British Pharmacopoeia 2016

Volume I
British Pharmacopoeia 2016

Volume I
The British Pharmacopoeia Commission has caused this British
Pharmacopoeia 2016 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 Eighth Edition of the European Pharmacopoeia
(2013), as amended by Supplements 8.1 to 8.5, 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 2016

see Notices

London: The Stationery Office

In respect of Great Britain:
THE DEPARTMENT OF HEALTH
In respect of Northern Ireland:

THE DEPARTMENT OF HEALTH, SOCIAL SERVICES AND

PUBLIC SAFETY
© Crown Copyright 2015
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: Dr S Atkinson,
MHRA, 5th Floor, 151 Buckingham Palace Road, London SW1W 9SZ.
First Published 2015

ISBN 978 Oil 3230 006

British Pharmacopoeia Commission Office:
MHRA
5th Floor
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FOREWORD

The British Pharmacopoeia (BP), after 150 years of publication, continues to help ensure the quality of
medicinal substances globally. One of its key attributes has been to take advantage of novel science and
this edition of the BP is no different.

Authentication of botanical constituents, to ensure the safety and quality of Traditional Herbal Medicines,
provides many challenges. With advances in molecular genetics, however, reliable methods of identifying
herbs are now available. This has enabled the BP and the National Institute for Biological Standards and
Control (NIBSC) to work collaboratively and successfully on a pilot project to determine the
deoxyribonucleic acid (DNA) profile of Ocimum tenuiflorum.

The BP 2016 introduces the species specific sequence of the selected barcode region in a new BP
Appendix as an identification method for Ocimum tenuiflorum. General guidance on how to conduct
DNA-based identification methods for herbal drugs is also included in this new Appendix. In addition to this
innovation, the BP 2016 publishes new and revised monographs for substances and products used in a
wide range of medicines. The collegial partnerships between the staff, the appointed experts of the British
Pharmacopoeia Commission and international partners have enabled these important quality standards to
be made publicly available.

The significant progress made jointly by the BP and NIBSC facilitates the reliable authentication of herbal
drugs and will enhance the protection of public health, the primary objective of the Medicines and
Healthcare products Regulatory Agency.

Professor Sir Michael Rawlins

Chairman
Medicines and Healthcare products Regulatory Agency
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 Tide
GENERAL NOTICES
MONOGRAPHS
Medicinal and Pharmaceutical Substances (A - 1)
Contents of Volume II
NOTICES
GENERAL NOTICES
MONOGRAPHS
Medicinal and Pharmaceutical Substances Q - Z)
Contents of Volume m
NOTICES
GENERAL NOTICES

MONOGRAPHS
Formulated Preparations: General Monographs
Formulated Preparations: Specific Monographs

I-vii
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
 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 eighth edition of the European Pharmacopoeia
comprising, unless otherwise stated, the main volume, published in 2013, 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 theừ inclusion in this
Pharmacopoeia neither conveySj nor implies, licence to manufacture.
Effective dates New and revised monographs of national origin enter into force on
1 January 2016. 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 Dừectorate 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 Dừectives 2001/82/ECj 2001/83/EC and 2003/63/ECj as
amended, on medicines for human and veterinary use.

I-ix
Preface

The British Pharmacopoeia Commission has caused this British

Pharmacopoeia 2016 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 2016 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 Pharmacopoeial standards are designed to
complement and assist the licensing and inspection processes and are part
of the overall system for safeguarding purchasers and users of medicinal
products in the U K
The British Pharmacopoeia Commission wishes to record its appreciation of
the services of all those who have contributed to this important work.
British Pharmacopoeia
Commission

The British Pharmacopoeia Commission is appointed, on behalf of the

Secretaiy of State for Health, by the Department of Health’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.

I-xi
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;
(2) To carry out members appraisals in accordance with Department of
Health policies and timelines;
(3) To participate in the process to appoint/re-appoint members of the
British Pharmacopoeia Commission.
Expert Advisory Groups, Panels
o f 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.

