examine in ultraviolet light at 254 nm.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
Results The principal spot in the chromatogram obtained
with the test solution is similar in position and size to the
principal spot in the chromatogram obtained with the
- reference solution.
Atenolol TESTS
(Ph. Bur. monograph 0703) Solution S
Dissolve 0.10 g in water R and dilute to 10 mL with the
H pH H same solvent.
O~N -CH3
9 ~ y-- andenantiomer
Appearance of solution
Solution S is clear (2.2.1) and not more intensely coloured
~ CH3
H2N than degree 6-of the range of reference solutions of the most
appropriate colour (2.2.2, Method II).
266.3 29122-68-7 Optical rotation (2.2. 7)
+ 0.10° to -0.10°, determined on solution S.
Action and use
Related substances
Beta-adrenoceptor antagonist.
Liquid chromatography (2.2.29).
Preparations
Test solution Dissolve 50 mg of the substance to be
Atenolol Injection
examined in 20 mL of the mobile phase and dilute to
Atenolol Oral Solution 25.0 mL with the mobile phase.
Atenolol Tablets Reference solution (a) Dissolve 2 mg of atenololfor system
Co-tenidone Tablets suitability CRS (containing impurities B, F, G, I and D in
PhEur _
1.0 mL of the mobile phase.
Reference solution (b) Dilute 1.0 mL of the test solution to
DEFINITION 100.0 mL with the mobile phase. Difute 1.0 mL of this
2-[4-[(2RS)-2-Hydroxy-3-[(propan-2-yl)amino] solution to 10.0 mL with the mobile phase.
propoxy] phenyl] acetamide. Column:
Content - size: I = 0.125 m,0 = 4.0 mm;
99.0 per cent to 101.0 per cent (dried substance). - stationary phase: end-capped octadecylsilyl silica gel for
chromatography R (5 urn).
CHARACTERS
Appearance Mobile phase Dissolve 1.0 g of sodium oetanesulfonate Rand
White or almost white powder. 0.4 g of tetrabutylammonium hydrogen sulfate R in 1 L of a
mixture of 20 volumes of tetrahydrofuran R, 180 volumes of
Solubility
methanol R2and 800 volumes of a 3.4 gIL solution of
Sparingly soluble in water, soluble in anhydrous ethanol,
potassium dihydrogen phosphate R; adjust the apparent pH to
slightly soluble in methylene chloride.
3.0 with phosphoric acid R.
IDENTIFICATION Flow rate 0.6 mUmin.
First identification: C.
Detection Spectrophotometer at 226 nm.
Second identification: A, B, D.
Injection 10 ilL.
A. Melting point (2.2.14): 152°C to 155 -c,
Run time 5 times the retention time of atenolol.
B. Ultraviolet and visible-absorption spectrophotometry Identification of impurities Use the chromatogram supplied
(2.2.25). with atenolol for system suitability CRS and the chromatogram

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 1-220 Atenolol 2020

H OH
obtained with reference solution (a) to identify the peaks due
to impurities B, F, G, I and J. 9 ~O~OH
Relative retention With reference to atenolol (retention ~ and enantiomer
H2N
= =
time about 8 min): impurity B about 0.3;
= =
impurity J about 0.7; impurity I about 0.8;
impurity F = about 2.0 (pair of peaks); B. 2- [4- [(2RS)- 2,3-dihydroxypropoxy]phenyl] acetamide,
impurity G = about 3.5.
H OH
System suitability Reference solution (a):
- resolution: minimum 1.4 between rhe peaks due to 9 ~O~CI
impurities J and 1. ~ and enantiomer
H2N
Limits:
- impurity B: not more than twice the area of the principal
D. 2- [4- [(2RS)- 3-chloro-2-hydroxypropoxy]phenyl] acetamide,
peak in the chromatogram obtained with reference
solution (b) (0.2 per cent);
- impurities F~ G~ I~ J: for each impurity, not more than
1.5 times the area of the principal peak in the
chromatogram obtained with reference solution (b)
(0.15 per cent);
- unspecified impurities: for each impurity, not more than the
area of the principal peak in the chromatogram obtained E. 2,2'-[(2-hydroxypropane-l,3-diyl)bis(oxy-4,I-phenylene)]
with reference solution (b) (0.10 per cent); diacetamide, ,
- total: not more than 5 times the area of the principal peak
in the chromatogram obtained withreference solution (b)
(0.5 per cent);
- disregard limit: 0.5 times the area of the principal peak in
the chromatogram obtained with reference solution (b)
(0.05 per cent).
Chlorides (2A.4)
Maximum 0.1 per cent. F. 2,2'-[[(propan-2-yl)azanediyl]bis[(2-hydroxypropane-3,1-
Dissolve 50 mg in a mixture of 1 mL of dilute nitric acid R diyl)oxy-4, l-phenylenej] diacetamide,
and 15 mL of water R. The solution, without further addition
of dilute nitric acid R, complies with the test. . H PHH

»
O~ N
Loss on drying (2.2.32) I. ~.. y CH
3 and enantiomer

Maximum 0.5 per cent, determined on 1.000 g by drying in H0 2C # CH3

an oven at 105 °C.
Sulfated ash (2.4.14) G.[4- [(2RS)-2-hydroxy-3- [(propan-2-yl) amino] propoxy]
Maximum 0.1 per cent, determined on 1.0 g. phenyl] acetic acid,
ASSAY , H pH H

»
Dissolve 0.200 g in 80 mL of anhydrous aceticadd R. Titrate
I ~
O~ N.y CH3
and enantiomer
with 0.1 M perchloric acid, determining the end-point
potentiometrically (2.2.20). NC . #. CH3 ..

1 mL of 0.1 M perchloric acid is equivalent to 26.63 mg of

C14H22N203'
H. [4-[(2RS)-2-hydroxy-3-[(propan-2-yl)amino]propoxy]
phenyl] acetonitrile,
IMPURITIES
Specified impurities B~ F, G~ I~ J.
Other detectable impurities (thefollowing substances would, if
present at a sufficient leuel, be detected by one or otherof the tests
in the monograph. They are limitedby the general acceptance
criterion for other/unspecified impurities and/or by the general
monograph Substances for pharmaceutical use (2034). It is 1. 2- [4- [(2RS)-3-( ethylamino)-2-hydroxypropoxy] phenyl]
therefore not necessary to identify these impurities for acetamide,
demonstration of compliance. See also 5.10. Control of impurities
in substances for pharmaceutical use) A~ D~ E~ H. H OH

· .I. ~ O~NH2
H2N
tJ) #
and enantiomer

J. 2- [4- [(2RS)- 3-amino-2-hydroxypropoxy] phenyl] acetamide.

. A. 2-(4-hydroXyphenyl) acetamide, _ _ _ _ _ _ _----'- '-- PhEur

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2020 Atomoxetine Hydrochloride 1-221

Flow rate 1.0 mUmin.

Atomoxetine Hydrochloride Detection Spectrophotometer at 273 nm.
(Ph. Bur. monograph 2640) Injection 10 ilL of the test solution and reference
solutions (b) and (c).
Run time 1.3 times the retention time of atomoxetine.
• Hel
Identification of impurities Use the chromatogram obtained
with reference solution (b) to identify the peaks due to
impurities Band D.
Relative retention With reference to atomoxetine (retention
time = about 12 min): impurity B = about 0.5;
291.8 82248-59-7 impurity D = about 0.6.
Action and use System suitability Reference solution (b):
Noradrenaline reuptake inhibitor; treatment of attention - resolution: minimum 1.8 between the peaks due to
deficit hyperactivity disorder (ADHD). impurities Band D.
Limits:
Ph Eur ---'- _
- impurity B:maximum 0.5 per cent;
DEFINITION - impurity D: maximum 0.15 per cent;
(3R)-N-l\1ethyl-3~(2-methylphenoxy)-
3-phenylpropan-1- - unspecified impurities: for each impurity, maximum
amine hyarochloride. 0.10 per cent;
- disregard limit: the area of the peak due to impurity B in
Content" the chromatogram obtained with reference solution (c)
98.0 percent to 102.0 per cent (dried substance).
(0.05 per cent); disregard any peak with a relative
CHARACTERS retention with reference to atomoxetine of about 0.7
Appearance (impurity A).
White or almost white powder. Related substances
Solubility Liquid chromatography (2.2.29).
Sparingly soluble in water, soluble in anhydrous ethanol, Solution A Dissolve 5.9 g of sodium octanesulfonate
practically insoluble in heptane. monohydrate R in 1000 mL of a 2.9 gIL solution of phosphoric
It shows polymorphism (5.9). acid R previously adjusted to pH 2.5 with a 280 gIL solution
of potassium hydroxide R.
IDENflFICATION
A. Infrared absorption spectrophotometry (2.2.24). Testsolution (a) Dissolve 25 mg of the substance to be
examined in the mobile phase and dilute to 10.0 mL with
Comparison atomoxetine hydrochloride CRS.
the mobile phase.
If the spectra obtained in the solid state show differences,
Test solution (b) Dissolve 25.0 mg of the substance to be
dissolve the substance to be examined and the reference
examined in the mobile phase and dilute to 100.0 mL with
substance separately in anhydrous ethanol R, evaporate to
the mobile phase.
dryness and record new spectra using the residues.
Reference solution (a) Dilute 1.0 mL of test solution (a) to
B. Isomeric purity (see Tests).
100.0 mL with the mobile phase. Dilute 1.0 mL of this
C. It gives reaction (a) of chlorides (2.3.1). solution to 10.0 mL with the mobile phase.
TESTS Reference solution (b) Dissolve 7.5 mg of 3-(methylamino)-1-
Isomeric purity phenylpropan-1-ol R (impurity H) and 5 mg of mandelic acidR
Liquid chromatography (2.2.29): use the normalisation (impurity E) in test solution (b) and dilute to 50 mL with
procedure. test solution (b).
Test solution Dissolve 35.0 mg of the substance to be Reference solution (c) Dissolve 5 mg of atomoxetine for
examined in 2.5 mL of anhydrous ethanol R, sonicate until impurityA identification CRS in the mobile phase and dilute
dissolution is complete and dilute to 10.0 mL with heptaneR. to 20 mL with the mobile phase.
Reference solution (a) Dissolve 3.5 mg of atomoxetine Reference solution (d) Dissolve 25.0 mg of atomoxetine
impurity B CRS and 1 mg of atomoxetine impurityD CRS in hydrochloride CRS in the mobile phase and dilute to
5 mL of anhydrous ethanol R, sonicate until dissolution is 100.0 mL with the mobile phase.
complete and dilute to 20.0 mL with heptane R. Column:
Reference solution (b) Dissolve 35.0 mg of the substance to = =
- size: l 0.15 m, (2) 4.6 mm;
be examined in 2.5 mL of anhydrous ethanol R. Add 1.0 mL - stationary phase: end-capped octylsilyl silica gelfor
of reference solution (a) and dilute to 10.0 mL with chromatography R (3.5 urn);
heptane R. - temperature: 40°C.
Reference solution (c) Dilute 1.0 mL of reference solution (a) Mobile phase propanol R, solution A (27:73 VIV).
to 100.0 mL with heptane R. Flow rate 1.0 mUmin.
Column: Detection Spectrophotometer at 215 nm.
- size: l = 0.25 m, (0 = 4.6 mm; Injection 10 ul, of test solution (a) and reference
- stationary phase: cellulose derivative of silica gelfor chiral solutions (a), (b) and (c).
separation R (5 !J.IIl).
Run time 2.5 times the retention time of atomoxetine.
Mobilephase Mix 1.5 mL of diethylamine R, 2.0 mL of
trifluoroacetic acid Rand 150.0 mL of 2-propanol R and dilute Identification of impurities Use the chromatogram obtained
to 1000 mL with heptane R. with reference solution (b) to identify the peaks due to

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1-222 Atomoxetine Hydrochloride 2020

impurities E and H; use the chromatogram supplied with

atomoxetine for impurity A identification CRS and the
chromatogram obtained with reference solution (c) to identify
the peak due to impurity A.
Relative retention With reference to atomoxetine (retention
= =
time about 10 min): impurity E about 0.2; C. (3R)-N-methyl-3-(4-methylphenoxy)-3-phenylpropan-1-
impurity H = about 0.3; impurity A = about 0.7. amine,
System suitabzlity Reference solution (b):
- resolution: minimum 5.0 between the peaks due to
impurities E and H.
Calculation of percentage contents:
- for each impurity, use the concentration of atomoxetine
hydrochloride in reference solution (a).
Limits: D. (3R)-N-methyl- 3-(3-methylphenoxy)-3-phenylpropan-1-
- impurity A: maximum 0.3 per cent; amine,
- unspecified impurities: for each impurity, maximum
HO H
0.10 per cent;
- total: maximum 0.5 per cent;
- reporting threshold: 0.05 per cent.
crzC~H
Loss on drying (2.2.32)
Maximum 0.5 per cent, determined on 1.000 g by drying in E. (2S)-2-hydroxy-2-phenylacetic acid (t-mandelic acid),
vacuo at 105°C for 2 h.
Sulfated ash (2.4.14)
Maximum 0.1 per cent, determined on 1.0 g.
ASSAY
Liquid chromatography (2.2.29) as described in the test for
related substances with the following modification.
Injection Test solution (b) and reference solution (d). F. (3S)- 3-(3-fluoro-2-methylphenoxy)-N-methyl-3-
Calculate the percentage content of C 17H 2 2CINO taking into phenylpropan-1-amine,
account the assigned content of atomoxetine
hydrochloride CRS.
IMPURITIES
Specifiedimpurities A, B, D.
Other detectable impurities (the following substances would, if
presentat a sufficient level, be detected by one or otherof the tests
in the monograph. They arelimited by the general acceptance
criterion for other/unspecified impurities and/or by the general G. 3,3'-[(2-methylbenzene-1,3-diyl)bis(oxy)]bis(N-methyl-3-
monograph Substances for pharmaceutical use (2034). It is phenylpropan-l-amine),
therefore not necessary to identify these impurities for
demonstration of compliance. See also 5.10. Control of impurities
in substances for pharmaceutical use) C, E, F, G, H.

H. 3-(methylamino)-1-phenylpropan-1-01.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _...,.-_ PhEur

A. N-methyl-3-phenoxy-3-phenylpropan-l-amine,

B. (3S)-N-methyl-3-(2-methylphenoxy)-3-phenylpropan-l-
amine,

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2020 Atorvastatin Calcium Trihydrate 1-223

Column:
Atorvastatin Calcium Trihydrate =
- size: I 0.25 m, 0 =
4.6 mm;
(Ph. Eur. monograph 2191) - stationary phase: amylose derivative of silica gel for
chromatography R (10 urn).
Mobile phase trifluoroacetic acid R, anhydrous ethanol R,
hexane R (0.1:6:94 VIVIV).
Flow rate 1.0 mlJmin.
,3 H20 Detection Spectrophotometer at 244 om.
Injection 20 IlL.
Run time 1.2 times the retention time of atorvastatin.
F Relative retention With reference to atorvastatin (retention
time = about 44 min): impurity E = about 0.8.
System suitability Reference solution (a):
344423-98-9
- resolution: minimum 2.0 between the peaks due to
Action and use impurity E and atorvastatin.
HMG Co-A reductase inhibitor; lipid-regulating drug. Limit:
- impurity E: not more than the area of the principal peak in
PhEur _--'----, _
the chromatogram obtained with reference solution (b)
DEFINmON (0.3 per cent).
Calcium (3R;5R)a ;'[2-(4-fluorophenyl)-5-(1-methylethyl)-3- Related substances
phenyl-4-(phenylcarbamoyl)-lH-pyrrol-1-yl]-3,5- Liquid chromatography (2.2.29).
dihydroxyheptanoate trihydrate. Test solution (a) Dissolve 40.0 mg of the substance to be
Content examined in dimethylformamide R and dilute to 100.0 mL
97.0 per cent to 102.0 per cent (anhydrous substance). with the same solvent.
CHARACTERS Test solution (b). Dissolve 50 mg of the substance to be
Appearance examined in dimethylformamide R and dilute to 50.0 mL with
White. or almost white powder. the same solvent.
Reference solution (a) Dissolve 40.0 mg of atoroastatin
Solubility
calcium trihydraie CRS in dimethylformamide R and dilute to
Very slightly soluble in water, slightly soluble in ethanol
(96 per cent), practically insoluble in methylene chloride. 100.0 mL with the same solvent.
Reference solution (b) Dilute 1.0 mL of test solution (b) to
It shows polymorphism (5.9).
100.0 mL with dimethylformamide R. Dilute 1.0 mL of this
IDENTIFICATION solution to 10.0 mL with dimethylformamide R.
A. Infrared absorption spectrophotometry (2.2.24). Reference solution (c) Dissolve 2.5 mg of atoruastatin
Comparison atoruastatin calcium trihydrate CRS. impurity A CRS, 2.5 mg of atoroastatin impurity B CRS,
If the spectra obtained in the solid state show differences, 2.5 mg of atoruastatin impurity C CRS, 2.5 mg of atoroastatin
dissolve the substance to be examined and the reference impurity D CRS and 2.5 mg of the substance to be examined
substance separately in methanol R, evaporate to dryness and in dimethylformamide R and dilute to 50.0 mL with the same
record new spectra using the residues. solvent.
B. Enantiomeric purity (see Tests). Column:
C. Water (see Tests). - size: I = 0.25 m, 0 = 4.6 mm;
- stationary phase: octylsilyl silica gelfor chromatography R
D~ Ignite. The residue gives reaction (b) of calcium (2.3.1).
(5 urn);
Filtration may be necessary in case the residue does not
- temperature: 35°C.
completely dissolve.
Mobile phase:
TESTS - mobile phase A: tetrahydrofuran R, acetonitrile R, 3.9 gIL
Enantiornericpurlty solution of ammonium acetate R adjusted to pH 5.0 with
Liquid chromatography (2.2.29). glacial acetic acid R (12:21:67 VIVIV);
Solvent mixture anhydrous ethanol R, methanol R - mobile phase B: tetrahydrofuran R, 3.9 gIL solution of
(50:50 VIV). ammonium acetate R adjusted to pH 5.0 with glacial acetic
Test solution Dissolve 10 mg of the substance to be acid R, acetonitrile R (12:27:61 VIVIV);
examined in 4 mL of the solvent mixture and dilute to
10.0 mL with hexane R. Time Mobile phase A Mobile phase B
(min) (per cent VIV) (per cent VIJI)
Reference solution (a) Dissolve 2 mg of atoroastatin
0-40 100 0
impurity E CRS in methanol R and dilute to 20.0 mL with the
same solvent (solution A). Dissolve 10 mg of the substance
40 - 70 100 ~ 20 o~ 80
70 - 85 20 ~ 0 80 ~ 100
to be examined in 1.25 mL of methanol R, add 0.75 mL of
solution A and 2 mL of anhydrous ethanol R and dilute to
10.0 mL with hexane R. Flow rate 1.5 mUmin.
Reference solution (b) To 2.0 mL of the test solution add Detection Spectrophotometer at 244 om.
40.0 mL of the solvent mixture and dilute to 100;0 mL with Injection 20!lL of test solution (b) and reference
hexane R. To 3.0 mL of this solution add 5 mL of the solutions (b) and (c).
solvent mixture and dilute to 20.0 mL with hexane R.