I-xiii
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
Chairs 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.
 Membership of the British
Pharmacopoeia Commission

The list below includes those members who served during the period 2014
to 2015.
Chair Professor Kevin M G Taylor BPharm PhD FRPharmS
Professor of Clinical Pharmaceutics, UCL School of Pharmacy
Vice-Chair Professor Alastair Davidson BSc PhD FRPharmS
Visiting Professor of Pharmaceutical Sciences, University of Strathclyde
Professor Donald Cairns BSc PhD MRPharmS CSci CChem FRSC
Head: School of Pharmacy and Life Sciences, Robert Gordon University,
Aberdeen
Mr Barry Capon CBE MA DL {Lay representative)
Former Non-executive Director, Norfolk and Suffolk NHS Foundation Trust
Dr Graham D Cook BPharm PhD MRPharmS
Senior Director, Process Knowledge!Quality by Design, Pfizer
Mr Andrew Coulson BVetMed MSc MRCVS
Member of the Royal College of Veterinary Surgeons; former Superintending
Inspector, Science & Research Group, The Home Office
Mr Christopher Goddard BSc DIS CSci EurChem CChem FRSC
Quality Control Technical Manager, Recipharm Limited
Dr Keith Helliwell BPharm PhD
Senior Technical Adviser, William Ransom & Son PLC
Dr Rodney L Horder BPharm PhD MRPharmS
Former Divisional Vice President, European Quality and Regulatory Strategy,
Abbott
Dr Gerard Lee BPharm PhD FRPharmS MRSC CChem
Former Group Manager, British Pharmacopoeia and Laboratory Services
(MHRA); former Secretary & Scientific Director of the British Pharmacopoeia
Commission
Dr Brian R Matthews BPharm PhD FRPharmS FTOPRA MRI
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
Dr Ronald Torano BSc PhD MRSC CChem
Pharmacopoeial Intelligence and Advisory Specialist; GlaxoSmithKline

I-xv
 Dr Lincoln Tsang BPharm LLB PhD FRSC FIBiol FRSA FRPharmS
Solicitor
Life Sciences Lawyer; Partner, Arnold & Porter LLP
Mrs Josephine Turnbull T-T R {Lay representative)
Former Chair of Tees, Esk and Wear Valley NHS Foundation Trust
Dr Paul Varley BSc PhD
Vice President of Biopharmaceutical Development, Medimmune Limited
Professor Elizabeth Williamson BPharm PhD MRPharmS
Former Professor of Pharmacy, University of Reading
Secretary and Scientific Dr Samantha Atkinson BSc MSc PhD MRSC
Director Visiting Fellow, University of Reading

I-xvi
 Membership 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 2014 to 2015.