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1-224 Atorvastatin Calcium Trihydrate 2020

Identification of impurities Use the chromatogram obtained demonstration of compliance. See also 5.10. Control of impurities
with reference solution (c) to identify the peaks due to in substances for pharmaceutical use) F, G, H.
impurities A, B, C and D.
Relative retention With reference to atorvastatin (retention
time = about 33 min): impurity A = about 0.8;
impurity B = about 0.9; impurity C = about 1.2;
impurity D = about 2.1.
If necessary, adjust the mobile phase by increasing or
decreasing the percentage of acetonitrile or the pH of the
ammonium acetate solution to achieve a retention time of
about 33 min for atorvastatin. For example, raising the pH A. (3R,5R)-3,5-dihydroxy-7- [5-(1-methylethyl)-2,3-diphenyl-
would decrease the retention time of atorvastatin. 4-(phenylcarbamoyl)-lH-pyrrol-l-y\]heptanoic acid
System suitability Reference solution (c): (desfluoroatorvastatin) ,
- resolution: minimum 1.5 between the peaks due to
impurity Band atorvastatin.
Limits:
- impurities A, B: for each impurity, not more than 3 times
the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.3 per cent);
- impurities C, D: for each impurity, not more than and enantiomer
1.5 times the area of the principal peak in the
chromatogram obtained with reference solution (b) F
- (0.15 per cent);
- unspecified impurities: for each impurity, not more than the B. (3RS,5SR)-7 -[2-(4-fluorophenyl)-5-(1-methylethyl)-3-
area of the principal peak in the chromatogram obtained phenyl-4-(phenylcarbamoyl)-lH-pyrrol-1-yl] -3,5-
with reference solution (b) (0.10 per cent); dihydroxyheptanoic acid,
- total: not more than 15 times the area of the principal
peak in the chromatogram obtained with reference
solution (b) (1.5 per cent);
-'- disregard limit: 0.5 times the area of the principal peak in
the chromatogram obtained with reference solution (b)
(0.05 per cent); disregard the peak due to
dimethylformamide.
Sodium F F
Maximum 0.4 per cent (anhydrous substance).
Atomic absorption spectrometry (2.2.23, Method 1). C. (3R,5R)-7 -[2,3-bis( 4-fluorophenyl)-5-(l-methylethyl)-4-
Solvent mixture hydrochloric acid R, water R, methanol R (phenylcarbamoyl)-lH-pyrrol-1-yl]-3,5-
(2:25:75 V/V/V). dihydroxyheptanoic acid (fluoroatorvastatin),
Test solution Dissolve 5.0mg in the solvent mixture and
dilute to 100.0 mL with the solvent mixture.
Reference solutions Prepare the reference solutions using
sodium standard solution (50 ppm Na) R, diluting with the
solvent mixture.
F
Source Sodium hollow-cathode lamp.
Wavelength S89.0nm.
Atomisation device Air-acetylene flame. D. 3-[( 4-fluorophenyl) carbonyl]-2-(2-methylpropanoyl)-N,3-
Water (2.5.12) diphenyloxirane-2-carboxamide,
3.5 per cent to 5.5 per cent, determined on 0.130 g.
ASSAY
Liquid chromatography (2.2.29) as described in the test for
related substances with the following modification.
Injection Test solution (a) and reference solution (a).
Calculate the percentage content of C6Jf6sCaF2N401O from
the declared content of atoruastatin calcium trihydrate CRS.
F
IMPURITIES
Specified impurities A, B, C, D, E. E. (3S,5S)-7 -[2-(4-fluorophenyl)-5-(1-methylethyl)-3-phenyl-
Other detectable impurities (the following substances would, if 4-(phenylcarbamoyD-1H-pyrrol-1-yfj-3,5-
present at a sufficientlevel, be detected by one.or other of the tests dihydroxyheptanoic acid (ent-atorvastatin),
in the monograph. They are limited by the generalacceptance
-criterion for other/unspecified impurities and/or by the general
monograph Substances for pharmaceutical use (2034). It is
therefore not necessary to identify these impurities for

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2020 Atovaquone 1-225

HO H H OH
H0 '.., ''': . CHARACTERS
2C
~NH Appearance
3C
oH C~3
H, OH 0
Yellow, crystalline powder.
, -'OH Solubility
~ N Practically insoluble in water, sparingly soluble in methylene
H
chloride, very slightly soluble in methanol.
It shows polymorphism (5.9).
IDENTIFICATION
F Infrared absorption spectrophotometry (2.2.24).
Comparison atovaquone CRS.
F. (3R,5R)-7-[[(3R,5R)-7-[2-(4-fluorophenyl)-5-( 1-
If the spectra obtained show differences, dissolve 0,1 g of the
methylethyl)-3-phenyl-4-(phenylcarbamoyl)-IH-pyrrol-l-
substance to be examined and 0.1 g of the reference
yl]-3,5-dihydroxyheptanoyl]amino]-3,5-
substance separately in 2.5 mL of a 50 gIL solution of
dihydroxyheptanoic acid,
potassium hydroxide R in methanol R. Filter the solutions and
add each filtrate dropwise to a mixture of 0.8 mL of acetic
acid Rand 1.5 mLof methanol R, stirring continuously.
Filter, wash the residues with methanol R and then with
water R, and dry under vacuum at 55°C. Record new
spectra using the, residues.
TESTS
Related substances
F Liquid chromatography (2.2.29). Carry out the test protected
from light.
G. (3R,5R)-7 -[2-(4-fiuorophenyl)-5-( l-methylethylj-Bephenyl-
Solvent mixture water R, acetonitrile R1 (20:80 VIV).
4-(pheny1carbamoyl)-IH-pyrrol-I-yl}-5-hydroxy-3-
methoxyheptanoic acid (3-G-methylatorvastatin), Test solution Dissolve 25.0 mg of the substance to be
examined in the solvent mixture and dilute to 100.0 mL with
o the solvent mixture.
oH C:i)H,
3C
, 0'
Reference solution (a) Dissolve 25.0 mg of atovaquone CRS
in the solvent mixture and dilute to 100.0 mL with the
~ N ,'-'OH
solvent mixture.
H H
Reference solution (b) Dissolve 2.5 mg of atovaquone for
system suitability CRS (containing impurities B and C) in the
solvent mixture and dilute to 10.0 mL with the solvent
mixture.
F
Reference solution (c) Dilute 1.0 mL of the test solution to
H. (4R,6R)-6-[2-[2-(4-fluorophenyl)-5-(I-methylethyl)-3- 100.0 mL with the solvent mixture. Dilute 1.0 mL of this
phenyl-4-(pheny1carbamoyl)-IH-pyrrol-I-yl] ethyl]-4- solution to 10.0 mL with the solvent mixture.
hydroxytetrahydro-2H-pyran-2-one. Column:
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur - size: I = 0.25 m, 0 =4.6 mm;
- stationary phase: end-capped oetadecylsilyl silica gel for
chromatography R (5 '/lm).
Mobile phase phosphoric acid R, methanol R2, water for
Atovaquone *** chromatography R, acetonitrile R1 (0.5:17.5:30:52.5 VIVIVIV).
** ** Flow rate 2.5 mUmin.
(Ph. Bur. monograph 2192) ***** Detection Spectrophotometer at 220 nm.

0 H
9tl
_~ H
Injection 20!1L of the test solution and reference
solutions (b) and (c).

«X
~.
~ I
o
1/
OH
~ I
CI
Run time Twice the retention time of atovaquone.
Identification of impurities Use the chromatogram supplied
with atovaquone for system suitability CRS and the
chromatogram obtained with reference solution (b) to
identify the peaks due to impurities Band C.
366.8 95233-18-4
Relative retention With reference to atovaquone (retention
Action and use =
time about 15 min): impurity B = about 0.85;
Antiprotozoal (malaria). =
impurity C about 0.90.
PhEur _ System suitability Reference solution (b):
- resolution: minimum 2.0 between the peaks due to
DEFINITION impurity C and atovaquone;
2-[ trans-4-(4-Chlorophenyl)cyclohexyl]-3- - peak-to-valley ratio: minimum 1.5, where H p height=
hydroxynaphthalene-I,4-dione. ' above the baseline of the peak due to impurity C and
Content H; = height above the baseline of the lowest point ~f the
97.5 per centto 102.0 per cent (anhydrous substance).

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1-226 Atracurium Besilate 2020

0
curve separating this peak from the peak due to
impurity B.
Calculation of percentage contents:
- for each impurity, use the concentration of atovaquone in
reference solution (c).
c¢ir
~
~
I
o
HY '
' H0

OCH3
~
~
I
CI

Limits: D. 2- [trans-4-(4-chlorophenyl) cyclohexyl]-3-

- impurity B: maximum 0.5 per cent; methoxynaphthalene-1,4-dione.
- impurity C: maximum 0.2 per cent; _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
- unspecified impurities: for each impurity, maximum
0.10 per cent;
- total: maximum 0.6 per cent;
- reporting threshold: 0.05 per cent.
Water (2.5.32)
Atracurium Besilate
Maximum 0.3 per cent, determined on 0.100 g using the (Ph. Bur. monograph 1970)
evaporation technique:
- temperature: 160 DC;
~ heating time: 3 min;
- flow rate: 50 mUmin.
Sulfated ash (2.4.14)
Maximum 0.1 per cent, determined on 1.0 g.
ASSAY
Liquid chromatography (2.2.29) as described in the test for
related substances with the following modification.
Injection Test solution and reference solution (a).
Calculate the percentage content of C22H19CI03 taking into
account the assigned content of atouaquone CRS.
IMPURITIES
Specified impurities B, C.
Other detectable impurities (the following substances would, if 1243 64228-81-5
present at a sufficient level, be detected by one or otherof the tests
in the monograph. They are limited by the general acceptance Action and use
Non-depolarizing neuromuscular blocker.
criterion for other/unspecified impurities and/or by the general
monograph Substances for pharmaceutical use (2034). It is PhEur _
therefore not necessary to identify these impurities for
demonstration of compliance. See also 5.10. Control of impurities DEFINITION
in substances for pharmaceutical use) A, D. Mixture of the cis-cis, cis-trans and trans-trans isomers of
2,2'- [pentane-1,5-diylbis [oxy(3-oxopropane-1,3-diyl) ]]bis [1-
(3,4-dinle~oxybe~D-6,7-dinnethoxy-2-methyl-1,2,3,4­

& ...
OH?0'H tetrahydroisoquinolinium] dibenzenesulfonate.
~

I " I, ' ,I Content

C0 2 H "'- CI 96.0 per cent to 102.0 per cent (anhydrous substance).
PRODUCTION
A. 2-[ trans-4-(4-chlorophenyl) cyclohexyl]-l-oxo-lH-indene- It is considered that alkyl benzenesulfonate esters are
3-carboxylic acid, genotoxic and are potential impurities in atracurium besilate.
The manufacturing process should be developed taking into

«X
0 ' H~H consideration the principles of quality risk management,
together with considerations of the quality of starting
"[ ["'\J""1""')
~ ~CI
materials, process capability and validation, The general
OH
method 2.5.41. Methyl, ethyl and iSOPropyl benzenesulfonate in
o active substances is available to assist manufacturers.

B. 2-[ cis-4-(4-chlorophenyl)cyclohexyl]-3- CHARACTERS

hydroxynaphthalene-1,4-dione, Appearance
White or yellowish-white, slightly hygroscopic powder.
Solubility
CI Soluble in water, very soluble in acetonitrile, in ethanol
(96 per cent) and in methylene chloride.
IDENTIFICATION
o A. Infrared absorption spectrophotometry (2.2.24).
C. 2-[(1 RS)-4-(4-chlorophenyl) cyclohex-3-en-1-yl] -3- Comparison atracurium besilate'CRS.
hydroxynaphthalene-1,4-dione, B. Examine the chromatograms obtained in the assay.
Results The 3 principal isomeric peaks in the chromatogram
obtained with test solution (a) are similar in retention time to

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2020 Atracurium Besilate 1-227

those in the chromatogram obtained with reference for impurity F identification CRS to identify the peak due to
solution (a). impurity F.
TESTS Relative retention With reference to the atracurium cis-cis
Solution S =
isomer (retention time about 30 min):
Dissolve 1.00 g in water R and dilute to 100 mL with the impurity E = about 0.2; impurity F = about 0.25;
same solvent. impurity G = about 0.3; impurity Dl = about 0.45;
impurity D2 = about 0.5; atracurium trans-
Appearance of solution trans isomer = about 0.8; atracurium cis-
Solution S is clear (2.2.1) and not more intensely coloured
trans isomer = about 0.9; impurity Al = about 1.04;
than reference solution Y7 (2.2.2, Method II).
= =
impurity 11 about 1.07; impurity HI about 1.07
Related substances (shoulder on the front of peak A2); impurity A2 (major
Liquid chromatography (2.2.29). isomer) = about 1.08; impurity Kl = about 1.09 (shoulder
Test solution (a) Dissolve 50.0 mg of the substance to be on the tail of peak A2); impurity 12 (major
examined in mobile phase A and dilute to 50.0 mL with isomer) = about 1.12; impurity H2 (major isomer) = about
mobile phase A. =
1.12; impurity K2 (major isomer) about 1.12;
Test solution (b) Dissolve 0.100 g of the substance to be impurity B = about 1.15; impurity Cl = about 1.2;
examined in mobile phase A and dilute to 10.0 mL with impurity C2 (major isomer) = about 1.3.
mobile phase A; System suitability:
Reference solution (a) Dissolve 50.0 mg of atracurium - resolution: minimum 1.5 between the peaks due to the
besilate CRS in mobile phase A and dilute to 50.0 mL with atracurium trans-trans isomer and the atracurium cis-trans
mobile phase A. isomer, and minimum 1.5 between the peaks due to the
atracurium cis-trans isomer and the atracurium cis-cis
Reference solution (b) Dilute 1.0 mL of test solution (a) to
isomer in the chromatogram obtained with reference
100.0 mL with mobile phase A.
solution (a);
Reference solution (c) Dissolve 20.0 mg of methyl - peak-to-valley ratio: minimum 1.2, where Hp = height
benzenesuljonate R in acetonitrile R and dilute to 100.0 mL above the baseline of the peak due to impurity Al and
with the same solvent. Dilute 50 JlL of the solution to H; = height above the baseline of the lowest point of the
100.0 mL with mobile phase A. curve separating this peak from the peak due to the
Reference solution (d) Dissolve 2.0 mg of atracuriumfor peak atracurium cis-cis isomer in the chromatogram obtained
identification CRS (containing impurities AI, A2, B, Cl, C2, with reference solution (d).
Dl, D2, E, G and K) in 2.0 mL of mobile phase A. Limits:
Reference solution (e) Dissolve 2.0 mg of atracurium for ~ correction factor. for the calculation of content, multiply the
impurity F identification CRS in 2.0 mL of mobile phase A. peak area of impurity G by 0.5;
Column: - impurity E: not more than 1.5 times the sum ofthe areas
=
- size: 1= 0.25 m, 0 4.6 mrn; of the peaks due to the atracurium cis-cis, trans-trans and
- stationaryphase: base-deactivated end-cappedoctadecylsilyl cis-trans isomers in the chromatogram obtained with
silica gelfor chromatography R (5 urn). reference solution (b) (1.5 per cent);
Mobile phase: - impurities A, D: for each impurity, for the sum of the areas
- mobile phase A: mix 5 volumes of methanol R, 20 volumes of the 2 isomer peaks, not more than 1.5 times the sum
of acetonitrile Rand 75 volumes of a 10.2 gIL solution of of the areas of the peaks due to the atracurium cis-cis,
potassium dihydrogen phosphate R previously adjusted to trans-trans and cis-trans isomers in the chromatogram
pH 3.1 with phosphoric acid R; obtained with reference solution (b) (1.5 per cent);
- mobile phase B: mix 20 volumes of acetonitrile R, - impurity C: for the sum of the areas of the 2 isomer peaks,
30 volumes of methanol Rand 50 volumes of a 10.2 giL not more than the sum of the areas of the peaks due to
solution of potassium dihydrogen phosphate R previously the atracurium cis-cis, trans-trans and cis-trans isomers in
adjusted to pH 3.1 with phosphoric acid R; the chromatogram obtained with reference solution (b)
(1.0 per cent);
Time Mobile phase A Mobile phase B - impurities F, G: for each impurity, not more than the sum
(min) (per cent VIV) (per cent VIV) of the areas of the peaks due to the atracurium cis-cis,
0-5 80 20 trans-trans and cis-trans isomers in the chromatogram
5 - 15 80 -+ 40 20 -+ 60 obtained with reference solution (b) (1.0 per cent);
15 - 25 40 60 - impurities H, I, K: for the sum of the areas of the isomer
25 - 30 40 -+ 0 60 -+ 100 peaks of these impurities, not more than the sum of the
30 - 45 0 100 areas of the peaks due to the atracurium cis-cis, trans-trans
and cis-trans isomers in the chromatogram obtained with
reference solution (b) (1.0 per cent);
Flow rate 1 mlJrnin.
- unspecified impurities: for each impurity, not more than
Detection Spectrophotometer at 280 nm. 0.1 times the sum of the areas of the peaks due to the
Injection 20 IlL of test solution (a) and reference atracurium cis-cis, trans-trans and cis-trans isomers in the
solutions (a), (b), (d) and (e). chromatogram obtained with reference solution (b)
Identification of impurities Use the chromatogram obtained (0.10 per cent);
with reference solution (d) and the chromatogram supplied - total: not more than 3.5 times the sum of the areas of the
with atracurium for peak identification CRS to identify the peaks due to the atracurium cis-cis, trans-trans and cis-trans
peaks due to impurities AI, A2, B, Cl, C2, Dl, D2, E, G isomers in the chromatogram obtained with reference
and K; use the chromatogram obtained with reference solution (b) (3.5 per cent);
solution (e) and the chromatogram supplied with atracurium

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1-228 Atracurium Besilate 2020

- disregard limit: 0.05 times the sum of the areas of the

peaks due to the atracurium cis-cis, trans-trans and cis-trans
isomers in the chromatogram obtained with reference
solution (b) (0.05 per cent).
Impurity J
Liquid chromatography (2.2.29) as described in the test for
related substances with the following modifications.
Mobile phase:

Time Mobile phase A Mobile phase B

(min) (per cent VIV) (per cent VIV)
0-5 80 20
5 - 15 80 -> 75 20 -> 25
15 - 25 75 25
25 - 30 75 -> 55 25 -> 45 A. 1-(3,4-dimethoxybenzyl)-2-[13-[1-(3,4-dimethoxybenzyl)-
30 - 38 55 -> 0 45 -> 100 6,7-dimethoxy-3,+·dihydroisoquinolin-2(1H)-yl]-3,11-
38 - 45 0 100 dioxo-4, 1O-dioxatridecyl] -6,7-dimethoxy-2-methyl-1,2,3,4-
tetrahydroisoquinolinium (AI = trans isomer, A2 =cis
Detection Spectrophotometer at 217 nm. isomer),
Injection 100 ul, of test solution (b) and reference
solution (c).
Retention time Impurity J = about 25 min; atracurium trans-
=
trans isomer about 38 min.
Limit:
- impurity J: not more than the area of the principal peak in
the chromatogram obtained with reference solution (c)
(10 ppm).
Isomer composition
Liquid chromatography (2.2.29) as described in the test for
related substances with the following modification. Use the
normalisation procedure.
Injection Test solution (a).
Limits:
- atracurium cis-Cis isomer: 55.0 per cent to 60.0 per cent, B. pentane-I,5-diyl bis[3-[1-(3,4-dimethoxybenzyl)-6,7-
- atracurium cis-trans isomer: 34.5 per cent to 38.5 per cent, dimethoxy-3,4-dihydroisoquinolin-2(11f)-yl]propanoate],
- atracurium trans-trans isomer: 5.0 per cent to 6.5 per cent.
Water (2.5.12)
Maximum 5.0 per cent, determined on 1.000 g.
Sulfated ash (2.4.14)
Maximum 0.1 per cent, determined on 1.0 g.
ASSAY
Liquid chromatography (2.2.29) as described in the test for
related substances with the following modification.
Injection Test solution (a) and reference solution (a).
Calculate the percentage content of C6sHszNzOlSSZ from
the sum of the .areas of the peaks due to the 3 isomers. C. 1-(3,4-dimethoxybenzyl)-2-(3, I1-dioxo-4, 1O-dioxatridec-
12-enyl)-6,7-dimethoxy-2-methyl-l,2,3,4-
STORAGE
tetrahydroisoquinolinium (C1 = trans isomer, C2 = cis
In an airtight container, protected from .light, at a
isomer),
temperature of 2 °C to 8°C.
IMPURITIES
Specifiedimpurities A, C, D, E, F, G, H, I, J, K.
Other detectable impurities (the following substances would, if
presentat a sufficient level, be detected by one or otherof the tests
in the monograph. They are limited by the general acceptance
criterion for other/unspecified impurities and/orby the general
monograph Substances for pharmaceutical use (2034). It is
therefore not necessary to identify these impurities for
demonstration of compliance. See also 5.10. Control of impurities D. 1-(3,4-dimethoxybenzyl)-2-[3-[(5-hydroxypentyl)oxy]-3-
in substances for pharmaceutical use) B. oxopropyl]-6,7-dimethoxy-2-methyl-1,2,3,4-
tetrahydroisoquinolinium (D 1 = trans isomer, D2 = cis
isomer),