EXPERT ADVISORY GROUPS

ABS: Antibiotics R L Horder (Chair), G Cook (Vice-Chair), P Ellis, E Flahive, A Gibson,
V Jaitely, A Livingstone, W Mann, J Miller, N Thomas, B White, I R
Williams
BIO: Biological and L Tsang (Chair), P Varley (Vice-Chair), L Bisset*, A F Bristow*, C Bums,
Biotechnological D H Calam, K Chidwick*, A Cook*, J Cook*, L Findlay*, S Gill,
Products E Griffiths, C Jones*, A Kippen*, B Patel, A M Pickett*, T Pronce,
L Randon, I Rees*, S Schepelmann*, D Sesardic, P Sheppard,
P Stickings*, W J Tarbit, A H Thomas, R Thorpe, M Wadhwa*
(Corresponding members A Onadipe, J N A Tettey)
HCM: Herbal and E Williamson (Chair), L A Anderson (Vice-Chair), P Anderson, A Bligh,
Complementary T Chapman, A Charvill, S Gibbons, K Helliwell, P Hylands, C Leon, R
Medicines Middleton, A C Moffat, B Moore, M Pires, M Rowan, K Strohfeldt-
Venables, J Sumal*, P Viner, C Welham, C Wright, K Zhao
(Corresponding members SS Handa, A Krauss, Z-T Wang)
MCI: Medicinal A G Davidson (Chair), D Cairns (Vice-Chair), M Ahmed, J C Berridge,
Chemicals M Broughton, A J Caws, P Fleming, A James, V Loh, W J Lough,
D Malpas
MC2: Medicinal G Cook (Chair), C T Goddard (Vice-Chair), M Cole, J Cowie, D Edwards,
Chemicals A Gibson, J Lim, J Miller, P Murray, J Qiu, A Ruggiero, M Turgoose,
N Wynne
(Corresponding members M Brits, W Sherwin)
MC3: Medicinal V Fenton-May (Chair), E Williamson (Vice-Chair), M Almond, S Arkle,
Chemicals J Beach, J Beaman, C T Goddard, P Hampshire, W K L Pugh,
B Rackstraw, R Torano, M Tubby, I R Williams
NOM: Nomenclature J K Aronson (Chair), L Tsang (Vice-Chair), M Ahmed, D Mehta,
G P Moss, R Thorpe
(Corresponding members R G Balocco Mattavelli, E M Cortes Montejano,
J S Robertson)
PCY: Pharmacy R L Horder (Chair), B R Matthews (Vice-Chair), M Ahmed*, M Aulton,
E Baker, J Beach, N Broad, G Davison, G Eccleston, D Elder, B Granell-
Villen, J Lim*, R Lowe, J MacDonald, J F McGuire, T Purewal,
L Randon, K Taylor
(Corresponding member J Churchill)
Denotes a specialist member.

I-xvii
 ULM: Unlicensed M G Lee (Chair), V Fenton-May (Vice-Chair), G Bennett, S Branch,
Medicines D Caulfield, A Charvill, W Goddard, N Hussain, S Jones, M A Oldcome,
N J Precious, J Rothwell, M Santdllo, J Smith, A Sully, P Weir

PANELS OF EXPERTS
BLP: Blood K Chidwick, A R Hubbard, J More, P Varley
Products
CX: Excipients B R Matthews (Chair), C Mroz (Vice-Chair), E Anno, C Cable, R
Cawthome, W Cook, D Deutsch, N Hussain, M I Robertson, K Slevin
IGC: Inorganic and C T Goddard (Chair), M Almond, S Atherton, S Boland, A C Cartwright,
General Chemicals D Caulfield, P Henrys, G Lay, D Malpas, C Mroz, D Riches
MIC: Microbiology V Fenton-May (Chair), S Denyer, D P Hargreaves, B R Matthews,
P Newby
RAD: Radioactive J Ballinger, J Brain, D Graham, S R Hesslewood, G Inwards, P Maltby, R
Materials D Pickett, R Smith, S Waters
VET: Veterinary E Williamson (Chair), A Coulson (Vice-Chair), A Cairns, S Cockbill,
Medicines D Evans, E Flahive, P Lees, B Ward
VIP: Veterinary A M Brady, R Banks, K Redhead, J Salt, P W Wells, R Woodland
Immunological
Products

WORKING PARTIES
AQbD: Analytical G Cook (Chair), S Brown, S Ellison, M Hanna-Brown, S Jones,
Quality by Design D Makohon, P Nethercote, E Razzano
(Corresponding member K Barnett)
DNA: Identification K Helliwell (Chair), J Hawkins, E Mee, A Slater, E Williamson
Techniques
MCS: Microscopy E Williamson (Chair), R Arroo, R Reck, K Helliwell, K MacLellan Gibson

I-xviii
 Current British Pharmacopoeia
Staff

Secretariat M VaUender (Editor-in-Chief)

S Young (.Head of Analytical Science)

M Barrett, H Corns, P Crowley, A Evans, J Francomb, A Gibb, P Holland,

G Li-Ship, R A Pask-Hughes, C Pitt, J Pound, F J Swanson, M Whaley
NIBSC-based Staff C Howard, C Lockie-Williams
Administrative B F Delahunty, W Jeffries, J Paine

ISO 9001
FS 27268
 Current British Pharmacopoeia
Laboratory Staff

K Courtney (Laboratory Manager)

A Ciesluk, D Colmer, J Elliot, C Galdino, P Gallagher, S Humphries, R
Mannan, C Marcolan, A Murphy, M Nanasi, A Panchal, E Sanderson,
N Vadukal, A Vasilaki, M Wallis, S Wilson

ISO 9001
FS 27613

I-xx
Current Staff of the Publisher of
the British Pharmacopoeia

J Hook (MD Public Sector EMEA)

A Prince (Client Services Director)
N Billington (Client Services Manager)
A Hughes (Account Manager)
C Hackett (.Project Manager)
P Allard, A Dampier, M Grant, A Hood, S Page, M Parka, N Pope,
M Rainbird, A Ray, P Relfe, C Spary, J Stoker, I Webb, K Williams

ISO 9001
FS 22428

I-xxi
2016 Introduction I-xxiii

Introduction

Triennial Review of the British Pharmacopoeia Commission

The Department of Health conducted a Triennial Review of the British
Pharmacopoeia Commission (BPC) to provide assurance to the Department
and the public that the functions of the Commission are required and that
it is operating effectively. The review of the BPC was undertaken in two
stages, the first examined the functions and form of the BPC and the
second examined the efficiency, governance and performance of the BPC.
The recommendation from the review of form and function is that “the BP
Commission should continue to deliver its existing functions as an Advisory
Non-Departmental Public Body” . This model facilitates the BP
Commission to be in a position close to the Medicines and Healthcare
products Regulatory Agency whilst being independent of it and facilitates
harnessing potential information, communication, laboratory and expertise
synergies.
The evidence gathered from the review of efficiency, governance and
performance suggested that “the BP commission operates efficiently, is
mostly compliant with the principles of good corporate governance and is
considered to be a leading pharmacopoeia. In particular, stakeholders
highlighted the BP Commission’s innovative work, the dedication and
expertise of members and the Secretariat, industry engagement and
transparency” .
The Triennial Review report, published in March 2015, makes a number of
m inor recommendations which will be taken forward by the BPC
Secretariat. The full report of the Review can be found on the website
www.gov.uk/government/consultations/british-pharmacopoeia-commission-
triennial-review.
British Pharmacopoeia 2016
The British Pharmacopoeia 2016 supersedes the British Pharmacopoeia
2015. 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 almost 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) 2016,
incorporates all the monographs of the 8th Edition of the European
Pharmacopoeia, as amended by Supplements 8.1 to 8.5. The user of the
British Pharmacopoeia thereby benefits by finding within this
comprehensively indexed compendium all current United Kingdom
pharmacopoeial standards for medicines for human use.
I-xxiv Introduction 2016

The BP 2016 comprises six volumes as follows.

Volumes I and II Medicinal Substances

Volume III Formulated Preparations: General
Monographs
Formulated Preparations: Specific
Monographs
Volume IV 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') 2016

Effective Date The effective date for British Pharmacopoeia monographs in this edition is
1 January 2016.

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.
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 2016 is given at the end of this introduction. It includes 37
new monographs of national origin and 25 new monographs reproduced
from the 8th Edition of the European Pharmacopoeia as amended by
Supplements 8.1 to 8.5.
Traditional H erbal M edicines; Homoeopathic Preparations
Work is continuing on the development of monographs for herbs used in
traditional herbal medicines and homoeopathic medicines. The Latin
scientific names cited in BP monographs for herbal drugs are consistent
with the advice provided by the Medicinal Plant Names Services at the
Royal Botanic Gardens, Kew. As stated in previous editions, the
requirements for the quality of the material are provided in the monograph
to set the standards for Traditional Herbal Medicines in the UK and to
assist the UK Traditional Herbal Medicines Registration Scheme. The
British Pharmacopoeia Commission, however, has not assessed the safety
and efficacy of the materials in traditional use.
2016 Introduction I-xxv