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2020 Atropine 1-229

o\\ II0
as'OCHO

J, methyl benzenesulfonate,

E. 2-(2-carboxyethyl)-1-(3,4-dimethoxybenzyl)-6,7-
dimethoxy-2-methyl-l,2,3,4-tetrahydroisoquinolinium,

H3CO~
I
H3CO #
H,CO _ . .,CH3
v I * N-CH 3

H3CO ~

F. 1-(3,4-dimethoxybenzyl)-6,7-dimethoxy-2,2-dimethyl-
1,2,3,4-tetrahydroisoquinolinium,

H3CO~""'::::
I
H3CO # K. 2,2'- [hexane-I,5-diylbis[oxy(3-oxopropane-I,3-diyl)]]bis
H,CO P" ... . CH3 [1-(3,4-dimethoxybenzyl)-6,7-dinnethoxy-2-methyl-
I N I,2,3,4-tetrahydroisoquinolinium].
H3CO ~ _ _ _ _ _ _ _ _ _ _--'- PhEur

Ci. 1-(3,4-dimethoxybenzyD-6,7-dimethoxy-2-methyl-I,2,3,4-
tetrahydrois 0 quinoline,
Atropine
(Ph. Bur. monograph 2056)

~.
~.# 0H>Q-.-------]
0" N-CH3 and enantiomer
\ • ---_.----
H ..
OH H

289.4 51-55-8

Action and use

Anticholinergic.
PhEur ~ _
H. 2,2'-[hexane-l ,6-diylbis[oxy(3-oxopropane-I,3-diyl)]]bis
[1~(3,4-dimethoxybenzyl)-6,7-dinlethoxy-2-methyl­ DEFINITION
1,2,3,4-tetrahydroisoquinolinium] (HI = cis-trans isomer, (IR,3R,5S)-8-Methyl-8-azabicyclo[3.2.I]oet-3-yl (2RS)-3-
=
H2 cis-cis isomer), hydroxy-2-phenylpropanoate.
Content
99.0 per cent to 101.0 per cent (dried substance).
CHARACTERS
Appearance
White or almost white, crystalline powder or colourless
crystals.
Solubility
Very slightly soluble in water, freely soluble in ethanol
(96 per cent) and in methylene chloride.
IDENTIFICATION
First identification: A, B, E.
Second identification: A, C, D, B.
A. Melting point (2.2.14): 115 °C to 119 °C.
1. 2,2'- [(3-methylpentane-1 ,5-diyl)bis[oxy(3-oxopropane- B. Infrared absorption spectrophotometry (2.2.24).
1,3-diyl)]]bis [1-(3,4-dimethoxybenzyl)-6,7-dimethoxy-2- Comparison atropine CRS.
=
methyl-l,2,3,4-tetrahydroisoquinolinium] (II cis-trans
C. Thin-layer chromatography (2.2.27).
isomer, 12 = cis-cis isomer),

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1-230 Atropine 2020

Test solution Dissolve 10 mg of the substance to be Time Mobile phase A Mobile phase B
(min) (per cent VIP) (per cent vlli')
examined in methanol R and dilute to 10 mL with the same
solvent. 0-2 95 5
2 - 20 95 --+ 70 5 --+ 30
Reference solution Dissolve 10 mg of atropine CRS in
methanol R and dilute to 10 mL with the same solvent.
Plate TLC sil~ca gelplate R. Flow rate 1 mUrnin.
Mobile phase concentrated ammonia R, .water R, acetone R Detection Spectrophotometer at 210 nm.
(3:7:90 V/V/V). Injection 10 ~.
Application 10 ~. Identification of impurities Use the chromatogram supplied
Development Over half of the plate. with atropine for peak identification CRS and the
chromatogram obtained with reference solution (c) to identify
Drying At 100-105. °C for 15 min.
the peaks due to impurities A, D, E, F, G and H; use the
Detection After cooling, spray with dilutepotassium chromatogram obtained with reference solution (b) to
iodobismuthate solution R. identify the peak due to impurity B; use the chromatogram
Results The principal spot in the chromatogram obtained obtained with reference solution (d) to identify the peak due
with the test solution is similar in position, colour and size to to impurity C.
the principal spot in the chromatogram obtained with the Relatioe retention With reference to atropine (retention
reference solution. time = about 11 min): impurity C = about 0.2;
D. Place about 3 mg in a porcelain crucible and add 0.2 mL impurity E = about 0.67; impurity D = about 0.73;
of fuming nitric acid R. Evaporate to dryness on a water-bath. impurity F = about 0.8; impurity B = about 0.89;
Dissolve the residue in 0.5 mL of a 30 gIL solution of =
impurity H about 0.93; impurity G about 1.1;=
potassium hydroxide R in methanol R; a violet. colour develops. impurity A = about 1.7.
E. Optical rotation (see Tests). System suitability Reference solution (b):
TESTS - resolution: minimum 2.5 between the peaks due to
impurity B and atropine.
Optical rotation (2.2. 7)
-0.70° to + 0.05° (measured in a 2 dm tube). Limits:
- correction factors: for the calculation of content, multiply
Dissolve 1.25 g in ethanol (96 per cent) R and dilute to
the peak areas of the following impurities by the
25.0 mL with the same solvent.
corresponding correction factor: impurity A 0.6; =
Related substances impurity C = 0.6;
Liquid chromatography (2.2.29). - impurities E~ H: for each impurity, not more than 3 times
Test solution Dissolve 24 mg of the substance to be the area of the principal peak in the chromatogram
examined in mobile phase A and dilute to 100.0 mL with obtained with reference solution (a) (0.3 per cent);
mobile phase A. - impurities A~ B~ C~ D~ F~ G: for each impurity, not more
Reference solution (a) Dilute 1.0 mL of the test solution to than twice the area of the principal peak in the
100.0 mL with mobile phase A. Dilute 1.0 mL of this chromatogram obtained with reference solution (a)
solution to 10.0 mL with mobile phase A. (0.2 per cent);
Reference solution (b) Dissolve 5 mg of atropine - unspecified impurities: for each impurity, not more than the
impurity B CRS in the test solution and dilute to 20.0 mL area of the principal peak in the chromatogram obtained
with the test solution. Dilute 5.0 mL of this solution to with reference solution (a) (0.10 per cent);
25.0 mL with mobile phase A. - total: not more than 5 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
Reference solution (c) Dissolve the contents of a vial of
(0.5 per cent);
atropine for peak identification CRS (containing impurities A,
- disregard limit: 0.5 times the area of the principal peak in
D, E, F, G and H) in 1.0 mL of mobile phase A.
the chromatogram obtained with reference solution (a)
Reference solution (d) Dissolve 5 mg of tropic acid R . (0.05 per cent).
(impurity C) in mobile phase A and dilute to 10.0 mL with
Loss on drying (2.2.32)
mobile phase A. Dilute 1.0 mL of the solution to 100.0 mL
Maximum 0.2 per cent, determined on 1.000 g by drying in
with mobile phase A. Dilute 1.0 mL of this solution to
an oven at 105 DC for 2 h.
10.0 mL with mobile phase A.
Column: ASSAY
- size: [.:= 0.10 m, (2) = 4.6 mm; Dissolve 0.250 g in 40 mL of anhydrous acetic acid R, heating
- stationaryphase: end-capped octadecylsilyl silica gelfor if necessary, and allow to cool. Titrate with 0.1 M perchloric
chromatography with embedded polargroups R (3 urn). acid, determining the end-point potentiometrically (2.2.20).
Mobile phase: 1 mL of 0.1 M perchloric acid is equivalent to 28.94 mg
- mobile phase A: dissolve 3.5 g of sodium dodecyl sulfate R in of C17H23N03..
606 II'..L of a 7.0 gIL solution of potassium dihydrogen STORAGE
phosphate R previously adjusted to pH 3.3 with a 5.8 gIL Protected from light.
solution of phosphoric acid R, and mix with 320 mL of
acetonitrile Rl; IMPURITIES
- mobile phase B: acetonitrile Rl; Specified impurities A~ B~ C~ D~ E~ F~ G~ H.

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2020 Atropine Sulfate 1-231

Atropine Sulfate
Atropine Sulphate
(ph. Bur. monograph 0068)

~~>q~:-:C~,]~
A. (lR,3r,5S)-8-methyl-8-azabicyclo[3.2.1]oct-3-yl
2-phenylpropenoate .(apoatropine),

H
I ~ 0 .---------

H ,H, S0H,O
4,

()
~ (:n----]
.1'o~-r
OH
H

H
and enantlomer
OH

695
2 and enantiomer

5908-99-6

B. (lR,3r,5S)-8-azabicyclo[3.2.1]oct-3-yl (2RS)-3-hydroxy-2-~
Action and use
phenylpropanoate (noratropine), Anticholinergic.
Preparations

~
Atropine Eye Drops
I.... CO H Atropine Eye Ointment
.# '. z and enantiomer
. ". H Atropine Injection
OH Atropine Tablets
PhEur _
C! (2RS)-3-hydroxy-2-phenylpropanoic acid (tropic acid),
DEFINITION
H OH Bis[(lR,3r,5S)-8-methyl-8-azabicydo[3.2.1]oct-3-yl (2RS)-3-
~.
" ".
~O""\.
~ H...r'c::t.
0
.: H and eplmer at C"
P hydroxy-2-phenylpropanoate] sulfate monohydrate.
Content
. \; ---H 99.0 per cent to 101.0 per cent (anhydrous substance).
CHARACTERS
Appearance
D. (lR,3S,5R, 6RS)-6~hydroxy-8-methyl-8-azabicyclo
[3.2.1]
White or almost white, crystalline powder or colourless
oct-3-yl (2S)-3-hydroxy-2-phenylpropanoate
crystals.
(6-hydroxyhyoscyamine),
Solubility
H Very soluble in water, freely soluble in ethanol (96 per cent).
~ .~HJ---b
~O'~"'H--H
IDENflFICATION
and eplmer at C" First identification: A, B, E.
Secondidentification: C, D, E, F.
\; H OH
A. Optical rotation (see Tests).
B. Infrared absorption spectrophotometry (2.2.24).
E. (1S,3R,5S, 6RS)-6-hydroxy-8-methyl-8-azabicydo[3.2.1]
oct-3-yl (2S)-3-hydroxy-2-phenylpropanoate Comparison atropine sulfate CRS.
(7-hydroxyhyoscyamine), C. Dissolve about 50mg inS mL of waterR and add 5 mL
of picric acid solution R. The precipitate, washed with waterR
and dried at 100-105 0 C for 2 h, melts (2.2.14) at 174 0 C to
~ ~ rH~.H .. 179°C.
~:.. \ . N-CH3 , 0 D. To about 1 mg add 0.2 mL ofjuming nitric acid Rand
\ H '
evaporate to dryness in a water-bath. Dissolve the residue in
OH H H 2 mL of acetone R and add 0.1 mL of a 30 gIL solution of
potassium hydroxide R in methanol R. A violet colour develops.
F. (lR,2R,4S,5S,7s)-9-methyl-3-oxa-9-azatricyclo [3.3.1.02 ,'1 E. It gives the reactions of sulfates (2.3.1).
non-7 -yl (2S)- 3-hydroxy-2-phenylpropanoate (hyoscine), F. It gives the reaction of alkaloids (2.3.1).
TESTS
pH (2.2.3)
4.5 to 6.2.
Dissolve 0.6 g in carbon dioxide-free tuater R and dilute to
30 mL with the same solvent.
Optical rotation (2.2.7)
G. (lR,3r,5S)-8-methyl-8-azabicyclo[3.2.1]oct-3-yl (2RS)-2-
-0.50° to + 0.05 0 (measured in a 2 dm tube).
hydroxy-3-phenylpropanoate (littorine),
Dissolve 2.50 g in water R and dilute to 25.0 mL with the
H. unknown structure.
same solvent.
_ _ _ _ _ _ _ _ _-'-- PhEur
Related substances
Liquid chromatography (2.2.29).

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1-232 Atropine Sulfate 2020

Test solution Dissolve 24 mg of the substance to be -'impurities A, B, C, D, F, G: for each impurity, not more
examined in mobile phase A and dilute to 100.0 mL with than twice the area of the principal peak in the
mobile phase A. chromatogram obtained with reference solution (a)
Reference solution (aJ Dilute 1.0 mL of the test solution to (0.2 per cent);
100.0 mL with mobile phase A. Dilute 1.0 mL ofthis - unspecified impurities: for each impurity, not more than the
solution to 10.0 mL with mobile phase A. area of the principal peak in the chromatogram obtained
Reference solution (b) Dissolve 5 rng of atropine with reference solution (a) (0.10 per cent);
impurity B CRS in the test solution and dilute to 20 mL with - total: not more than 5 times the area of the principal peak
the test solution. Dilute 5 mL of this solution to 25 mL with in the chromatogram obtained with reference solution (a)
mobile phase A. (0.5 per cent);
- disregard limit: 0.5 times the area of the principal peak in
Reference solution (c) Dissolve the contents of a vial of the chromatogram obtained with reference solution (a)
atropine for peak identification CRS (containing impurities A, (0.05 per cent).
D, E, F, G and II) in 1 mL of mobile phase A.
Water (2.5.12)
Reference solution (d) Dissolve 5 mg of tropic acidR
2.0 per cent to 4.0 per cent, determined on 0.500 g.
(impurity C) in mobile phase A and dilute to 10 mL with
mobile phase A. Dilute 1 mL of the solution to 100 mL with Sulfated ash (2.4.14)
mobile phase A. Dilute 1 mL of this solution to 10 mL with Maximum 0.1 per cent, determined on 1.0 g.
mobile phase A. ASSAY
Column: Dissolve 0.500 gin 30 mL of anhydrous acetic acidR,
= =
- size: l 0.10 m, 0 4.6 mm; warming if necessary. Cool the solution. Titrate with 0.1 M
- stationary phase: end-capped octadecylsilyl silica gelfor perchloric acid, determining the end-point potentiometrically
chromatography with embedded polargroups R (3 urn). (2.4.20).
Mobile phase: 1 mL of 0.1 M perchloric acid is equivalent to 67.68 mg
- mobile phase A: dissolve 3.5 g of sodium dodecyl sulfate R in of C34H4SNzOlOS,
606 mL of a 7.0 gIL solution of potassium dihydrogen
STORAGE
phosphate R previously adjusted to pH 3.3 with a 5.8 gIL
Protected from light.
solution of phosphoric acid R, and mix with 320 mL of
acetonitrile Rl; IMPURITIES
- mobile phase B: acetonitrile Rl; Specified impurities A, B, C, D, E, F, G, ·H.

TiIne Mobile phase A Mobile phase B H

~ 0H>G·········]
Cl;
(min) (per cent VIJI) (per cent VIJI)
0-2 95 5 A • N-CH 3
b" O. . ...•.•..
2 - 20 95 -> 70 5 -> 30
CHz H

Flow rate 1 mIJmin.

A. (IR,3r,5S)-8-methyl-8-azabicyclo[3.2.1]oct-3-yl
Detection Spectrophotometer at 210 nm. 2-phenylpropenoate (apoatropine),
Injection 10 JlL.

() IH~""']
Identification of impurities Use the chromatogram supplied
with atropine for peak identification CRS and the
chromatogram obtained with reference solution (c) to identify ·O··"--fN~.
. --./ (H and enantiomer
the peaks due to impurities A, D, E, F, G and H. Use the
chromatogram obtained with-reference solution (b) to OH H
identify the peak due to impurity B, and use the
chromatogram obtained with reference solution (d) to B. (IR,3r,5S)-8-azabicyc10[3.2.1]oct':'3-yl (2RS)-3-hydroxy-2-
identify the peak due to impurity C. phenylpropanoate (noratropine),
Relative retention With reference to atropine (retention

~
time = about 11 min): impurity C = about 0.2;
= =
impurity E about 0.67; impurity D about 0.73; I. # ·CO H
\H 2 and enantiorner
impurity F = about 0.8; impurity B= about 0.89;
impurity H = about 0.93; impurity G = about 1.1;
OH
=
impurity A about 1.7.
System suitabzlity Reference solution (b): C. (2RS)-3-hydroxy-2-phenylpropanoic acid (tropic acid),
- resolution: minimum 2.5 between the peaks due to
impurity B and atropine.

WI ~ .....1'--H~?H
. H
Limits: 0
- correction factors: for the calculation of content, multiply #,' : ...."\... N-CH; andepimeratC*
the peak areas of the following impurities by the
corresponding correction factor: impurity A = 0.6; \: H
impurity C = 0.6;
- impurities E, H: for each impurity, not more than 3 times D. (IR,3S,5R,6RS)-6-hydroxy-8-methyl-8-azabicyc10[3.2.1]
the area of the principal peak in the chromatogram oct-3-yl (2S)- 3-hydroxy-2-phenylpropanoate
obtained with reference solution (a) (0.3 per cent); (6-hydroxyhyoscyamine),

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2020 Attapulgite 1-233

H
TESTS
~ ~HJ---b Acidity or alkalinity
~o.···\._--H-'--H and epimer at C· pH of a 5%w/v suspension in carbon dioxide-free water, after

\~
shaking for 5 minutes, 7.0 to 9.5, Appendix V L.
H OH
Adsorptive capacity
Moisture adsorption, 5 to 14% when determined by the
E. (lS,3R,5S,6RS)-6-hydroxy-8-methyl-8-azabicyclo[3.2.1] following method. Dry in air and powder a sufficient quantity
oct-3-yl (2S)- 3-hydroxy-2-phenylpropanoate of the substance being examined and pass through a sieve
(7-hydroxyhyoscyamine), with a nominal mesh aperture of 150 um. Spread 0.5 g as a
thin layer on a previously weighed piece of aluminium foil

~ ~ "rb
(60 mm x 50 mm) of nominal gauge 17.5 urn and transfer
to a desiccator containing a dish of sodium chloride crystals
~~"\ ~O partially immersed in saturated brine at 25°. After 4 hours,
remove from the desiccator and weigh immediately. Dry in
\ H H
OH an oven at 110° for 4 hours, allow to cool in a desiccator and
weigh. The moisture adsorption is the gain in weight of the
F. (lR,2R,4S,5S, 7s)-9-methyl-3-oxa-9-azatricyclo [3.3.1.02,4] substance being examined expressed asa percentage of its
non-7-yl (2S)-3-hydroxy-2-phenylpropanoate (hyoscine), oven-dried weight.
H _ Arsenic
To 0.13 g add 5mL of water, 2 mL of sulfuric acid and

'.".~.".'.
U ~r-~~-~~;]
""-t-------
0
H#'ClH
and enantiomer
.
10 mL of sulfur dioxide solution and evaporate on a water bath
until the sulfur dioxide solution is removed and the volume
reduced to about 2 mL. Transfer the solution to the
generator flask with the aid of 5 mL of water. The resulting
G. (lR,3r,5S)-8-methyl-8-azabicyclo[3.2.1] oct-3-yl (2RS)-2- solution complies with the limit testfor arsenic, Appendix VII
hydroxy-3-phenylpropanoate (littorine). (8 ppm).
H. unknown structure. Acid-soluble matter
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur Boil 2 g with 100 mL of O.2M hydrochloric acid under a reflux
condenser for 5 minutes, cool and filter. Evaporate 50 mL of
the filtrate to dryness. The residue, after ignition at about
600° for 30 minutes, weighs not more than 0.25 g.
Water-soluble matter
Attapulgite Boil 109 with 100 mL of water under a reflux condenser for
Action and use 5 minutes, cool and filter. Evaporate 50 mL of the filtrate to
Excipient. dryness. The residue, after ignition at 600° for 30 minutes,
weighs not more than 50 mg.
DEFINITION
Loss on drying
Attapulgite is a purified native hydrated magnesium
When- dried to constant weight at 105°, loses not more than
aluminium silicate essentially consisting of the clay mineral
17.0% of its weight. Use 1 g.
palygorskite.
Loss on ignition
CHARACTERISTICS When ignited at 600°, loses 15.0 to 27.0% of its weight.
A light, cream or buff, very fine powder, free or almost free Use 1 g.
from gritty particles.
IDENTIFICATION
A. Ignite 0.5 g with 2 g of anhydrous sodium carbonate for
20 minutes, cool and extract with 25 mL of boiling water. Activated Attapulgite
Cool, filter, wash the residue with water and add the
Action and use
washings to the filtrate. Reserve the residue for test B.
Antidiarrhoeal.
Cautiously acidify the combined filtrate and washings with
hydrochloric acid, evaporate to dryness, moisten the residue DEFINITION
with 0.2 mL of hydrochloric acid, add 10 mL of water and stir. Activated Attapulgite is a purified native hydrated magnesium
A white, gelatinous precipitate is produced. aluminium silicate essentially consisting of the clay mineral
B. Wash the residue reserved in test A with water and palygorskite that has been carefully heated to increase its
dissolve in 10 mL of 2M hydrochloric acid. To 2 mL of the adsorptive capacity.
solution add a 10% w/v solution of ammonium thiocyanate.
An intense red' colour is produced.
CHARACTERISTICS
A light, cream or buff, very fine powder, free or almost free
C.To 2 mL of the solution obtained in test B add 1 mL of from gritty particles.
strongsodium hydroxide solution and filter. To the filtrate add
3 mL of ammonium chloride solution. A gelatinous white IDENTIFICATION
precipitate is produced. A. Ignite 0.5 g with 2 g of anhydrous sodium carbonate for
D. To 2 mL of the solution obtained in test B add 20 minutes; cool and extract with 25 mL of boiling water.
ammonium chloride and an excess of 13.5M ammonia and Cool, filter, wash the residue with water and add the
filter. To the filtrate add 0.15 mL of magneson reagent and an washings to the filtrate. Reserve the residue for test B.
excess of 5M sodium hydroxide. A blue precipitate is produced. Cautiously acidify the combined filtrate and washings with

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1-234 Azathioprine 2020

hydrochloric acid, evaporate to dryness, moisten the residue

with 0.2 mL of hydrochloric acid, add 10 mL of water and stir.
Azathioprine
A white, gelatinous precipitate is produced. (ph. Bur. monograph 0369)
B. Wash the residue reserved in test A with water and
dissolve in 10 mL of 2M hydrochloric acid. To 2 mL of the
solution add a 10% w/v solution of ammonium thiocyanate.
An intense red colour is produced.
C. To 2 mL of the solution obtained in test B add 1 mL of
strongsodium hydroxide solution and filter. To the filtrate add
3 mL of ammonium chloride solution. A gelatinous white
precipitate is produced.
D. To 2 mL of the solution obtained in test B add
277.3 446-86-6
ammonium chloride and an excess of 13.5M ammonia and
filter. To the filtrate add 0.15 mL of magneson reagent and an Action and use
excess of 5M sodium hydroxide. A blue precipitate is produced. Immunosuppressant.
TESTS Preparations
Acidity or alkalinity Azathioprine Tablets
pH of a 5% w/v suspension in carbon dioxide-free water, after Azathioprine Oral Suspension
shaking for 5 minutes, 7.0 to 9.5, Appendix V L.
PhEur - - - - - - - - - -C.-_ _
Arsenic
To 0.13 g add 5 mL of water, 2 mL of sulfuric acid and DEFINITION
10 mL of sulfur dioxide solution and evaporate on a water bath 6-[ (l-Methyl-4-nitro-1H-imidazol-5-yl)sulfanyl]-7H-purine.
until the sulfur dioxide solution is removed and the volume
Content
reduced to about 2 mL. Transfer the solution to the
98.5 per cent to 101.0 per cent (dried substance).
generator flask with the aid of 5 mL of water. The resulting
solution complies with the limit testfor arsenic, Appendix VII CHARACTERS
(8 ppm). Appearance
Acid-soluble matter Pale-yellow powder.
Boil 2 g with 100 mL of 0.2M hydrochloric acid under a reflux Solubility
condenser for 5 minutes, cool and filter. Evaporate 50 mL of Practically insoluble in water and in ethanol (96 per cent).
the filtrate to dryness. The residue, after ignition at about It is soluble in dilute solutions of alkali hydroxides and
600° for 30 minutes, weighs not more than 0.25 g. sparingly soluble in dilute mineral acids.
Water-soluble matter IDENI1FICATION
Boil 10 g with 100 mL of water under a reflux condenser for Infrared absorption spectrophotometry (2.2.24).
5 minutes, cool and filter. Evaporate 50 ml, of the filtrate to Comparison azathioprine CRS.
dryness. The residue, after ignition at 600° for 30 minutes,
weighs not more than 50 mg. TESTS
Related substances
Adsorptive capacity
Liquid chromatography (2.2.29).
In a stoppered bottle shake 1.0 g, in very fine powder, with
50 mL of a 0.12% w/v solution of methylene blue for SolutionA 2.76 gIL solution of sodium dihydrogen phosphate
5 minutes, allow to settle and centrifuge. The colour of the monohydrate R adjusted to pH 2.5 with phosphoric acid R.
clear supernatant solution is not more intense than that of a Test solution Dissolve 10 mg of the substance to be
0.0012% w/v solution of methylene blue. examined in 35 mL of a 0.8 gIL solution of sodium
Loss on drying hydroxide R and dilute to 100.0 mLwith solution A.
When dried to constant weight at 105°, loses not more than Reference solution (a) Dissolve 5 mg of azathioprine
4.0% of its weight. Use 1 g. impurity A CRS and 5 mg of mercaptopurine R (impurity B) in
8.75 mL of a 0.8 gIL solution of sodium hydroxide Rand
Loss on ignition
dilute to 25.0 mL with solution A. To 1.0 mL of this
When ignited at 600°, loses not more than 9.0% of its
solution, add 35 mL of a 0.8 gIL solution of sodium
weight. Use 1 g.
hydroxide R and dilute to 100.0 mL with solution A.
Reference solution (b) Dissolve 2.5 mg of azathioprine
impurity G CRS and 2.5 mg of the substance to be examined
in 8.8 mL of a 0.8 gIL solution of sodium hydroxide Rand
dilute to 25.0 mL with solution A. To 1.0 mL of this
solution, add 17.5 mL of a 0.8 gIL solution of sodium
hydroxide Rand dilute to 50.0 mL with solution A.
Reference solution (c) Dilute 1.0 mL of the test solution to
100.0 mL with solution A. Dilute 1.0 mL of this solution to
10.0 mL with solution A.
Column:
"'-'- size: l =.0.15 m, (2) = 4.6 mm;
- stationary phase: phenylsilyl silica gelfor chromatography R
(5 J.l.111);
- temperature: 30 "C.

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2020 Azathioprine 1-235

Mobzle phase:
- mobile phase A: methanol R, solution. A (5:95 UlV);
- mobile phase B: solution A, methanolR (40:60 V/V);

TiIne Mobile phase A Mobile phase B

(min) (per cent V/JI) (per cent VIV)
A. 1-methyl-4-nitro-IH-imidazol-5-amine,
0-5 100 0
5 - 15 100 -+ 0 o -+ 100 SH

~I )
15 - 20 0 100 H
N:7 . N

Flow rate 1.0 mIJmin.

l N N
Detection Spectrophotometer at 240 om.
Injection 20 ilL. B. 7H-purine-6-thiol (mercaptopurine),
Identification of impurities Use the chromatogram obtained
with reference solution (a) to identify the peaks due to
impurities A and B. Use the chromatogram obtained with
reference solution (b) to identify the peak due to impurity G.
Relatiue retention With reference to azathioprine (retention
time = about IS min): impurity A = about 0.3;
impurity B= about 0.4; impurity G = about 0.97. C. 5-cWoro-1-methyl-4-nitro-1H-imidazole,
System su£tcibz1£ty:
- resolution: minimum 2.0 between the peaks due to
impurities A and B in the chromatogram obtained with
reference solution (a); minimum 2.0 between the peaks
due to impurity G and azathioprine in the chromatogram
obtained with reference solution (b).
Limits: D. 1-methyl-4-nitro-IH-imidazole-;-thiol,
- impurities A, B: for each impurity, not more than
1.5 times the area of the principal peak in the
chromatogram obtained with reference solution (c)
(0.15 per cent);
- unspecified impurities: for each impurity, notmore than the
area of the principal peak in the chromatogram obtained
with reference solution (c) (0.10 per cent); E. 1-methyl-4-nitro-IH-imidazol-5-01,
- total: not more than 5 times the area of the principal peak
in the chromatogram obtained with reference solution (c) o
(0.5 per cent); H
HN N
- disregard limit: 0.5 times the area of the principal peak in
the chromatogram obtained with reference solution (c)
l:N \
I )
N
(0.05 per cent).
Loss on drying (2.2.32) F. 1,7-dihydro-6H-purin-6-one (hypoxanthine),
Maximum 1.0 per cent, determined on 0.500 g by drying in
an oven at 105 "C.
Sulfated ash (2.4.14)
Maximum 0.1 per cent, determined on 1.0 g.
ASSAY
Dissolve 0.250 gin 25 mL of dimethylformamide R. Titrate
with 0.1 M tetrabutylammonium hydroxide, determining the
end-point potentiometrically (2.2.20).
1 mL of 0.1 M tetrabutylammonium hydroxide is equivalent G. 6-[(I-methyl-4-nitro-IH-imidazol-5-yl)sulfanyl]-7H-purin-
to 27.73 mg of C 9H7N70 ZS. 2-amine (thiamiprine).
- - - - -_ _-'- PhEur
STORAGE
Protected from light.
IMPURITIES
Specifiedimpurities A, B.
Other detectable impurities (thefollowing substances would, if
presentat a sufficientlevel, be detected by one or other of the tests
in the monograph. They are limitedby the general acceptance
criterion for other/unspecified impurities and/or by the general
monograph Substances for pharmaceutical use (2034). It is
therefore not necessary to identify these impurities for
demonstration of compliance. See also 5.10. Control of impurities
in substances for pharmaceutical use) C, D, E, F, G.

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1-236 Azelastine Hydrochloride 2020

- stationary phase: cyanosilyl silica gelfor chromatography R

Azelastine Hydrochloride (fi um); -
(Ph. Eur. monograph 1633) - temperature: 30 "C.
Mobilephase Dissolve 2.16 g of sodium octanesulfonate Rand
0.68 g of potassium dihydrogen phosphate R in 740 mL of water
for chromatography R; adjust to pH 3.0-3.1 with dilute
phosphoric acid R, add 260 mL of acetonitrile R1 and mix.
• HCI Flow rate 2.0 mLlmin.
Detection Spectrophotometer at 210 nm.
and enantiomer Injection 10 ilL.
CI
Run time 1.5 times the retention time of azelastine.
Identification of impurities Use the chromatogram obtained
418.4 79307-93-0 with reference solution (b) to identify the peaks due to
impurities A, B, C, D and E. The elution order may vary,
Action and use
especially for impurity E, but the peak area of each
Histamine HI receptor antagonist; antihistamine.
impurity in reference solution (b) is different, so a clear
PhEur -,- _ identification of the impurities is possible.
DEFINITION Relative retention With reference to azelastine (retention
time = about 10 min): impurity A = about 0.2;
4- [(4-Chlorophenyl)methyl]-2-[ (4RS)-I-methylhexahydro-
impurity B = about 0.3; impurity C = about 0.4;
IH-azepin-4-yl]phthalazin-l (2H)-one hydrochloride.
impurityD = about 0.6; impurity E = about 0.8.
Content
System suitability:
99.0 per cent to 101.0 per cent (dried substance).
- resolution: minimum 2.0 between the peaks due to
CHARACTERS impurity E and azelastine and minimum 1.5 between the
Appearance peaks due to impurities C and D in the chromatogram
White or almost white, crystalline powder. obtained with reference solution (b);
Solubility - signal-to-noise ratio: minimum 90 for the principal peak in
Sparingly soluble in water, soluble in anhydrous ethanol and the chromatogram obtained with reference solution (a).
in methylene chloride. Calculation of percentage contents:
- correction factors: multiply the peak areas of the following
IDENfIFICATION
impurities by the corresponding correction factor:
A. Infrared absorption spectrophotometry (2.2.24).
=
impurity A 2.6; impurity B = 4.5; impurity C 2.0; =
Comparison azelastine hydrochloride CRS. impurity E = 2.8;
B. Solution S (see Tests) gives reaction (a) of chlorides - for each impurity, use the concentration of azelastine in
(2.3.1). reference solution (a).
TESTS Limits:
Solution S - impurities A, B, C, E: for each impurity, maximum
Dissolve 1.0 g in carbon dioxide-free waterR and dilute to 0.15 per cent;
100 mL with the same solvent. - unspecified impurities: for each impurity, maximum
0.10 per cent;
Acidity or alkalinity
- total: maximum 0.2 per cent;
To 10 mL of solution S add 0.2 mL of bromothymol blue
- reporting threshold: 0.05 per cent.
solutionRl. Not more than 0.1 mL of 0.01 M hydrochloric
acid or 0.01 M sodium hydroxide is required to change the Loss on drying (2.2.32)
colour of the indicator. Maximum 0.5 per cent, determined on 1.000 g by drying in
an oven at 105 "C.
Related substances
Liquid chromatography (2.2.29). ASSAY
Solvent mixture acetonitrile R1, waterfor chromatography R In order to avoid overheating in the reaction medium, mix
(45:55 VIV)o thoroughly throughout and stop the titration immediately after the
end-point has been reached.
Test solution Dissolve 0.125 g of the substance to be
examined in the solvent mixture and dilute to 50.0 mL with Dissolve 0.300 gin 5 mL of anhydrous formic acid R.
the solvent mixture. Add 30 mLof acetic anhydride R. Titrate quickly with 0.1 M
perchloric acid, determining the end-point potentiometrically
Reference solution (a) Dilute 1.0 mL of the test solution to
(2.2.20).
100.0 mL with the solvent mixture. Dilute 1.0 mL of this
solution to 10.0 mL with the solvent mixture. 1,0 mL of 0.1 M perchloric acid is equivalent to 41.84 mg of
C22H25C12N30.
Reference solution (b) Dissolve 1 mg of benzohydrazide R
(impurity A), 1 mg of azelastine impurity B CRS, 1 mg of IMPURITIES
2-[2-(4-chlorophenyl)acetyl]benzoic acid R (impurity C), 1 mg Specified impurities A, B, C, E.
of azelastine impurity D CRS and 1 mg of azelastine Other detectable impurities (the following substances would, if
impurity E CRS in the test solution and dilute to 20.0 mL present at a sufficient level, be detected by one or otherof the tests
with the test solution. in the monograph. They arelimited by the general acceptance
Column: criterion for other/unspecified impurities and/or by the general
- size: 1 = 0.25 m, 0 = 4.6 mm; monograph Substances for pharmaceutical use (2034). It is
therefore not necessary to identify these impurities for

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2020 Azithromycin 1-237

demonstration of compliance. See also 5.10. Control of impurities

in substancesfor pharmaceutical use) D.
Azithromycin
(ph.· Eur. monograph 1649)

A. benzohydrazide (benzoyldiazane),

()
~ H'-~~
o
~,~n, CH
3
and enantiorner

".
B.. N'- [(4R$)-I-methylhexahydro-1H-azepin-4-
yl]benzohydrazide,

C3sH72N2012>xH20 749 (anhydrous substance)

8
CO,H
~ I 0 =
with x lor 2

Azithromycin monohydrate 121470-24-4

~I
Azithromycin dihydrate 117772-70-0
ci ~
Action and use
C. 2-[2-(4-chlorophenyl)acetyl]benzoic acid, Macrolide antibacterial.
Preparations
o Azithromycin Capsules
Azithromycin Eye Drops
Azithromycin for Infusion
Azithromycin Oral Suspension
Azithromycin Tablets
CI PhEur ---' _

D. 4- [(4-chlorophenyl)methyl]phthalazin-l (21f)-one, DEFINITION

(2R,3S,4R,SR,SR,10R,IIR,12S,13S,14R)-13-[(2,6-Dideoxy-
o 3-C-methyl-3-0-methyl-ct-L-ribo-hexopyranosyl)oxy]-2-ethyl-
3,4,1 0-trihydroxy-3,S,6,S,1 0,12,14-heptamethyl-l1-[[3,4,6-
trideoxy- 3-(dimethyl amino )-B-D-xylo-hexopyranosyl] oxy]-I-
oxa-6-azacyclopentadecan-IS-one. The degree of hydration is
and (Z)-isomer
1 or 2.
Semi-synthetic product derived from a fermentation product.
Content
CI
96.0 per cent to 102.0 per cent (anhydrous substance).

E. (3E)- 3-[(4-chlorophenyl)methylidene]-2-benzofuran-l CHARACTERS

(31f)-one. Appearance
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur White or almost white powder.
Solubility
Practically insoluble in water, freely soluble in anhydrous
ethanol and inmethylene chloride.
IDENTIFICATION
Infrared absorption spectrophotometry (2.2.24).
Comparison azithromycin CRS.
If the spectra obtained in the solid state show differences,
prepare further spectra using 90 gIL solutions in methylene
chloride R.
TESTS
Solution S
Dissolve 0.500 g in anhydrous ethanol R and dilute to
SO.O mL with the same solvent.
Appearance of solution
Solution S is clear (2.2.1) and colourless (2.2.2, Method II).

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1-238 Azithromycin 2020

pH (2.2.3) impurity 0 = =
about 1.23; impurity G about 1.26;
9.0 to 11.0. impurity B = about 1.31.
Dissolve 0.100 gin 25.0 mL of methanolR and dilute to System suitability Reference solution (b);
50.0 mL with carbon dioxide-free waterR. - peak-to-valley ratio: minimum 1.4, where H p = height
Specific optical rotation (2.2.7) above the baseline of the peak due to impurity J and
-49 to -45 (anhydrous substance), determined on solution S. H; = height above the baseline of the lowest point of the
curve separating this peak from the peak due to
Related substances
impurity F.
Liquid chromatography (2.2.29).
Limits:
Solvent mixture Prepare a 1.73 gIL solution of ammonium
- correction factors: for the calculation of content, multiply
dihydrogen phosphate R adjusted to pH 10.0 with ammonia R
the peak areas of the following impurities by the
Transfer 350 mL of this solution to a suitable container.
corresponding correction factor: impurity F = 0.3;
Add 300 mL of acetonitrile Rand 350 mL of methanolR.
impurity G = 0.2; impurity H = 0.1; impurity L = 2.3;
Mix well.
impurity M = 0.6; impurity N = 0.7;
Test solution Dissolve 0.200 g of the substance to be - impurityB: not more than twice the area of the principal
examined in the solvent mixture and dilute to 25.0 mL with peak in the chromatogram obtained with reference
the solvent mixture. solution (a) (2.0 per cent);
Reference solution (a) Dilute 1.0 mL of the test solution to - impurities A, C, E, F,H, I, L, M, N, 0, P: for each
100.0 mL with the solvent mixture. impurity, not more than 0.5 times the area of the
Reference solution (b) Dissolve the contents of a vial of principal peak in the chromatogram obtained with
azithromycin for system suitability CRS (containing impurities reference solution (a) (0.5 per cent);
F, Hand D in 1.0 mL of the solvent mixture and sonicate - sum of impurities D, J and Q: not more than 0.5 times the
for 5 min. area of the principal peak in the chromatogram obtained
with reference solution (a) (0.5 per cent);
Reference solution (c) Dissolve 8.0 mg of azithromycin for
peak identification CRS (containing impurities A, B, C, E, F, - impurity G: not more than 0.2 times the area of the
principal peak in the chromatogram obtained with
G, I, J, L, M, N, 0 and P) in 1.0 mL of the solvent mixture.
reference solution (a) (0.2 per cent);
Column: - any other impurity: for each impurity, not more than
- size: 1 = 0.25 m, (2) = 4.6 mm; 0.2 times the area of the principal peak in the
- stationary phase: end-capped octadecylsilyl amorphous chromatogram obtained with reference solution (a)
organosilica polymerfor chromatography R (5 urn); (0.2 per cent);
- temperature: 60 DC. - total: not more than 3 times the area of the principal peak
Mobilephase: in the chromatogram obtained with reference solution (a)
- mobile phaseA: 1.80 gIL solution of anhydrous disodium (3.0 per cent);
hydrogen phosphate R adjusted to pH 8.9 with dilute - disregard limit: 0.1 times the area of the principal peak in
phosphoric acid R or with dilute sodium hydroxide solution R; the chromatogram obtained with reference solution (a)
- mobile phase B: methanolR2, acetonitrile Rl (25:75 V/V); (0.1 per cent); disregard the peaks eluting before
impurity L and after impurity B.
Time Mobile phase A Mobile phase B
(min) (per cent V/Ii') (per cent V/Ii') Water (2.5.12) .
1.8 per cent to 6.5 per cent, determined on 0.200 g.
0-25 50 45 50 55
25 - 30 45 40 55 60 Sulfated ash (2.4.14)
30 - 80 40 25 60 75 Maximum 0.2 per cent, determined on 1.0 g.
80 - 81 25 50 75 50 ASSAY
81 - 93 50 50 Liquid chromatography (2.2.29).
Solution A Mix 60 volumes of acetonitrile Rand 40 volumes
Flow rate 1.0 mUmin. of a 6.7 gIL solution of dipotassium hydrogen phosphate R
Detection Spectrophotometer at 210 nm. adjusted to pH 8.0 with phosphoric acid R.
Injection 50 ilL. Test solution Dissolve 53.0 mg of the substance to be
Identification of impurities Use the chromatogram supplied examined in 2 mL of acetonitrile R and dilute to 100.0 mL
with azithromycin for peak identification CRS and the with solution A.
chromatogram obtained with reference solution (c) to identify Reference solution (a) Dissolve 53.0 mg of azithromycin CRS
the peaks due to impurities A, B, C, E, F, G, I, J, L, M, N, in 2 mL of acetonitrile R and dilute to 100.0 mL with
o and P; use the chromatogram supplied with azithromycin solution A.
for system suitabz1ity CRS and the chromatogram obtained Reference solution (b) Dissolve 5 mg of the substance to be
with reference solution (b) to identify the peak due to examined and 5 mg of azithromycin impurityA CRS in
impurity H. 0.5 mL of acetonitrile R and dilute to 10 mL with solution A.
Relative retention With reference to azithromycin (retention Column:
time = 45-50 min): impurity L = about 0.29; - size: 1= 0.25 m, (2) = 4.6 mm;
impurity M = about 0.37; impurity E = about 0.43; - stationary phase: octadecylsilyl vinyl polymer for
impurity F = about 0.51; impurity D = about 0.54; chromatography R (5 11m); ;
impurity J = about 0.54; impurity Q = about 0.54; -,- temperature: 40 DC. .
impurity I = about 0.61; impurity C = about 0.73;
Mobile phase Mix 60 volumes of acetonitrile R1 and
impurity N = about 0.76; impurity H = about 0.79;
40 volumes of a 6.7 gIL solution of dipotassium hydrogen
impurity A = about 0.83; impurity P = about 0.92;