Likewise, the British Pharmacopoeia Commission has not assessed the

safety and efficacy of materials for use in homoeopathic preparations for
which monographs are published.
This edition sees the first publication of a DNA-based identification method
for Holy Basil. The user benefits from modem technology for the
identification of herbal drugs, providing a direct measure of the species in
use (see under Appendices).
Unlicensed Medicines
With this new edition, a further 5 monographs for unlicensed formulations
have been added. These individual monographs are characterised by a
statement that they are not currently licensed in the United Kingdom. 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.

Revisions A significant number (141 comprising 87 technical revisions and 54

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.
For those texts reproduced from the European Pharmacopoeia, the
European Directorate for the Quality of Medicines & Healthcare (EDQM)
database (see below, under Websites) provides information on revisions of
the monographs or other texts on a historical basis, beginning from the 5th
Edition of the European Pharmacopoeia.
Title Changes
Four monograph titles have been amended in this edition. The list of
changes is appended at the end of this Introduction.
Anti-epiletic Drugs (AEDs)
The Commission on Human Medicines (CHM) reviewed reports of
spontaneous adverse reactions received by the Medicines and Healthcare
products Regulatory Agency (MHRA) and publications that reported
potential harm arising from switching of oral preparations of AEDs in
patients previously stabilised on manufacturer’s product. Following this
review, CHM concluded that reports of loss of seizure control and/or
worsening of side effects around the time of switching between products
could be explained as chance associations, but that a causal role of
switching could not be ruled out in all cases. This includes switching
between branded original and generic products, and between different
generic products of a particular drug.
I-xxvi Introduction 2016

As a consequence of the conclusion by CHM, the Medicines and

Healthcare products Regulatory Agency circulated guidance to stakeholders
on the non-interchangeability of anti-epileptic drugs. The guidance can be
found on the website www.gov.uk/drug-safety-update/antiepileptic-drugs.
In considering the published monographs for AEDs, the BP Commission
recommended that statements would be included in monographs for orally
administered category 1 AEDs, that is, those where products were not
interchangeable and category 2 AEDs, that is, products that were changed
based on clinical judgement. Statements that category 1 products “are not
interchangeable” and category 2 products “may not be interchangeable”
have been included in AEDs monographs in this edition.
A suitable statement has also been added to one monograph for
antiepileptic unlicensed medicine to reflect that different formulations may
vary in bioavailability and patients should be adequately monitored.
Inhaled Products
A programme of revision to BP monographs for inhaled products has been
established and the changes will be introduced in future editions of the
British Pharmacopoeia.

Omissions Thirty five monographs have been omitted from the British Pharmacopoeia
2016. The list of omissions is appended at the end of this Introduction.
In line with recommendations from the Commission on Human Medicines
and the British Pharmacopoeia Commission, reference to chloroform as an
ingredient in licensed medicines has been removed from all affected
monographs in this publication. This has been through either removing the
monograph from the publication or deleting the formula and/or method of
preparation.

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

Appendices Two new Appendices to harmonise with the European Pharmacopoeia were
first published in the British Pharmacopoeia 2015 electronic updates. These
have been consolidated in the new edition as follows.
Appendix VUIX: Methyl, Ethyl and Isopropyl Toluenesulfonate in Active
Substances (Ph. Eur. method 2.5.40)
Appendix XV K: Carrier Proteins for the Production of Conjugated
Polysaccharide Vaccines for Human Use (Ph. Eur. method 5.2.11)
A new BP method, Appendix XI V: Deoxyribonucleic Acid Based
Identification Techniques for Herbal Drugs, is introduced in this edition
and is accompanied by a worked example for Holy Basil.