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2020 Azithromycin 1-239

phosphate R adjusted to pH 11.0 with a 560 g/L solution of

HaC
potassium hydroxide R.
H ..··
Plow rate 1.0 mUmin. HaC
Detection Spectrophotometer at 210 nm. CHa HO·..·
1_ 0 0 ,
Injection 10 JlL. HaCe(CHa :'
Run time 1.5 times the retention time of azithromycin. :-""N/ Ho~Ho.
CHa H:' , 0
Retention time Azithromycin = about 10 min. OH OCHa H ',- OH
System suitability Reference solution (b):
- resolution: minimum 3.0 between the peaks due to CHa
impurity A and azithromycin.
Calculate the percentage content of C3sH72NzOIZ taking into D. 14-demethyl-14-(hydroxymethyl)azithromycin
account the assigned content of azithromycin CRS. (azithromycin F),
STORAGE
In an airtight container.
HaC
IMPURITIES H....
Specifiedimpurities A., B., C., D., E., P., G., H., I., J., L., M.,N., HaC

~ H~oH~:,
0; P.,Q. CHa 00 HO.....
Other dete~'table impurities (the following substances uiould, if
presentata.sufficient leoel, be detected by one or otherof the tests NH
2

in the monograph. They are limited by.the general acceptance CHa H' o
criterion for otherlunspecified impurities and/or by the general OH OCHa H 'cH a
monograph.Substances for pharmaceutical use (2034). It is
therefore not necessary to identify these impurities for CHa
demonstration of compliance. See also 5.10. Control of impurities
in substances for pharmaceutical use) K. E. 3'-(N,N-didemethyl)azithromycin (aminoazithromycin),
HaC
H····
HaC
CHa HO····
hoo
Hac~N ...... cHa H'
:'
HO~O
CHa .
H:'''' 0
OH OCHa H CHa

CHa

A. 6-demethylazithromycin,
F. 3' -N-demethyl-3'-N-formylazithromycin,
HaC
H····
HaC HaC
CHa HO.... H..··

hoo
Hac.(~/cH' ~ H
:' CHa
HaC
HO....
o L....
.'L..{ H~01 Hi
0

D
\S~
~ "" CHaO 0
OH P ...
HCH a
0

HaC~
. I N HO
~u~ H-'
Hi
0 0 -'

CHa OH?

CHa
B. 3-deoxyazithromycin (azithromycin B),

G. 3'-N-demethyl-3' -N- [(4-methylphenyl)

sulfonyl] azithromycin,

C. 3"-O-demethylazithromycin (azithromycin C),

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1-240 Azithromycin 2020

H.3'-N-[[4-(acetylamino)phenyl]sulfonyl]-3'-N- M.3'-(N,N-didemethyI)-3'-N-fonnylazithromycin,
demethylazitbromycin,

H3C
H3C H····
H···· H3C

H,cQ /
H3C
HO"'.~.
CH3
0 0

W
CH3 HO····

H~oHo
~NH H~
,
CH Hi...
3 0
OH l H CH, a o OH OCH3 H Cti3

CH3
CH3

N. 3'-de(dimethylamino)-3'-oxoazitbromycin,
I. 3'-N-demethylazithromycin,
CH3
H3C Nt .CH3
H3C / H
H-"-
H3C
H···· H3C pH
CH3 HO···· .. CH3
CH3
1_ 0
HO""
0 . hoo
H3C(t:I/
CH3
~
, ····H

H3C~?3 H/
L-r H~01H/
H/

OH
HO ...
H'
H
o
? OH
"
H 'CH3

CH3
J. 13-0-dec1adinosylazithromycin,
O. 2-desethyl-2-propylazithromycin,
P. unknown structure,

H3C
H....
H3C
CH3 HO··--
o o/-
I V:~oo .-
O H~u1l
o ~ 'NH OH

H,J'N " o
H OHP HCH3

K. 0 14,1 "-epoxyazithromycin (azitbromycin E), CH3

Q. 3'-N-[[4-(acetylamino)phenyl] sulfonyl]-3'-(N,N-
didemethyl)azithromycin.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _-----,-----, PhEur

L. azithromycin 3'-N-oxide,

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2020 Bacampicillin Hydrochloride 1-241

contents of the tube by swirling; the solution is practically

Bacampicillin Hydrochloride colourless. Place the test-tube on a water-bath for 1 min;
(Ph. Bur. monograph 0808) a dark yellow colour develops.
D. Dissolve about 25 mg in 2 mL of water R. Add 2 mL of
dilute sodium hydroxide solution R and shake. Wait a few
minutes and add 3 mL of dilute nitric acid Rand 0.5 mL of
silvernitrate solution Rl. A white precipitate is formed.
Add 0.5 mL of concentrated ammonia R. The precipitate
dissolves.
TESTS
502.0 37661-08-8 Appearance. of solution
Dissolve 0.200 gin 20 mL of water R; the solution is not
Action and use more opalescent than reference suspension II (2.2.1).
Penicillin antibacterial. Dissolve 0.500 gin 10 mL of water R; the absorbance
(2.2.25) of the solution at 430 om is not greater than 0.10.
PhEur _
pH (2.2.3)
DEFINITION 3.0 to 4.5.
(IRS)-I-[(Ethoxycarbonyl)oxy]ethyl (2S,5R,6R)-6-[[(2R)-2- Dissolve 1.0 g in carbon dioxide-free water R and dilute to
amino-2-phenylacetyl] amino]-3,3-dimethyl-7-oxo-4-thia-l- 50 mL with the same solvent.
azabicyclo[3.2.0]heptane-2-carboxylate hydrochloride.
Specific optical rotation (2.2.7)
Semi-synthetic product derived from a fermentation product. + 175 to + 195(anhydrous substance).
Content Dissolve 0.250 g in waterR and dilute to 25.0 mL with the
95.0 per cent to 102.0 per cent (anhydrous substance). same solvent.
CHARACTERS Related substances
Appearance Liquid chromatography (2.2.29). Prepare the test solution and
White or almost white powder or granules, hygroscopic. reference solutions (a), (b) and (d) immediately before use.
Solubility Phosphate bufferA Dissolve 1.4 g of sodium dihydrogen
Soluble in water, freely soluble in ethanol (96 per cent), phosphate monohydrate R in water R and dilute to about
soluble in methylene chloride. 800 mL with the same solvent. Adjust to pH 3.0 with dilute
IDENTIFICATION phosphoric acid R and dilute to 1000.0 mL with waterR.
First identification: A, D. Phosphate bufferB Dissolve 2.75 g of sodium dihydrogen
phosphate monohydrate Rand 2.3 g of disodium hydrogen
Secondidentification: B, C, D.
phosphate dihydrate R in water R and dilute to about 1800 mL
A. Infrared absorption spectrophotometry (2.2.24). with the same solvent. Adjust to pH 6.8, if necessary, using
Comparison bacampicillin hydrochloride CRS. dilute phosphoric acid R or dilute sodium hydroxide solution R
B. Thin-layer chromatography (2.2.27). and dilute to 2000.0 mL with water R.
Test solution Dissolve 10 mg of the substance to be Test solution Dissolve 30.0 mg of the substance to be
examined in 2 mL of methanolR. examined in phosphate buffer A and dilute to 100.0 mL with
Reference solution (a) Dissolve 10 mg of bacampicillin phosphate buffer A.
hydrochloride CRS in 2 mL of methanolR. Reference solution (a) Dissolve 30.0 mg of bacampicillin
Reference solution (b) Dissolve 10 mgof bacampicillin hydrochloride CRS in phosphate buffer A and dilute to
hydrochloride CRS, 10 mg of talampicillin hydrochloride CRS 100.0 mL with phosphate buffer A.
and 10 mg of pivampicillin CRS in 2 mL of methanolR. Reference solution (b) Dilute 1.0 mL of reference solution (a)
Plate TLC silanised silica gelplate R. to 100.0 mL with phosphate buffer A.
Mobile phase Mix 10 volumes of a 272 gIL solution of Reference solution (c) Dissolve 30 mg of the substance to be
sodium acetate R adjusted to pH 5.0 with glacialacetic acid R, examined in phosphate buffer B and dilute to 100 mL with
40 volumes of water Rand 50 volumes of ethanol phosphate buffer B. Heat at 80 DC for about 30 min.
(96 per cent) R. Reference solution (d) Dissolve 20 mg of ampicillin
Application 1 ilL. trihydrate CRS (impurity I) in phosphate buffer A and dilute
to 250 mL with phosphate buffer A. Dilute 5 mL of this
Development Over a path of 15 em.
solution to 100 mL with phosphate buffer A.
Drying In a current of warm air.
Column:
Detection Spray with ninhydrin solution Rl and heat at 60 DC - size: l = 0.05 m, 0 = 3.9 mm;
for 10 min. - stationary phase: octadecylsilyl silica gelfor chromatography R
System suitability Reference solution (b): (5 urn).
- the chromatogram shows 3 clearly separated spots. Mobilephase Mix 30 volumes of acetonitrile R1 and
Results The principal spot in the chromatogram obtained 70 volumes of a 0.06 per cent mlm solution of
with the test solution is similar in position, colour and size to tetrahexylammonium hydrogen sulfate R in phosphate buffer B.
the principal spot in the chromatogram obtained with Flow rate 1.0 mLlmin.
reference solution (a).
Detection Spectrophotometer at 220 nm.
C. Place about 2 mgin a test-tube about 150 mm long and
Injection 20 ul, of the test solution and reference
15 mm in diameter. Moisten with 0.05 mL of water Rand
solutions (b), (c) and (d).
add 2 mL of sulfuric acid-formaldehyde reagent R. Mix the

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1-242 Bacampicillin Hydrochloride 2020

Run time 3.5 times the retention time of bacampicillin.

System suitability:
- the peak. due to impurity I is separated from the peaks
due to the solvent in the chromatogram obtained with
reference solution (d); B. (2R)-2-amino-2-phenylacetic acid (n-phenylglycine),
- relative retention with reference to bacampicillin:
degradation product eluting just after
bacampicillin = 1.12 to 1.38 in the chromatogram
obtained with reference solution (c); if necessary, adjust
the concentration of tetrahexylammonium hydrogen
sulfate in the mobile phase.
Limits:
- any impurity: for each impurity, not more than 1.5 times C. (2RS,4S)-2-[[ [(2R)-2-amino-2-phenylacetyl]
the area of the principal peak in the chromatogram amino]methyl]-5,5-dimethylthiazolidine-4-carboxylic acid
obtained with reference solution (b) (1.5 per cent); (penilloic acids of ampicillin),
- total: not more than 3 times the area of the principal peak
in the chromatogram obtained with reference solution (b)
(3 per cent);
- disregard limit: 0.1 times the area of the principal peak in
the chromatogram obtained with reference solution (b)
(0.1 per cent).
Butyl acetate and ethyl acetate (2.4.24, System A)
Maximum 2.0 per cent of butyl acetate, maximum D. (4S)-2-[[[(2R)-2-amino-2-phenylacetyl]
4.0 per cent of ethyl acetate and maximum 5.0 per cent for amino]carboxymethyl]-5,5-dimethylthiazolidine-4-
the sum of the contents. carboxylic acid (penicilloic acids of ampicillin),
Sample solution Dissolve 50.0 mg of the substance to be
examined in water R and dilute to 10.0 mL with the same
solvent.
Use the method of standard additions.
Static head-space conditions that may be used:
- equilibration temperature: 60°C;
- equilibration time: 20 min.
N,N-Dimethylaniline (2.4.26, Method A)
Maximum 20 ppm. E. (4S)-2-(3,6-dioxo-5-phenylpiperazin-2-yl)-5,5-
Water (2.5.12) dimethylthiazolidine-4-carboxylic acid (diketopiperazines
Maximum 0.8 per cent, determined on 0.300 g. of ampicillin),
Sulfated ash (2.4.14)
Maximum 1.5 per cent, determined on 1.0 g.
ASSAY
Liquid chromatography (2.2.29) as described in the test for
related substances with the following modifications.
F. (2RS)-2-amino- 3-methyl-3-sulfanylbutanoic acid (DL-
Injection Test solution and reference solution (a). penicillamine),
System suitability Reference solution (a):
-repeatability: maximum relative standard deviation of
1.0 per cent after 6 injections.
Calculate the percentage content of C21H2SCIN307S from
the declared content of bacampicillin hydrochloride CRS.
STORAGE G. methyl (2R)-2-amino-2-phenylacetate (methyl D-
In an airtight container. phenylglycinate),
IMPURITIES

A. (2S,SR,6R)-6-amino-3,3-dimethyl-7-oxo-d-thia-I> H. (lRS)-l-[ (ethoxycarbonyl)oxy] ethyl (2S,5R,6R)-6- [[(2R)-

azabicyclo[3.2.0]heptane-2-carboxylic acid 2-(acetylamino)-2-phenylacetyl]amino]-3,3-dimethyl-7-
(6-aminopenicillanic acid), oxo-I-thia-l-azabicyclo[3.2.0]heptane-2-carboxylate (N-
acetylbacampicillin),

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2020 Bacitracin 1-243

Solubility
Freely soluble in water and in ethanol (96 per cent).
IDENTIFICATION
First identification: B, C.
Secondidentification: A, C.
1. (2S,5R,6R)-6-[[(2R)-2-arnino-2-phenylacetyl]amino]-3,3- A. Thin-layer chromatography (2.2.27).
dimethyl-7-oxo-d-thia-I-azabicyclo [3.2.0]heptane-2- Test solution Dissolve 10 mg of the substance to be
carboxylic acid (ampicillin). examined in a 3.4 gIL solution of hydrochloric acid Rand
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur dilute to 1.0 mLwith the same solution.
Reference solution Dissolve 10 mg of bacitracin zinc CRS in a
3.4 gIL solution of hydrochloric acid R and dilute to 1.0 mL
with the same solution.
Bacitracin ***** Plate TLC silica gel plate R.
* *
(Ph. Bur. monograph 0465) ***** Mobilephase glacial acetic acid R, . water R, butanol R
(14:29:57 V/V/V).
Application 10 ilL.
H2~~.
Hl ..
CH.3•
H
" Development Over half of the plate.
Drying At 100-105 "C.
s ~N
y.. -_.r L-LeU-o-GIU-V-L-LYS-D-Om-X-D-PheJ
t L-Asn--D-Asp--L-HIS.
Detection Spray with ninhydrin solution R1 and heat at
110 °e for 5 min.
H
° Results The spots in the chromatogram obtained with the
test solution are similar in position, size and colour to the
Name Mol. Formula X Y R
spots in the chromatogram obtained with the reference
Bacitracin A C66H103N17016S L-lle L-lle CH3 solution.
Bacitracin B1 C65H101N17016S L-lle L-lle H B. Composition (see Tests).
Bacitracin B2 C65H101N17016S L-Val t-Ile CH3
Bacitracin B3 C65H101N17016S L-lle L-Val CH3
e. Ignite 0.2 g. There is no significant yellow-coloured
residue at high temperature. Allow to cool. Dissolve the
residue in 0.1 mL oidiluie hydrochloric acid R. Add 5 mL of
1405-87-4 water Rand 0.2 rnL of strong sodium hydroxide solution R.
No white precipitate is formed.
Action and use
TESTS
Polypeptide antibacterial.
Solution S
PhEur _ Dissolve 0.25 g in carbon dioxide-free waterR and dilute to
25 mL with the same solvent.
DEFINITION
Mixture of antimicrobial polypeptides produced by certain Appearance of solution
strains of Bacillus licheniformis or Bacillus subtilis, the main Solution S is clear (2.2.1).
components being: pH (2.2.3)
- 4,10-fulhydro[N-[[(4R)-2-[(IS,2S)-I-amino-2- 6.0 to 7.0 for solution S.
methylbutyl] -4,5-dihydro-1,3-thiazol-4-yl] carbonyl]-L- Composition
leucyl-n-ex-glutamyl-L-isoleucyl-L-Iysyl-n-ornithyl-L- Liquid chromatography (2.2.29): use the normalisation
isoleucyl-n-phenylalanyl-L-histidyl-n-ex-aspartyl-L- procedure. Prepare the solutions immediately before use.
asparagine] (bacitracin A);
Solution A 40 gIL solution of sodium edetate R adjusted to
- 4,10-anhydro[N-[[(4R)-2-[(IS)-I-amino-2-methylpropyl]-
pH 7.0 with dilute sodium hydroxide solution R.
4,5-dihydro-1,3-thiazol-4-yl] carbonyl] -L-Ieucyl-n-ex-
glutamyl-L-isoleucyl-L-Iysyl-n-ornithyl-L-isoleucyl-n- Solution B In a volumetric flask, dissolve 54.4 g of potassium
phenylalanyl-L-histidyl-n-ex-aspartyl-L-asparagine] dihydrogen phosphate R in waterfor chromatography Rand
(bacitracin B I); dilute to 2000 rnL with the same solvent. Adjust to pH 6.0
- 4,10-anhydro[N-[[(4R)-2-[(IS,2S)-I-amino-2- with a 34.8 gIL solution of dipotassium hydrogen phosphate R
methylbutyl] -4,5-dihydro-1 ,3-thiazol-4-yl] carbonyl] -L- and filter through a membrane filter (nominal pore size
leucyl-n-ex-glutamyl-L-isoleucyl-L-Iysyl-n-ornithyl-L-valyl- 0.45 urn),
n-phenylalanyl-L-histidyl-n-ex-aspartyl-L-asparagine] Test solution Dissolve 0.100 g of the substance to be
(bacitracin B2); examined in the mobile phase and dilute to ?O.O mL with
- 4,10-anhydro[N-[[(4R)-2-[(IS,2S)-1-amino-2- the mobile phase.
methylbutyl] -4,5-dihydro-1 ,3-thiazol-4-yl] carbonyl] -L- Reference solution (a) Dissolve 20.0 mg of bacitracin for
leucyl-n-ex-glutamyl-L-valyl-L-Iysyl-n-ornithyl-L-isoleucyl- system suitability CRS in solution A and dilute to 10.0 mL
n-phenylalanyl-L-histidyl-n-ex-aspartyl-L-asparagine] with the same solution.
(bacitracin B3). Reference solution (b) Dilute 5.0 mL of reference solution (a)
Content to 100.0 rnL with solution A. Dilute 1.0 mL of this solution
Minimum 60 IV/mg (dried substance). to 10.0 mL with solution A.
CHARACTERS Reference solution (c) In order to prepare impurities E, F, G
Appearance and H in situ, heat about 4 mL of reference solution (a) in a
White or almost white, hygroscopic powder. water-bath for 30 min. Cool to room temperature.

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1-244 Bacitracin 2020

0.20 10

0.15

~0.10

0.05

0.00 h..:;::;:~:;::;~~:;:;...;;.~~:::-r~::;:::;::::;:::;:::;::;::;:::;::;::;:::::;::::;::;:::::;::;::;;::::;:::::;::::;::::;:::;:.=;:::;:::;:::;:::;::::;::::;::::;:::;:::;:::;::::;::;::;:;::;:~
o 5 10 15 20 25 30 35 40 45 50 55 60 min

0.010 8
0.008

0.006
7
~ 0.004

0.002 16

0.000

-0.002+-r-r-..--r--r-r-.--,....,.............-r-T-.--r-r--r-r-r-,.-..,--r-r....,.....,~..---r...,.....,~..-.--r-r-r-,....,...-.--.--r-,....,...'T"""""T'"....,.....,~..,...,....,.......,~..,...,....,.......,-.--,.-..,--r-1
o 5 10 15 20 25 30 35 40 45 50 55 60 min

1. impurity A 5. bacitracin B2 9. impurity L 13. impurity F

2. impurity B 6. bacitracin B3 10. bacitracin A 14. impurity G
3. impurity C 7. impurity M 11. impurity 0 15. impurity H
4. bacitracin Bl 8. impurity N 12. impurities P and Q 16. impurity E

Figure 0465.-1. - Chromatogram for the testfor composition of bacitracin: testsolution

Column: Systemsuitability:
- size: 1 = 0.15 m, 0 = 4.6 mm; - peak-to-valley ratio: minimum 1.2, where Hp = height
- stationary phase: end-capped, charged surface, ethylene-bridged above the baseline of the peak due to bacitracin B2 and
octadecylsilyl silica gelfor chromatography (hybrid material) R H; = height above the baseline of the lowest point of the
(3.5 1Jll1); curve separating this peak from the peak due to bacitracin
- temperature: 28 ± 2 DC. B1 in the chromatogram obtained with reference
Mobile phase acetonitrile R, solution B, waterfor solution (a);
chromatography R, methanolRl (43:100:300:557 VIVIVIV). - peak-to-valley ratio: minimum 1.1; where H p = height
above the baseline of the peak due to impurity M and
Flow rate 1.0 mIJrnin.
H; = height above the baseline of the lowest point of the
Detection Spectrophotometer at 254 nm. curve separating this peak from the peak due to bacitracin
Injection 100 ul, of the test solution and reference B3 in the chromatogram obtained with reference
solutions (a) and (b). solution (a);
Run time 3 times the retention time .of bacitracin A. - signal-to-noise ratio: minimum 50 for the peak due to
Identification ofpeaks Use the chromatogram obtained with bacitracin A in the chromatogram obtained with reference
reference solution (a) to identify the peaks due to impurity M solution (b).
and bacitracins A, B1, B2 and B3 (see Figure 0465.-1). Limits:
Relativeretention With reference to bacitracin A (retention - bacitracin A: minimum 45.0 per cent;
time = about 20 min): impurity A = about 0.44; - sum of bacitracins A, Bl, B2 and B3: minimum
impurity B = about 0.52; impurity C = about 0.55; 77.0 per cent.
bacitracin B1 = about 0.65; bacitracin B2 = about 0.67; Related substances
bacitracin B3 = about 0.81; impurityM = about 0.87; Liquid chromatography (2.2.29) as described in the test for
impurity N = about 0.90; impurity L = about 0.93; composition with the following modifications. Use the
impurity 0 = about 1.2; impurities P and Q = about 1.3; normalisation procedure.
impurity F = about 1.6; impurity G = about 1.8; Injection Test solution and reference solutions (a), (b) and
impurity H = about 2.1; impurity E = about 2.8. (c).
If necessary, adjust the composition of the mobile phase by Identification of impurities Use the chromatogram obtained
changing the amount of organic modifier whilst keeping the with reference solution (a) to identify the peaks due to
ratio constant between methanol and acetonitrile. impurities A, B, C, L, M, N, 0, P and Q (see
Figure 0465.-1); use the chromatogram obtained with

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2020 Bacitracin 1-245

0.080

0.070

0.060

0.050

0.040
~
0.030

0.020

0.010

0.000

-0.0 1 O-;---'r-r--r-r-,--,--.--r-r~-'-"""T""'-r"-,--,r-r--r-T--r-1....--r--r-r""""-'--'--r-T'--r-1r-r-""""'--r-1r-r--r-r-,--,...,.....-r-r-,---r-,-......,.....,....,.--,-......,.....,....,.-,...,,--,-,
o

1. impurity F 2. impurity G 3. impurity H 4. impurity E

Figure 0465.-2. - Chromatogram for the testfor related substances of bacitracin: reference solution (c)

reference solution (c) to identify the peaks due to necessary to identify these impurities for demonstration of
impurities E, F, G and H (see Figure 0465.-2). compliance. See also 5.10. Control of impurities in substances for
Limits: pharmaceutical use) D~ I, J~ K.
- sum of impurities Land N: maximum 8.0 percent;
- impurity E: maximum 4.0 per cent;
- impurityA: maximum 3.5 per cent;
- impurities B~ M: for each impurity, maximum 3.0 per cent;
- impurity C: maximum 2.5 per cent;
- sum of impurities O~ P and Q: maximum 2.5 per cent;
- sum of impurities F and G: maximum 2.0 per cent;
- impurity H: maximum 1.0 per cent;
- any other impurity: for each impurity, maximum
2.0 per cent; A. 4,10-anhydro[N-[[(4R)-2-[(IS)-1-amino-2-methylpropyl]-
- total: maximum 23.0 per cent; 4,5-dihydro-1 ,3-thiazol-4-yl]carbonyl]-t-leucyl-n-«-
- reporting threshold: 0.25 per cent. glutamyl-L-isoleucyl-L-lysyl-D-ornithyl-L-valyl-D-
phenylalanyl-L-histidyl-D-et-aspartyl-L-asparagine]
The thresholds indicated under Related substances
(bacitracin DI, bacitracin en,
(Table 2034.-1) in the general monograph Substances for
pharmaceutical use (2034) do not apply.
Loss on drying (2.2.32) H~~vt3
Maximum 5.0 per cent, determined on 1.000 g by drying at
60 "C over diphosphorus pentoside R at a pressure not
Hl CH 3

s ~N
exceeding 0.1 kPa for 3 h.
'--j.'l L-Leu-- o-Glu - L-Val- L-Lys-- o-Orn -- L-lle -- o-PheJ
Sulfated ash (2.4.14)
H 0 L-Asn-o-AsP-L-HiS
Maximum 1.0 per cent, determined on 1.0 g.
ASSAY B. 4,10-anhydro[N-[[(4R)-2-[(IS)-I-amino-2-methylpropyl]-
Carry out the microbiological assay of antibiotics (2.7.2). 4,5-dihydro-1 ,3-thiazol-4-yl]carbonyl]-t-Ieucyl-n-«-
Use bacitracin zinc CRS as the reference substance. glutamyl-L-valyl-L-lysyl-D-omithyl-L-isoleucyl-D-
STORAGE phenylalanyl-L-histidyl-n-et-aspartyl-L-asparagine]
In an airtight container at 2 "C to 8 "C. If the substance is (bacitracin D2, bacitracin C3),
sterile, the container is also sterile and tamper-proof.
IMPURITIES
Specified impurities A, B~ C~ E, F, G, H~ L, M~ N~ 0, P, Q.
Other detectable impurities (the following substances would, if
present at a sufficient leoel, be detected by one or otherof the tests
in the monograph. They are limitedby the general acceptance
criterion for other/unspecified impurities. It is therefore not

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1-246 Bacitracin 2020

H. CH3
HzN H... CH 3

H~CH'
S ~N s
aYCCH'
~N
y_.. i-Leu-s- o-Glu-L-Val- L-Lys -- a-Orn-e- L-Val-O-Phe]
'=)- L-Leu - o-"'u - L-Val- t-Lys - o-Om - t-Ile - O-PheJ
H [ tL-ASn-O-ASP-L-HiS
t L-Asn -- o-Asp - t-His

C, 4,lO-anhydro[N-[[(4R)-2-[(lS,2S)-1-amino-2-
methylbutyl] -4,5-dihydro-l,3-thiazol-4-yl] carbonyl]-L- H.4,lO-anhydro[N-[[2-[(2S)-2-methyl-l-oxobutyl]-1,3-
leucyl-n-ct-glutamyl-L-valyl-L-lysyl-D-ornithyl-L-valyl-D- thiazol-4-yl] carbonyl]-L-Ieucyl-D-a.-glutamyl-L-valyl-L-Iysyl-
phenylalanyl-L-histidyl-n-a.-aspartyl-L"-asparagine] D-ornithyl-L-isoleucyl-D-phenylalanyl-L-histidyl-D-a.-
(bacitracin D3, bacitracin CIa), aspartyl-t.-asparagine] (bacitracin H3),

CH3

H~~vt3 arc~
Hl CH 3
s ~N
s ~N
y.. L-Leu- o-Glu- L-Val- L-Lys-- c-Orn - L-Val- o-PheJ
'=)- L-LeO- 0-"'0 - L-lie- L-Lys - c-Orn- L-Val- o-Phe]
H [ t-Asn-O-AsP-L-HiS
t L-Asn - n-Asp -- L-His

D.4,lO-anhydro[N-[[(4R)-2-[(1S)-1-amino-2-methylpropyl]-
4,5-dihydro-l,3-thiazol-4-yl]carbonyl]-L-Ieucyl-n-a.- 1. 4,1O-anhydro [N-[[2-(2-methyl-l-oxopropyl)-1,3-thiazol-4-
yl]carbonyl]-L-leucyl-D-a.-glutamyl-L-isoleucyl-L-lysYI-D-
glutamyl-L-valyl-L-Iysyl-D-ornithyl-L-valyl-n-phenylalanyl-
ornithyl-L-valyl-D-phenylalanyl-L-histidyl-D-a.-aspartyl-L-
L-histidyl-n-cc-aspartyl-t-asparagine] (bacitracin E),
asparagine] (bacitracin II),

aXCH~.... 3
arCH,
CH3

S ~N
s ~N

'=)- L-Leu- 0-<310 - L"'e- L-Lys - c-Orn - t-ile - O-PheJ

t
'=)- t-Leu - o-<3lu - L-Val- i-Lys - o-Om - L-II. - o-Phe
L-Asn -- o-Asp- L-His
t L-Asn - o-Asp-- L-HisJ

E. 4,lO-anhydro[N-[[2-[(2S)-2-methyl-l-oxobutyl]-1,3-
J. 4,lO-anhydro[N-[[2-(2-methyl-l-oxopropyl)-1,3-thiazol-4-
thiazol-4-yl]carbonyl]-L-Ieucyl-D-a.-glutamyl-L-isoleucyl-L-
yl]carbonyl]-L-Ieucyl-D-ct-glutamyl-L-valyl-L-Iysyl-D-
lysyl-D-ornithyl-L-isoleucyl-D-phenylalanyl-L-histidyl-n-a.-
ornithyl-L-isoleucyl-D-phenylalanyl-L-histidyl-n-a.-aspartyl-
aspartyl-L-asparagine] (bacitracin F),
L-asparagine] (bacitracin 12),
CH 3

arCH, arCfI, H. CH3

s ~N s ~N

'=}-
o
L-Leu- n-Glu - L-lie- L-Lys - c-Orn - L-lle - o-PheJ
t . .
L-Asn- o-Asp -- L-HIS
'=)- t-Leu - o-Glu - L-V~ - L-Lys - o-Om - L

t
-ver- o-Phe]
L-Asn -- o-Asp- L-His

F. 4,1O-anhydro [N-[[2-(2-methyl-l-oxopropyl)-1,3-thiazol-4- K. 4,1 O-anhydro[N-[[2-[(2S)-2-methyl-l-oxobutyl]-1,3-

yl]carbonyl]-L-leucyl-D-a.-glutamyl-L-isoleucyl-L-Iysyl-D- thiazol-4-yl] carbonyl]-L-Ieucyl-D-ct-glutamyl-L-valyl-L-Iysyl-
ornithyl-L-isoleucyl-n-phenylalanyl-L-histidyl-n-a.-aspartyl- n-ornithyl-L-valyl-D-phenylalanyl-L-histidyl-D-a.-aspartyl-L-
L-asparagine] (bacitracin HI), asparagine] (bacitracin 13),

arCH, H. CH 3 HzN

H~CH3
~" CH3

s ~N S ~N

'=}- L-Leu- O-,,'U- L-lie - L-Lys - c-Orn - L-Val- O-Phe]

y...~H
L-Leu- n-Glu-- L-lie- L-Lys-- c-Orn-- t-ile -- o-PheJ
t .
o .
o ti-Asn -- o-Asp - .
L-HIS
L-Asn-o-Asp-L-His

L. 4,1O-anhydro[N-[[(4R).,.2-[(lR,2S)-1-amino-2-
G.4,lO-anhydro[N-[[2-[(2S)-2-methyl-l-oxobutyl]-1,3- methylbutyl] -4,5-dihydro-l,3-thiazol-4-yl] carbonyl] -L-
thiazol-4-yl]carbonyl]-L-Ieucyl-D-a.-glutamyl-L-isoleucyl-L- leucyl-D.,.a.-glutamyl-L-isoleucyl-L-Iysyl-n-ornithyl-L-
lysyl-D-ornithyl-L-valyl-D-phenylalanyl-L-histidyl-D-a.- isoleucyl-D-phenylalanyl-L-histidyl-n-'a.-aspartyl-L-
aspartyl-L-asparagine] (bacitracin H2), asparagine]' (bacitracin X),

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2020 Bacitracin Zinc 1-247

H2N H", CH3

Bacitracin Zinc ***
H~CH' *** ***
S ~N (Ph. Bur. monograph 0466) ***
~ o-Om~L-IIe- 2-

~:rcR
L-Leu-o-Glu- L-IIe-L-Lys- o-Phe]

° t
L-Asn- ·
o-Asp- L-His

S ~N
M.4,10-anhydro[N-[[2-[(1S,2S)-1-amino-2-methylbutyl]-1,3-
thiazol-4-yl]carbonyl]-L-leucyl-D-lX-glutamyl-L-isoleucyl-L- L ~ . L-Leu-- o-Glu -
~"l
Y - L-Lys- o-Orn- X - o-Phe. J
lysyl-D-omithyl-L-isoleucyl-D-phenylalanyl-L-histidyl-D-lX- H ° tL-Asn--o-AsP-L-HiS
aspartyl-r.-asparagine] (bacitracin Y),
Name Mol. Formula X Y R
H2N H, CH3

H~CH'
BacitracinA CSSH103N17016S L-lle L-lle CH3
BacitracinB1 CSSH101 N1701SS t-lle L-ne H
BacitracinB2 CSSH101N1701SS L-Val L-lle CH3
S ~N .
BacitracinB3 CSSH101N17016S t-Ile L-Val CH3
~L-Leu- o-Glu- L-lle- L-Lys- o-Orn- L-lle-. o-PheJ

. H ° t-Asn,-o-AsP-L-HiS
1405-89-6
N. 4,1 O-anhydro[N-[[(4S)-2-[(lS,2S)-1-amino-2- Action and use
methylbutyl] -4,5-dihydro-1 ,3-thiazol-4-yl]carbonyl] -L- Polypeptide antibacterial.
leucyl"'D-lX-gluta.myl-L-isoleucyl-L-lysyl-D-ornithyl-L-
Preparation
isoleucyl-D-phenylalanyl-L-histidyl-D-lX-aspartyl-L-
Polymyxin and Bacitracin Ointment
asparagine] (bacitracin Z),
PhEur _
H H, CH3
~CH3
2N
H, .. DEFINITION
Zinc complex of bacitracin, which consists of a mixture of
S
~.
N antimicrobial polypeptides produced by certain strains of
y... L-Leu;' o-Glu-- MiI-- L-Lys- o-Orn-- t-Ile -- O-Phe] Bacillus licheniformis or Bacillus subtilis, the main components
H
°r t ~
L-Asn-o-Asp-L-His
being:
- 4,10-anhydro[N-[[(4R)-2-[(1S,2S)-1-amino-2-
methylbutyl] -4,5-dihydro-1,3-thiazol-4-yl] carbonyl] -L-
O. Mil = 5-methylene-L-isoleucine: 4,10-anhydro[N-[[(4R)-2- leucyl-D-lX-glutamyl-L-isoleucyl-L-lysyl-D-ornithyl-L-
[(1S,2S)-1-amino-2-methylbutyl]-4,5-dihydro-1,3-thiazol- isoleucyl-D-phenylalanyl-L-histidyl-D-lX-aspartyl-L-
4-yl] carbonyl]-L-leucyl-D-lX-glutamyl-5-methylene-L- asparagine] (bacitracin A);
isoleucyl-L-lysyl-D-ornithyl-v·isoleucyl-D-phenylalanyl-L- - 4,1O-anhydro [N- [[ (4R)-2-[ (1S)-1-amino-2-methylpropyl]-
histidyl-n-c-aspartyl-t-asparagine] (bacitracin J1), 4,5-dihydro-1 ,3-thiazol-4-yl] carbonyl] -t-leucyl-n-«-
glutamyl-L-isoleucyl-L-lysyl-D-ornithyf'::L-isoleucyl-D-
H2N ~" CH3 phenylalanyl-L-histidyl-D-lX-aspartyl-L-asparagine]
H~CH' (bacitracin B1);
- 4,10-anhydro[N-[[(4R)-2-[(lS,2S)-1-amino-2-
S ~N
methylbutyl] -4 ,5-dihydro-1 ,3-thiazol-4-yl] carbonyl] -L-
L J. i-Leu-» o-Glu" L-lIe- L-Lys- c-Orn-s- MiI-- o-PheJ
1"1 leucyl~D-lX-glutamyl-L-isoleucyl-L-lysyl-D-ornithyl-L-valyl­
H ° t-Asn,-o-AsP-L-HiS D-phenylalanyl-L-histidyl-D-lX-aspartyl-L-asparagine]
(bacitracin B2);
P. Mil = 5-methylene-L-isoleucine: 4,1 O-anhydro [N- [[ (4R)-2- - 4,10-anhydro[N-[[(4R)-2-[(1S,2S)-1-amino-2-
[(1S,2S)-1-amino-2-methylbutyl]-4,5-dihydro-1,3-thiazol- methylbutyl] -4,5-dihydro-1,3-thiazol-4-yl] carbonyl] -L-
4-yl]carbonyl]-L-leucyl-D-lX-glutamyl-L-isoleucyl-L-lysyl-D- leucyl-D-lX-glutamyl-L-valyl-L-lysyl-D-ornithyl-L-isoleucyl-
omithyl-5-methylene-L-isoleucyl-D-phenylalanyl-L-histidyl- D-phenylalanyl-L-histidyl-D-lX-aspartyl-L-asparagine]
n-c-asparryl-t-asparagine] (bacitracin J2), (bacitracin B3).
Content
H2~.~'
H····
CH
3
~
Minimum 60 ill/mg (dried substance).
CH2 CHARACTERS
S ~N . Appearance
y..
H rL-Leu- o-Glu- L-lle-. L-Lys- c-Orn-s- L-ne-- O-Phe]

tL-Asn-o-AsP-L-HiS
White or light yellowish-grey, hygroscopic powder:
Solubility
Slightly soluble in water and in ethanol (96 per cent).
Q.4,10-anhydro[N-[[(4R)-2-[(1S,2S)-1-amino-2-methylpent- IDENTIFICATION
4-en-1-yl] -4,5-dihydro-1,3-thiazol-4-yl] carbonyl] -L-Ieucyl- First identification: B~ C.
D-tt-glutamyl-L-isoleucyl-L-Iysyl-D-ornithyl-L-isoleucyl-D- Second identification: A~ G.
phenylalanyl-L-histidyl-D-lX-aspartyl-L-asparagine]
A. Thin-layer chromatography (2.2.27).
(bacitracin J3).
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur

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1-248 Bacitracin Zinc 2020

0.20 10

-0.15

~0.10

0.05

0.00
0 5 10 15 20 25 30 35 40 45 50 55 60 min

0.010
8
0.008

0.006
7
~ 0.004

0.002 12 16

0.000

-0.002
0 5 10 15 20 25 30 35 40 45 50 min

1. impurity A 5. bacitracin B2 9. impurity L ' 13. impurity F

2. impurity B 6. bacitracin B3 10. bacitracin A 14. impurity G
3. impurity C 7. impurity M 11. impurity 0 15. impurity H
4. bacitracin Bl 8. impurity N 12. impurities P and Q 16. impurity E

Figure 0466.-1. - Chromatogram for the testfor composition of bacitracin zinc: test solution

Test solution Dissolve 10 mg of the substance to be Composition

examined in 0.5 mL of dilute hydrochloric acid R and dilute to Liquid chromatography (2.2.29): use the normalisation
1.0 mL with waterR. procedure. Prepare the solutions immediately before use.
Reference solution Dissolve '10 mg of bacitracin zinc CRS in Solution A 40 gIL solution of sodium edetate R adjusted to
0.5 mL of dilute hydrochloric acid R and dilute to 1.0 mL with pH 7.0 with dilute sodium hydroxide solution R.
waterR. Solution B In a volumetric flask, dissolve 54.4 g of potassium
Plate TLC silica gelplate R. dihydrogen phosphate R in water for chromatography Rand
Mobilephase glacial acetic acid R, waterR, butanolR dilute to 2000 mL with the same solvent. Adjust to pH 6.0
(14:29:57 V/V/V). with a 34.8 g/L solution of dipotassium hydrogen phosphate R
Application 10 J1L. and filter through a membrane filter (nominal pore size
0.45 /lm ).
Development Over half of the plate.
Test solution Dissolve 0.100 g of the substance to be
Drying At 100-105 DC. examined in solution A and dilute to 50.0 mL with the same
Detection Spray with ninhydrin solution R1 and heat at solution.
110 DC for 5 min. .
Reference solution (a) Dissolve 20.0 mg of bacitracin for
Results The spots in the chromatogram obtained with the system suitability CRS in solution A and dilute to 10.0 mL
test solution are similar in position, size and colour to the with the same solution.
spots in the chromatogram obtained with the reference Reference solution (b) Dilute 5.0 mL of reference solution (a)
solution. to 100.0 mL with solution A. Dilute 1.0 mL of this solution
B. Composition (see Tests). to 10.0 mL with solution A.
C. Ignite about 0.15 g, allow to cool and dissolve the residue Reference solution (c) In order to prepare impurities E, F, G
in 1 mL of dilute hydrochloric acid R. Add 4 mL of waterR. and H in situ, heat about 4 mL of reference solution (a) in a
The solution gives the reaction of zinc (2.3.1). water-bath for 30 min. Cool to room temperature.
TESTS Column:
pH (2.2.3) - size: 1= 0.15 m, 0 = 4.6 mm;
6.0 to 7.5. - stationary phase: end-capped, charged surface, ethylene-bridged
Shake 1.0g for about 1 min with 10 mL of carbon dioxide-free octadecylsilyl silica gelfor chromatography (hybrid material) R
waterR and filter. (3.5 urn);
- temperature: 28 ± 2 "C.