Supplementary One new Supplementary Chapter to harmonise with the European

Chapters Pharmacopoeia was first published in the British Pharmacopoeia 2015
electronic updates. It has been consolidated in the new edition as follows.
2016 Introduction I-xxvii

Supplementary Chapter VII C: Monographs on Herbal Drugs (Ph. Eur.

General Chapter 5.23)
Supplementary Chapter I O: Inhaled Products This Supplementary
Chapter has been reintroduced in this edition to reflect the revised BP
policy on monographs for inhaled products.
Supplementary Chapter I E: Dissolution Testing o f Solid Oral
Dosage Forms This Supplementary Chapter has been updated to reflect
the monographs that are omitted from this edition.
Supplementary Chapter V: Unlicensed Medicines This Supplementary
Chapter has been amended to recommend that use of chloroform as an
ingredient in unlicensed medicines should be avoided.

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 2015 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) 2016.
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 lPh. Eur. monograph xxxx\ Where the title in the British
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. Eur.\
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.
I-xxviii Introduction 2016

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

be taken in isolation. A valid interpretation of any particular requirement
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
(iv) where appropriate, 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 phaimacopoeial 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.
Expert Advisory G roups; Panels of Experts; Working Parties
The British Pharmacopoeia Commission has reviewed the membership of
all of its Expert Advisory Groups, Panels of Experts and Working Parties.
The tenure of appointed members runs for a period of 4 years from 1
January 2015 and is renewable. Criteria for the appointment of experts are
available on the consolidated website (www.pharmacopoeia.com) .
Panels of Experts The British Pharmacopoeia Commission has changed
the status of the former Working Party on Excipients to a Panel of Experts
based on its current workload.
Working Parties The British Pharmacopoeia Commission has established
3 new Working Parties as follows. Two of the Working Parties,
DNA: Identification Techniques and MCS: Microscopy, relate to the
programme of work on herbal analysis. The third Working Party, AQbD:
Analytical Quality by Design, has been established to support the work of
the joint MHRA-BP Analytical Quality by Design project. The joint project
examines the application of Quality by Design concepts to analytical
methods and includes representation from the MHRA Licensing Division,
the Good Manufacturing Practice Inspectorate and industry.

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 (www.pharmacopoeia.com).

Websites B ritish Pharm acopoeia Websites

The British Pharmacopoeia websites, www.pharmacopoeia.co.uk and
www.pharmacopoeia.com, have been consolidated and the new website,
https://www.pharmacopoeia.com, is now available, containing information
relating to the British Pharmacopoeia and allowing subscribers to access the
British Pharmacopoeia 2016 and British Pharmacopoeia (Veterinary) 2016
online and British Approved Names publications.
The consolidated website provides new and improved functionality together
with greater access to improved BP-supporting resources. These resources
include freely available example chromatograms, herbal micrographs and
omitted surgical materials monographs for all users. Draft new and revised
texts of the BP will continue to be placed on the website in an improved
and more accessible way.
2016 Introduction I-xxix

Subscribers to the BP online will find that these resources will also be
linked with relevant texts and directly accessible from the BP online
content.
Access to previous editions of the BP will be available as a BP archive
product for purchase by new and existing BP online subscribers. The
content of the archive will start from the BP 2014 onwards and will grow
year-on-year as superseded editions are added to the archive.
Improvements and new features have also been made to the BPCRS
catalogue and ordering facility. Users will be able to receive notifications on
the status of out-of-stock items and view order histories. BPCRS products
will also be linked with relevant BP monographs and subscribers to the BP
online will be able to purchase these directly from the BP online content.
BPCRS customers will continue to be able to make purchases through
invoice or credit card orders.
An email subscription feature will allow users to keep abreast with BP news
and BPCRS updates.
In line with a policy of continuous improvement, users of the newly
consolidated www.pharmacopoeia.com website are invited to provide the
Secretariat with feedback on their experience.
European Pharm acopoeia Websites
httpss://extranet.edqm.eu/publications/recherches_sw.shtml For those texts
reproduced from the European Pharmacopoeia, the EDQM website
provides access to a database (the Knowledge database) 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 of reagents 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.
https://pharmeuropa.edqm.eu/home The European Pharmacopoeia Forum,
Pharmeuropa, is published quarterly as an aid for the elaboration of
monographs and as a vehicle for information on pharmacopoeial and related
matters. Pharmeuropa is available as a free on-line publication.