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2020 Bacitracin Zinc 1-249

0.080

0.070

0.060

0.050

0.040
:::>
«
0.030

0.020 4

0.010

0.000

1. impurity F 2. impurity G 3. impurity H 4. impurity E

Figure 0466.-2. - Chromatogram for the testfor related substances of bacitracin zinc: reference solution (c)

Mobile phase acetonitrile R, solution B, waterfor - signal-to-noise ratio: minimum 50 for the peak due to
chromatographyR, methanol Rl (43:100:300:557 V/V/VIV). bacitracin A in the chromatogram obtained with reference
Flowrate 1.0 mIJmin. solution (b). .
Detection Spectrophotometer at 254 om. Limits:
Injection 100 ~lL of the test solution and reference - bacitracin A: minimum 45.0 per cent;
solutions (a) and (b). . - sum of bacitracins A, Bl, B2 and B3: minimum
77.0 per cent.
Run time 3 times the retention time of bacitracin A.
Related substances
Identification of peaks Use the chromatogram obtained with
Liquid chromatography (2.2.29) as described in the test for
reference solution (a) to identify the peaks due to impurity M
composition with the following modifications. Use the
andbacitracins A, Bl, B2 and B3 (see Figure 0466.-1)~
normalisation procedure.
Relative retention With reference to bacitracin A (retention
Injection Test solution and reference solutions (a), (b) and
= =
time about 20 min): impurity A about 0.44;
(c).
impurity B = about 0.52; impurity C = about 0.55;
bacitracin Bl = about 0.65; bacitracin B2 = about 0.67; Identification of impurities Use the chromatogram obtained
= =
bacitracin B3 about 0.81; impurity M about 0.87; with reference solution (a) to identify the peaks due to
impurities A, B, C, L, M, N, 0, P and Q (see Figure
impurity N = about 0,90; impurity L = about 0.93;
impurity 0 = about 1.2; impurities P and Q = about 1.3; 0466.-1); use the chromatogram obtained with reference
= =
impurity F about 1.6; impurity G about 1.8; solution (c) to identify the peaks due to impurities E, F, G,
= =
impurity H about 2.1; impurity E about 2.8. and H (see Figure 0466.-2).
If necessary, adjust the composition of the mobile phase by Limits:
changing the amount of organic modifier whilst keeping the - sum of impurities Land N: maximum 8.0 per cent;
ratio constant between methanol and acetonitrile. - impun'ty E: maximum 4.0 per cent;
- impurityA: maximum 3.5 per cent;
System suitability:
- ~mpu~ties B, M: for each impurity, maximum 3.0 per cent;
- peak-to-valley ratio: minimum 1.2, where Hp = height
- zmpunty C: maximum 2.5 per cent;
above the baseline of the peak due to bacitracin B2 and
- sum of impurities 0, P and Q: maximum 2.5 per cent;
H; = height above the baseline of the lowest point of the
- sum of impurities F and G: maximum 2.0 per cent;
curve separating this peak from the peak due to bacitracin
- impurityH: maximum 1.0 per cent;
B 1 in the chromatogram obtained with reference
- any other impurity: for each impurity, maximum
solution (a);
2.0 per cent;
- peak-to-valley ratio: minimum 1.1, where Hp = height
- total: maximum 23.0 per cent;
above the baseline of the peak due to impurity M and
- reporting threshold: 0.25 per cent.
H; = height above the baseline of the lowest point of the
curve separating this peak from the peak due to bacitracin The thresholds indicated under Related substances
B3 in the chromatogram obtained with reference (Table 2034.-1) in the general monograph Substances for
solution (a); pharmaceutical use (2034) do not apply.
Zinc
3.5 per cent to 5.5 per cent (dried substance).

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1-250 Bacitracin Zinc 2020

Dissolve 0.200 g in a mixture of 2.5 mL of dilute acetic acid R H

2N
H.. CH3
and 2.5 mL of water. Add 50 mL of water R, 50 mg of HycCH'
xylenol orange triturate R and sufficient
5 ~N
hexamethylenetetramine R to produce a red colour. Add 2 g of
hexamethylenetetramine R in excess. Titrate with 0.01 M y.. L-Leu- o-Glu- L-Val- L-Lys- o-Orn- L-Val- O-PheJ
sodium edetate until a yellow colour is obtained. H IT
o t L-Asn-o-Asp-L-H.IS
1 mL of 0.01 M sodium edetate is equivalent to 0.654 mg of
Zn. C.4,10-anhydro[N-[[(4R)-2-[(lS,2S)-1-amino-2-
Loss on drying (2.2.32) methylbutyl] -4,5-dihydro-l ,3-thiazol-4-yl] carbonyl] -L-
Maximum 5.0 per cent, determined on 1.000 g by drying at leucyl-D-a.-glutamyl-L-valyl-L-Iysyl-o-ornithyl-L-valyl-D-
60 DC over diphosphorus pentoxideR at a pressure not phenylalanyl-L-histidyl-D-a.-aspartyl-L-asparagine]
exceeding 0.1 kPa for 3 h. (bacitracin D3, bacitracin CIa),
ASSAY
Suspend 50.0 mg in 5 mL of waterR, add 0.5 mL of dilute
hydrochloric acid R and dilute to 100.0 mL with water R.
H~~vt3
Hl CH3
Allow the solution to stand for 30 min. Carry out the
s ~N
microbiological assay of antibiotics (2.7.2).
y. L-LeU-O-GIU-L-Val-L-Lys-o-orn-L-val-o-PheJ
STORAGE
In an airtight container. If the substance is sterile, the H l tL-Asn-o-AsP-L-HiS

container is also sterile and tamper-proof.

D.4,10-anhydro[N-[[(4R)-2-[(1S)-I-amino-2-methylpropyl]-
IMPURITIES
4,5-dihydro-1 ,3-thiazol-4-yl] carbonyl] -L-Ieucyl-D-a.-
Specified impurities A, B, C, E, F, G, H, L, M, N, 0, P, Q.
glutamyl-L-valyl-L-Iysyl-D-ornithyl-L-valyl-D-phenylalanyl-
Other detectable impurities (the following substances would, if t-hisridyl-n-e-aspartyl-t-asparagine] (bacitracin E),
present at a sufficientlevel, be detected by one or otherof the tests
in the monograph. They are limited by the general acceptance H. CH3
criterion for other/unspecified impurities. It is therefore not
necessary to identify these impurities for demonstration of O~CH,
compliance. See also 5.10. Control of impurities in substances for 5~N
pharmaceutical use) D, 1, J, K.

H~~vt3
'=)- L-Leu - 0-<3~ - L-lIe- L-Lys - o-Om- L-lie - o-PheJ

tL-Asn-- o-Asp-- L-Hls

H l - _ CH3
5 ~N E. 4,10-anhydro[N-[[2-[(2S)-2-methyl-l-oxobutyl]-1,3-
y. L-LeU-O-GIU-L-lIe-L-LYS-O-orn-OO-L-val-O-PheJ thiazol-4-yl]carbonyl]-L-Ieucyl-D-a.-glutamyl-L-isoleucyl-L-
H l tL-Asn-O-AsP-L-HiS
lysyl-D-ornithyl-L-isoleucyl-o-phenylalanyl-L-histidyl-D-a.-
aspartyl-L-asparagine], (bacitracin F),

A. 4,10-anhydro[N-[[(4R)-2-[(1S)-1-amino-2-methylpropyl]-
4,5-dihydro-l,3-thiazol-4-yl]carbonyl] -L-Ieucyl-D-a.-
glutamyl-L-isoleucyl-L-Iysyl-D-ornithyl-L-valyl-D-
phenylalanyl-L..:histidyl-D-a.-aspartyl-L-asparagine]
(bacitracin Dl, bacitracin C2),

H~~vt3
H l - _ CH3
5 ~N F. 4,10-anhydro[N-[[2-(2-methyl-l-oxopropyl)-1,3-thiazol-4-
y... L-Leu- o-Glu- L-Val- L-Lys- c-Orn -- L-lle -00- O-PheJ yl]carbonyl]-L-Ieucyl-D-a.-glutamyl-L-isoleucyl-L-Iysyl-o-
H ~ tL-Asn-O-ASP-L-HiS
ornithyl-L-isoleucyl-D-phenylalanyl-L-histidyl-D-a.-aspartyl-
L-asparagine] (bacitracin HI),

B. 4,10-anhydro[N-[[(4R)-2-[(1S)-1-amino-2-methylpropyl]- H CH3
4,5-dihydro-l,3-thiazol-4-yl] carbonyl] -L-Ieucyl-D-a.-
glutamyl-L-valyl-L-Iysyl-D-ornithyl-L-isoleucyl-D-
O~CH'
phenylalanyl-L-histidyl-D-a.-aspartyl-L-asparagine] ·5 ~N

(bacitracin D2, bacitracin C3),

'=)- L-Leu - o-Gtu- L-lle- L-Lys - c-Orn- L-Val- o-Phe

tL-Asn-- D-Asp -- t-His J

G.4,10-anhydro[N-[[2-[(2S)-2-methyl-l-oxobutyl]-1,3-
thiazol-4-yl] carbonyl]-L-Ieucyl-D-a.-glutamyl-L-isoleucyl-L-
lysyl-D-ornithyl-L-valyl-D-phenylalanyl-L-histidyl-o-a.-
asp artyl-L-asp aragine] (bacitracin H2),

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2020 Bacitracin Zinc 1-251

H~~X
~ CH3

O~CH' Hl. CH
3

S ~N s ~N

' 1 -L-Leu - o-Glu- L-Val- L-Lys - c-Orn - L-lle- O-PheJ '1-L-Leu- o-Glu> t-lle - L-Lys- o-om-L-IIe-o-PheJ

t i-Asn -- o-Asp - L-Hls t L-Asn-- o-Asp- - L-His

H.4,IO-anhydro[N-[[2-[(2S)-2-methyl-I-oxobutyl]-1,3- M.4,IO-anhydro[N-[[2-[(IS,2S)-I-amino-2-methylbutyl]-1,3-
thiazol-4-yl] carbonyl]-L-Ieucyl-n-cx-glutamyl-L-valyl-L-Iysyl- . thiazol-4-yl] carbonyl]-L-Ieucyl-n-cx-glutamyl-L-isoleucyl-L-
n-omithyl-L-isoleucyl-n...;phenylalanyl-L-histidyl-n-cx- lysyl-n-ornithyl-L-isoleucyl-n-phenylalanyl-L-histidyl-n-cx-
aspartyl-t-asparagine]: (bacitracin H3), aspartyl-t-asparagine] (bacitracin Y),

CH3 H2N ~\ CH3

)!'CH, HYCCH'
S ~N
S ~N
~ L-Leu -to- o-Glu'" L-lle - L-Lys- o-Orn - L-lle - o-PheJ

' 1 - i-Leu - o-Glu- L-lie - L-Lys - o-Orn- L-Val- o-PheJ H 0 t-Asn--o-AsP-L-HiS

tL-Asn-- o-Asp -- L-His N.4,10-anhydro[N-[[(4S)-2-[(IS,2S)-I-amino-2-

methylbutyl] -4,5-dihydro-1,3-thiazol-4-yl] carbonyl] -L-
1. ·4,1 O-anhydro [N- [[2-(2-methyl-I-oxopropyl)-1,3-thiazol-4-
leucyl-n-cx-glutamyl-L-isoleucyl-L-lysyl-D-omithyl-L-
yl]carbonyl]-L~leucyl-n-cx-glutamyl-L-isoleucyl-L-Iysyl-n­
isoleucyl-n-phenylalanyl-L-histidyl-n-cx-aspartyl-L-
omithyl-L-valyl-n-phenylalanyl-L-histidyl-n-cx-aspartyl-L-
asparagine] (bacitracin Z),
asparagine] (bacitracin II),

CH3

s
°rCH, ~N

' 1 - t-Leu - o-Glu- L-Val- L-Lys - o-Om - L-lie - O-PheJ

t L-Asn-- o-Asp-- L-His

o. Mil = 5-methylene-L-isoleucine: 4,IO-anhydro[N-[[(4R)-2-
[(IS,2S)-I-amino-2-methylbutyl]-4,5-dihydro-I,3-thiazol-
J. 4,IO-anhydro[N-[[2-(2-methyl-l-oxopropyl)-1,3-thiazol-4- 4-yl] carbonyl]-L-Ieucyl-n-cx-glutamyl-5-methylene-L-
yl]carbonyl]-L-Ieucyl-n-cx-glutamyl-L-valyl-L-Iysyl-n- isoleucyl-L-Iysyl-n-omithyl-L-isoleucyl-n-phenylalanyl-L-
omithyl-L-isoleucyl-n-phenylalanyl-L-histidyl-n-cx-aspartyl- histidyl-n-c-aspartyl-t-asparagine] (bacitracin jl ),
L-asparagine] (bacitracin 12),
H N H.. CH3

Hrz:CH'
2
H CH3

O~CH' S ~N .
S ~N
'--I-"r L-Leu- o-Glu'" L-lle - L-Lys - o-Orn -- M~-: o-PheJ
H 0 t-Asn--o-ASP-L-HiS
. ' 1 - L-Leu - o-Glu- L-Val- L-Lys - n-Om - L-Val- O-PheJ

tL-Asn-- o-Asp- L-His P. Mil = 5-methylene-L-isoleucine: 4,IO-anhydro[N-[[(4R)-2-

[(IS,2S)-I-amino-2-methylbutyl]-4,5-dihydro-I,3-thiazol-
K.4,IO-anhydro[N-[[2-[(2S)-2-methyl-l-oxobutyl]-1,3- 4-yl]carbonyl]-L-Ieucyl-n-cx-glutamyl-L-isoleucyl-L-Iysyl-n-
thiazol-4-yl]carbonyl]-L-Ieucyl-n-cx-glutamyl-L-valyl-L-Iysyl- omithyl-5-methylene-L-isoleucyl-n-phenylalanyl-L-histidyl-
D-omithyl-L-valyl-n-phenylalanyl-L-histidyl-n-cx-aspartyl-L":' n-o-asparryl-t-asparagine] (bacitracin J2),
asparagine] (bacitracin 13),
H2r
.
~'" CH3
H~.H3
H···· ~ -

H 1. CH
3
S ~N
CH2

S ~N '--1-'1 L-Leu - o-Glu - L-lle - L-Lys - o-Orn -- L-lie -io- O-PheJ

'--1-., L-Leu -- o-Glu - L-lle - L-Lys - o-Orn -- L-lle -- O-PheJ H 0 tL-Asn--o-ASP--L-HiS
H [ tL-Asn--o-AsP--L-HiS
Q.4,IO-anhydro[N-[[(4R)-2-[(lS,2S)-I-amino-2-methylpent-
L. 4,IO-anhydro[N-[[(4R)-2-[(lR,2S)-I-amino-2- 4-en-l-yl] -4,S-dihydro-1,3- thiazol-4-yl] carbonyl] -r.-leucyl-
methylbutyl] -4,5-dihydro-l,3-thiazol-4-yl] carbonyl]-L- n-cx-glutamyl-L-isoleucyl-L-lysyl-n-omithyl-L-isoleucyl-n-
leucyl-n-cx-glutamyl-L-isoleucyl-L-Iysyl-n-omithyl-L- phenylalanyl-L-histidyl-n-cx-aspartyl-L-asparagine]
isoleucyl-n-phenylalanyl-L-histidyl-n-cx~aspartyl-L­ (bacitracin J3).
asparagine] (bacitracin X), _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur

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1-252 Baclofen 2020

Reference solutionDissolve 10 mg of baclofen CRS in the

Bacloten mobile phase and dilute to 10 mL with the mobile phase.
(Ph. Bur. monograph 0653) Plate TLC silica gel G plate R.
Mobile phase anhydrous formic acid R, water R, methanol R,
NH2 chloroform R, ethyl acetate R (5:5:20:30:40 V/V/V/V/V).
( H
~co,H and enantlorner Application 5 JlL.
Development Over a path of 12 cm.
cl N Drying Allow the solvents to evaporate.
Detection Spray with ninhydrin solution R3 until the plate is
213.7 1134-47-0 slightly wet. Place in an oven maintained at 100°C for
10 min. Examine in daylight.
Action and use Results The principal spot in the chromatogram obtained
Skeletal muscle relaxant. with the test solution is similar in position, colour and size to
Preparations the principal spot in the chromatogram obtained with the
Baclofen Oral Solution reference solution.
Baclofen Tablets TESTS
PhEur '--- - _ Appearance of solution
The solution is not more opalescent than reference
DEFINITION suspension IT (2.2. 1) and not more intensely coloured than
(3RS)-4-Amino-3-( 4-chlorophenyl)butanoic acid. reference solution BYs (2.2.2, Method II).
Content Dissolve 0.50 gin 1 M sodium hydroxide and dilute to 25 mL
98.0 per cent to 101.0 per cent (anhydrous substance). with the same solvent.
CHARACTERS Related substances
Appearance Liquid chromatography (2.2.29).
White or almost white powder. Test solution Dissolve 25.0 mg of the substance to be
Solubility examined in the mobile phase and dilute to 10.0 mL with
Slightly soluble in water, very slightly soluble in ethanol the mobile phase.
(96 per cent), practically insoluble in acetone. It dissolves in Reference solution (a) Dissolve 25.0 mg of baclofen
dilute mineral acids and in dilute solutions of alkali impurity A CRS in the mobile phase and dilute to 10.0 mL
hydroxides. with the mobile phase.
It shows polymorphism (5.9). Reference solution (b) Dilute 1.0 mL of reference solution (a)
to 100.0 mL with the mobile phase.
IDENTIFICATION
First identification: B. Reference solution (c) Dilute 2.0 mL of the test solution to
100.0 mL with the mobile phase.
Second identification: A, C.
Reference solution (d) Dilute 2.0 mL of the test solution and
A. Ultraviolet and visible absorption spectrophotometry
2.0 mL of reference solution (a) to 100.0 mL with the
(2.2.25).
mobile phase.
Test solution Dissolve 70 mg in water R and dilute to
Column:
100.0 mL with the same solvent.
=
- size: 1 0.25 m, 0 = 4.0 mm;
Spectral range ·220-320 nm. '--- stationary phase: octadecylsilyl silica gel for chromatography R
Absorption maxima At 259 nm, 266 nm and 275 nm. (10 J.IlIl).
Resolution (2.2.25): minimum 1.5 for the absorbance ratio. Mobile phase Dissolve 1.822 g of sodium hexanesulfonate R in
Specific absorbance at the absorption maxima: 1 L of a mixture of 560 volumes of water R, 440 volumes of
- at 259 nm: 9.8 to 10.8; methanol R and 5 volumes of glacial acetic acid R.
- at 266 nm: 11.5 to 12.7; Flow rate 2.0 mUmin.
- at 275 nm: 8.4 to 9.3. Detection Spectrophotometer at 266 nm.
B. Infrared absorption spectrophotometry (2.2.24). Injection 20 J.LL of the test solution and reference
Preparation Discs prepared using 3·mg of substance and solutions (b), (c) and (d).
300 mg of potassium bromide R. Run time 5 times the retention time of baclofen.
Comparison baclofen CRS. System suitability Reference solution (d):
If the spectra obtained in the solid state show differences, - resolution: minimum 2.0 between the peaks due to
dissolve 0.1 g of each of the substances separately in 1 mL of baclofen and impurity A.
dilute sodium hydroxide solution R and add 10 mL of ethanol Limits:
(96 per cent) Rand 1 mL of dilute acetic acid R. Allow to - impurity A: not more than the area of the principal peak
stand for 1 h. Filter, wash the precipitate with ethanol in the chromatogram obtained with reference solution (b)
(96 per cent) R and dry in vacuo. Prepare new discs and (1.0 per cent);
record the spectra. - total: not more thanthe area of the principal peak in the
C. Thin-layer chromatography (2.2.27). chromatogram obtained with reference solution (c)
Test solution Dissolve 10 mg of the substance to be (2.0 per cent).
examined in the mobile phase and dilute to 10 mL with the Water (2.5.12)
mobile phase. Maximum 1.0 per cent, determined on 1.000 g.

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2020 Bambuterol Hydrochloride 1-253

Sulfated ash (2.4.14) Solubility

Maximum 0.1 per cent, determined on 1.0 g. Freely soluble in water, soluble in ethanol (96 per cent).
ASSAY It shows polymorphism (5.9).
Dissolve 0.1500 gin 50 mL of anhydrous acetic acid R. IDENTIFICATION
Titrate with 0.1 M perchloric acid, determining the end-point A. Infrared absorption spectrophotometry (2.2.24).
potentiometrically (2.2.20). Preparation Discs.
1 mL of 0.1 M perchloric acid is equivalent to 21.37 mg Comparison bambuterol hydrochloride CRS.
of C lO H 12ClN02 •
If the spectra obtained show differences, dissolve the
IMPURITIES substance to be examined and the reference substance
Specified impurities A. separately in a mixture of 1 volume of water Rand 6 volumes
Other detectableimpurities (the following substances toould, if of acetone R, cool in ice to precipitate and dry both
present at a sufficient leuel, be detected by one or otherof the tests precipitates in vacuo at 50°C to constant weight. Record new
in the monograph. They are limited by the general acceptance spectra using the residues.
criterion for other/unspecified impurities and/or by the general B. It gives reaction (a) of chlorides (2.3.1) ..
monograph Substances for pharmaceutical use (2034). It is
TESTS
therefore not necessary to identify these impurities for
demonstration of compliance. See also 5.10. Controlof impurities Solution S
Dissolve 4.0 g in carbon dioxide-free water R and dilute to
insubstances for pharmaceutical use) B.
20.0 mL with the same solvent.

"'rfr°
H Acidity or alkalinity
To 10 mL of solution S add 0.2 mL of methyl red solution R
and enantlorner
and 0.2 mL of 0.01 M hydrochloric acid. The solution is red.
Add 0.4 mL of 0.01 M sodium hydroxide. The solution is
CI~ yellow.
Optical rotation (2.2.7)
A. (4RS)-4-( 4-chlorophenyl)pyrrolidin-2-one, -0.10° to + 0.10°.
o Dilute 1 mL of solution S to 10 mL with carbon dioxide-free
)-:NH
'. 2
water R.
, H Related substances

if
.CO Hand enantiomer
~ 2 Liquid chromatography (2.2.29).
I Test solution Dissolve 5.0 mg of the substance to be
CI ~ examined in the mobile phase and dilute to 10.0 mL with
. the mobile phase.
B. (3RS)~5-amino-3-(4-chlorophenyl)-5-oxopentanoic acid.
Reference solution (a) Dissolve 1.0 mg oi formoterol fumaraie
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _- PhEur
dihydrate CRS in the mobile phase and dilute to 10.0 mL
with the mobile phase. Mix 0.8 mL of this solution with
0.4 mL of the test solution and dilute to. 100.0 mL with the
mobile phase.
Bambuterol Hydrochloride Reference solution (b) Dilute 1.0 mL of the test solution to
50.0 mL with the mobile phase. Dilute 2.0 mL of this
(Ph. Bur. monograph 1293) solution to 20.0 mL with the mobile phase.
Column:
- size: 1= 0.15 m, 0 = 4.6 mm;
- stationaryphase: base-deactivated octadecylsilyl silica gelfor
chromatography R (5 JlII1).
Mobile phase Dissolve 1.3 g of sodium octanesulfonate R in
430 mL of a mixture of 25 volumes of acetonitrile Rl and
75 volumes of methanolR; then mix this solution with
570 mL of 0.050 M phosphate buffer pH 3.0 prepared as
403.9 81732-46-9 follows: dissolve 6.90 g of sodium dihydrogen phosphate
monohydrate R in water R and dil~lte to 1000 mL with
Action and use water R, adjust to pH 3.0 with a 50 gIL solution of dilute
Betaj-adrenoceptor agonist; bronchodilator. phosphoric acid R.
PhEur _ Flow rate 1.5 mUmin.
Detection Spectrophotometer at 214 nm.
DEFINITION
Injection 20!ll-; inject the mobile phase as a blank.
5-[(lRS)-2-[(1,1-Dimethylethyl)amino]-I-hydroxyethyl]-1,3-
phenylene bis(dimethylcarbamate) hydrochloride. Run time 1.5 times the retention time of bambuterol.
Content
=
Retention time F onnoterol about 7 min;
bambuterol = about 9 min. If necessary, adjust the
98.5 per cent to 101.5 per cent (anhydrous substance).
composition of the mobile phase; increase the content of
CHARACTERS phosphate buffer to increase the retention time.
Appearance System suitability Reference solution (a):
White or almost white, crystalline powder.

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1-254 Barbital 2020

- resolution: minimum 5.0 between the peaks due to

bambuterol and formoterol.
Limits:
- impurities A~ B~ C~ D~ B~ F: for each impurity, not more
than the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.2 per cent);
- total: not more than 3 times the area of the principal peak
in the chromatogram obtained with reference solution (b) E. 5-acetyl-1,3-phenylene bis(dimethylcarbamate),
(0.6 per cent);
- disregard limit: 0.25 times the area of the principal peak in
the chromatogram obtained with reference solution (b)
(0.05 per cent); disregard any peak due to the mobile
phase.
Water (2.5.12)
Maximum 0.5 per cent, determined on 0.500 g.
Sulfated ash (2.4.14)
Maximum 0.1 per cent, determined on 1.0 g. F. 5-[[(l,1-dimethylethyl)amino]acetyl]-1,3-phenylene bis
ASSAY (dimethylcarbamate).
Dissolve 0.320 gin 50 mL of ethanol (96 per cent) R and add _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
5 mL of 0.01 M hydrochloric acid. Carry out a potentiometric
titration (2.2.20), using 0.1 M sodium hydroxide. Read
the volume added between the 2 points of inflexion.
1 mL of 0.1 M sodium hydroxide is equivalent to 40.39 mg of Barbital
ClsH30C1N30S'
(Ph. Bur. monograph 0170)
IMPURITIES
Specified impurities A~ B~ C~ D~ B~ F.

H... OH H
HO~NX.CH3
Y H,C CH,
and enantiomer

OH 184.2 57-44-3

A. (lRS)-1-(3,5-dihydroxyphenyl)-2-[(1,1- Action and use

dimethylethyl) amino ]ethanol (terbutaline), Barbiturate.
PhEur ~ _

DEFINITION
Barbital contains not less than 99.0 per cent and not more
than the equivalent of 101.0 per cent of
5,5-diethylpyrimidine-2,4,6(lH,3H,5H)-trione, calculated
with reference to the dried substance.
CHARACTERS
B. 5-[( 1RS)-1 ,2-dihydroxyethyl] -1,3-phenylene bis A white or almost white, crystalline powder or colourless
(dimethylcarbamate), crystals, slightly soluble in water, soluble in boiling water and
in alcohol. It forms water-soluble compounds with alkali
CH3 H OH hydroxides and carbonates and with ammonia.
H'C'~YO~. ~XCH3
o Y H,C CH,
and enantiomer
IDENTIFICATION
First identification: A~ B.
Secondidentification: A~ C~ D.
OH
A. Determine the melting point (2.2.14) of the substance to
C. 3-[(lRS)-2-{(1,1-dimethylethyl)amino]-1-hydroxyethyl]-5- be examined. Mix equal parts of the substance to be
hydroxyphenyl dimethylcarbamate, examined and barbital CRS and determine the melting point
of the mixture. The difference between the melting points
(which are about 190°C) is not greater than 2°C.
B. Examine by infrared absorption spectrophotometry
and enantiomer
(2.2.24), comparing with the spectrum obtained with
barbital CRS.
C. Examine by thin-layer chromatography (2.2.27), using
silica gel GF254R as the coating substance.
Test solution Dissolve 75 mg of the substance to be
D. 5-[(lRS)-1-hydroxyethyl]-1,3-phenylene bis examined in alcohol R and dilute to 25 mL with the same
(dimethylcarbamate), solvent.

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2020 Barium Sulfate 1-255

Reference solution Dissolve 75 mg of barbital CRS in

alcohol R and dilute to 25 mL with the same solvent.
Barium Sulfate
Apply separately to the plate 10 JlL of each solution. Develop (ph. Bur. monograph 0010)
over a path of 18 ern using the lower layer of a mixture of
5 volumes of concentrated ammonia R, 15 volumes of alcohol R BaS04 233.4 7727-43-7
and 80 volumes of chloroform R. Examine immediately in
Action and use
ultraviolet light at 254 nm. The principal spot in the
Radio-opaque substance used in the investigation of the
chromatogram obtained with the test solution is similar in
gastro-intestinal tract.
position and size to the principal spot in the chromatogram
obtained with the reference solution. Preparation
Barium Sulfate for Suspension
D. It gives the reaction of non-nitrogen substituted
barbiturates (2.3.1). PhEur _

TESTS CHARACTERS
Appearance of solution Appearance
Dissolve 1.0 g in a mixture of 4 mL of dilute sodiumhydroxide Fine, white or almost white powder, free from gritty particles.
solution Rand 6 mL of water R. The solution is clear (2.2.1)
Solubility
and not more intensely coloured than reference solution Y 6
Practically insoluble in water and in organic solvents. It is
(2.2.2, Method II).
very slightly soluble in acids and in solutions of alkali
Acidity hydroxides.
Briill.0 g with 50mL of waterR for 2 min, allow to cool
and filter. To 10 mL of the filtrate add 0.15 mL of methylred
IDENTIFICATION
solution R. The solution is orange-yellow. Not more than A. Boil a suspension of 0.2 g with 5 mL of a 500 gIL
0.1 mL of 0.1 M sodium hydroxide is required to produce a solution of sodium carbonate R for 5 min; add 10 mL of
pure yellow colour. waterR, filter and acidify a part of the filtrate with dilute
hydrochloric acid R. The solution gives the reactions of sulfates
Related substances (2.3.1).
Examine by thin-layer chromatography (2.2.27), using silica
B. Wash the residue collected in the preceding test with
gel GF254 R as the coating substance.
3 successive small quantities of water R. To the residue add
Test solution Dissolve 1.0 g of the substance to be examined 5 mL of dilute hydrochloric acid R, filter and add to the filtrate
in alcohol R and dilute to 100 mL with the same solvent. 0.3 mL of dilute sulfuric acid R. A white precipitate is formed
Reference solution Dilute 0.5 mL of the test solution to that is insoluble in dilute sodium hydroxide solution R.
100 mL with alcohol R.
TESTS
Apply separately to the plate 20 IlL of each solution. Develop
Solution S
over a path of 15 em using the lower layer of a mixture of
To 20.0 g add 40 niL of distilled water Rand 60 mL of dilute
5 volumes of concentrated ammonia R, 15 volumes of alcohol R
acetic acid R. Boil for 5 min, filter and dilute the cooled
and 80 volumes of chloroform R. Examine immediately in
filtrate to 100 mL with distilled water R.
ultraviolet light at 254 nm. Spray with diphenylcarbazone
mercuric reagent R. Allow the plate to dry in air and spray Acidity or alkalinity _
with freshly prepared alcoholic potassium hydroxide solution R Heat 5.0 g with 20 mL of carbon dioxide-free water R on a
diluted 1 in 5 with aldehyde-free alcohol R. Heat at 100 DC to water-bath for 5 min and filter. To 10 mL of the filtrate add
105 DC for 5 min and examine immediately. When examined 0.05 mL of bromothymol blue solution R1. Not more than
in ultraviolet light and after spraying, any spot in the 0.5 mL of 0.01 M hydrochloric acid or 0.01 M sodium
chromatogram obtained with the test solution, apart from the hydroxide is required to change the colour of the indicator.
principal spot, is not more intense than the spot in the Acid-soluble substances
chromatogram obtained with the reference solution Maximum 0.3 per cent.
(0.5 per cent). Evaporate 25 mL of solution S to dryness on a water-bath
Loss on drying (2.2.32) and dry to constant mass at 100-105 DC. The residue weighs
Not more than 0.5 per cent, determined on 1.00 g by drying a maximum of 15 mg.
in an oven at 105 DC. Oxidisable sulfur compounds
Sulfated ash (2.4.14) Shake 1.0 g with 5 mL of water R for 30 s and filter. To the
Not more than 0.1 per cent, determined on 1.0 g. filtrate add 0.1 mL of starchsolution R, dissolve 0.1 g of
potassium iodideR in the mixture, add 1.0 mL of a freshly
ASSAY
prepared 3.6 mgIL solution of potassium iodate Rand 1 mL
Dissolve 85.0 mg in 5 mL of pyridineR. Add 0.5 mLof
of 1 M hydrochloric acid and shake well. The colour of the
thymolphthalein solution Rand 10 mL of siluer nitrate solution
solution is more intense than that of a standard prepared at
in pyridineR. Titrate with 0.1 M ethanolic sodium hydroxide
the same time and in the same manner,but omitting the
until a pure blue colour is obtained. Carry out a blank
potassium iodate.
titration.
Soluble barium salts
1 mL of 0.1 M ethanolic sodium hydroxide is equivalent to
Maximum 10 ppm.
9.21 mg ofCsH12N203'
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur To 2.5 mL of a 0.2 mgIL solution of barium nitrate R in a
mixture of 30 volumes of ethanol (96 per cent) Rand
70 volumes of uiaterR, add 10 mL of dilute sulfuric acid R.
Shake and allow to stand for 5 min. To 1 mL of this solution
add 10 mL of solution S. Prepare a standard in the same

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1-256 Barium Sulfate for Suspension 2020

manner using 10 mL of barium standard solution (2 ppm Add 10 mL of a 40% w/v solution of ammonium acetate,.
Ba) R instead of solution S. 25 mL of a 10% w/v solution of potassium dichromate and
After 10 min, any opalescence in the test solution is not 10 g of urea. Cover and digest in a hot-air oven at 80° to 85°
more intense than that in the standard. for 16 hours. Filter whilst still hot through a sintered-glass
filter (ISO 4793, porosity grade 4, is suitable), washing the
Loss on ignition
precipitate initially with a 0.5% w/v solution of potassium
Maximum 2.0 per cent, determined on 1.0 g at
dichromate and finally with 2 mL of water. Dry to constant
600 ± 50°C.
weight at 105°. Each g of the residue is equivalent to
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
0.9213 g of barium sulfate, BaS04'

Barium Sulfate for Suspension Beclometasone Dipropionate

Barium Sulphate for Suspension
Anhydrous Beclometasone Dipropionate
Action and use
Radio-opaque preparation used in the investigation of the (Ph. Bur. monograph 0654)
gastro-intestinal tract.
Preparation
Barium Sulfate Oral Suspension

DEFINITION
Barium Sulfate for Suspension is a dry mixture of Barium
Sulfate with a suitable dispersing agent and may contain
suitable flavours and suitable antimicrobial preservatives.
o
Content of barium sulfate, BaS04
90.0 to 110.0% of the stated amount.
521.0 5534-09-8
CHARACTERISTICS
A fine, white or creamy white powder. Action and use
Glucocorticoid.
IDENTIFICATION
Preparations
A. Ignite 1 g to constant weight. To 0.2 g of the residue add
Beclometasone Cream
5 mL of a 50% w/v solution of sodium carbonate and boil for
5 minutes. Add 10 mL of water and filter. Reserve the Beclometasone Aqueous Nasal Spray
residue for test B. Acidify a portion of the filtrate with Beclometasone Inhalation Powder
2M hydrochloric acid. The solution yields the reactions Beclometasone Inhalation Powder, pre-metered
characteristic of sulfates, Appendix VI. Beclometasone Ointment
B. Wash the residue reserved in test A with water, add 5 mL Beclometasone PressurisedInhalation
of 2M hydrochloric acid, mix well and filter. Add 0.3 mL of 1M
sulfuric acid to the filtrate. A white precipitate is produced PhEur _
which is insoluble in 2M hydrochloric acid.
DEFINITION
TESTS 9-Chloro-11 ~-hydroxy-16~-methyl-3,20-dioxopregna-1,4­
Acidity or alkalinity diene-17,21-diyl dipropanoate.
pH of an aqueous suspension containing the equivalent of Content
60% w/w of Barium Sulfate or, for lower strengths, the 96.0 per cent to 102.0 per cent (dried substance).
aqueous suspension at the strength of intended use, 3.5 to
8.5, Appendix V L. CHARACTERS
Appearance
Loss on drying
White or almost white, crystalline powder.
When dried at -105° for 4 hours, loses not more than 1.0% of
its weight. Use 1 g. Solubility
Practically insoluble in water, freely soluble in acetone,
ASSAY sparingly soluble in ethanol (96 per cent).
To a quantity containing 0.6 g of Barium Sulfate in a
platinum dish add 5 g of sodium carbonate and 5_g of IDENTIFICATION
potassium carbonate sesquihydrate and mix. Heat to 1000° and A. Infrared absorption spectrophotometry (2.2.24).
maintain at this temperature for 15 minutes. Allow to cool Comparison anhydrous beclometasone dipropionate CRS.
and suspend the residue in 150 mL of water. Wash the dish B. Treat 25 mg by the oxygen-flask method (2.5.10). Use a
with 2 mL of 6M acetic acid and add the washings to the mixture of 1 mL of 1 M sodium hydroxide and 20 mL of
suspension. Cool in ice and decant the supernatant liquid, waterR to absorb the combustion products. The solution
transferring as little of the solid matter as possible to the gives reaction (a) of chlorides (2.3.1).
filter. Wash the residue with successive quantities of a
C. Loss on drying (see Tests).
2% w/v solution of sodium carbonate until the washings are
free from sulfate and discard the washings. Add 5 mL of 2M TESTS
hydrochloric acid to the filter, wash through into the vessel Specific optical rotation (2.2.7)
containing the bulk of the solid matter with water, add 5 mL + 108 to + 115 (dried substance).
of hydrochloric acid and dilute to 100 mL with water.

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2020 Beclometasone Dipropionate 1-257

Dissolve 0.100 g in ethanol (96 per cent) R and dilute to - peak-to-valley ratio: minimum 1.5, where Hp = height
10.0 mL with the same solvent. above the baseline of the peak due to impurity D and
Related substances H; = height above the baseline of the lowest point of the
Liquid chromatography (2.2.29). CUIVe separating this peak from the peak due to
bec1ometasone dipropionate.
Solvent mixture Mobile phase A, mobile phase B
(45:55 VIV)o Limits:
- correction factors: for the calculation of content, multiply
Test solution (a) Dissolve 50.0 mg of the substance to be