International Therapeutic Goods A dm inistration, A ustralia The British

Collaboration Pharmacopoeia Commission is pleased to continue its long-standing co­
operation with the Australian Department of Health Therapeutic Goods
Adm inistration (TGA). The TGA continues to provide advice to British
Pharmacopoeia Commission Expert Advisory Groups, participate in inter­
laboratory evaluation of British Pharmacopoeia monographs and review data
jointly. This collaboration has enabled the production of robust, high
quality monographs for users.
Chinese Pharm acopoeia The British Pharmacopoeia Commission is
pleased to continue its collaboration with the Chinese Pharmacopoeia on
the development of monographs and mutually agreed projects. Three Joint
Working Groups have been established to cover Traditional Herbal
I-xxx Introduction 2016

Medicines, Biological Products and Active Pharmaceutical Ingredients and

Excipients and associated formulated products.
The Croatian Agency for M edicinal Products and M edical Devices
(“ HALMED” ) Following the signing of a Collaboration Agreement in
January 2015, the Medicines and Healthcare products Regulatory Agency
has granted HALMED a licence to use the information in tile British
Pharmacopoeia on unlicensed medicines.
State Pharm acopoeia of the Republic o f Kazakhstan Following the
signing of a Collaboration Agreement in April 2014, die Medicines and
Healthcare products Regulatory Agency has granted die Committee on
Surveillance of Medical and Pharmaceutical Activities of tile Ministry of
Health of the Republic of Kazakhstan a licence to use relevant contents of
the British Pharmacopoeia in the State Pharmacopoeia of the Republic of
Kazakhstan.
U nited States Pharm acopeia The informal harmonisation between the
British Pharmacopoeia and die United States Pharmacopeia continues with
a projected work programme. Both Pharmacopoeias are committed to
harmonising analytical procedures for such monographs wherever possible.
W orld Health O rganization Following the renewal of a Collaboration
Agreement in April 2014, the British Pharmacopoeia Commission is pleased
to exchange information with the International Pharmacopoeia and to
collaborate on the development of monographs for formulated preparations.

Forward Look Electronic Updates The British Pharmacopoeia 2016 online updates will
be published on tile website, www.pharmacopoeia.comj to enable users to
keep up to date with monographs published in tile 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 die members

of its Expert Advisory Groups, Panels of Experts and Working Parties for
theừ dedicated enthusiasm and assistance in the preparation of this edition.
Close co-operation has continued with many organisations at home and
overseas. These include the Medicines and Healthcare products Regulatory
Agency, the Veterinary Medicines Dừectorate, the Royal Pharmaceutical
Society, the Association of the British Pharmaceutical Industry, the British
Association of Homoeopathic Manufacturers, die United Kingdom Herbal
Forum, The China Food and Drug Administration, the Chinese
Pharmacopoeia Commission, tile European Pharmacopoeia Commission
and the European Dừectorate for the Quality of Medicines & Healthcare,
tile Therapeutic Goods Adminisưation (Australia), the Health Products and
Food Branch of Health Canada, the United States Pharmacopeia, tile
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
Dừectorate for the Quality of Medicines & Healthcare for theừ support
and assistance in tile reproduction of the European Pharmacopoeia texts
and monographs. The British Pharmacopoeia Commission acknowledges
2016 Introduction I-xxxi

the importance of the work of the European Pharmacopoeia (Ph. Eur.)

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 is especially indebted to the Chinese
Pharmacopoeia for facilitating the training of two members of staff at the
Institute of Chinese Materia Medica on molecular methods of identification
for herbal drugs. The Commission appreciates the training provided by
Professor Chen Shilin, Professor Xu Jiang and their colleagues.
The British Pharmacopoeia Commission is grateful for the contribution of
Dr Ann Tough, Dr Graeme Kay and Ernie John Janus and Pamela
Methven, from the Robert Gordon University for their collaboration and
advice in the practical evaluation of monographs for this edition. The
British Pharmacopoeia is also grateful for the contribution of Ms Harjit
Jagpal for administrative and events management, Mr S Maddox, formerly
of the BP Laboratory, and for the contribution of Rebecca Hendry, Marta
Hemandez-Rueda and Walyd Mohammed of the Medicines and Healthcare
products Regulatory Agency Laboratory for their collaboration and practical
evaluation of monographs in this edition.
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 of the publishing team at The Stationery Office, in particular, Ms
Nichola Billington, Mr Colin Hackett, Mr Paul Allard, Mr Paul Relfe and
Mr Ian Webb, in the production of this edition.
The British Pharmacopoeia Commission is grateful for the commitment and
contribution of the website team at the Stationery Office, in particular, Mr
Terry Blake, Mr Andrew Hood and Mr Vinod Sathyamoorthy, in the
consolidation of the two websites.

Additions The following monographs of the British Pharmacopoeia 2016 were not
included in the British Pharmacopoeia 2015.
Medicinal and Pharm aceutical Substances
Eplerenone*
Glucosamine Sulfate Potassium Chloride*
Imatinib Mesilate*
High-molecular-mass Macrogols*
Macrogol Isotridecyl Ether*
Meldonium Dihydrate*
Methane*
Permethrin*
Polyoxypropylene Stearyl Ether*
Pullulan*
Rosuvastatin Calcium*
Sulfadimethoxine*
Tizanidine Hydrochloride*
* denotes a monograph of the European Pharmacopoeia
I-xxxii Introduction 2016

Tolterodine Tartrate*
Zanamivir Hydrate*
Formulated Preparations: Specific Monographs
Abacavir and Lamivudine Tablets
Abacavir, Zidovudine and Lamivudine Tablets
Gastro-resistant Acamprosate Tablets
Bendroflumethiazide Oral Suspension
Carvedilol Tablets
Ceftazidime Eye Drops
Clotrimazole Eye Drops
Clotrimazole Vaginal Tablets
Diamorphine Tablets
Estradiol Vaginal Tablets
Etynodiol Tablets
Fluconazole Capsules
Fluconazole Infusion
Fluconazole Oral Suspension
Flumetasone and Clioquinol Ear Drops
Flupentixol Tablets
Fluticasone and Salmeterol Pressurised Inhalation Powder, pre-dispensed
Fluticasone and Salmeterol Pressurised Inhalation, Suspension
Interferon Beta-la Injection
Ketoconazole Cream
Ketoconazole Shampoo
Lorazepam Oral Solution
Miconazole Eye Drops
Olanzapine Tablets
Orodispersible Olanzapine Tablets
Olmesartan Tablets
Soluble Prednisolone Tablets
Rizatriptan Tablets
Orodispersible Rizatriptan Tablets
Prolonged-release Sodium Valproate Capsules
Prolonged-release Sodium Valproate Tablets
Terbinafine Tablets
Vecuronium Bromide for Injection
Zidovudine Infusion
Herbal Drugs, Herbal Drug Preparations and Herbal Medicinal
Products
Agnus Castus Fruit Dry Extract*
Barbary Wolfberry Fruit*
Holy Basil Leaf
Nettle Root*
Phellodendron Amurense Bark
Phellodendron Chínense Bark
Materials for use in the Manufacture of Homoeopathic Preparations
Coated Homoeopathic Pillules*
Agaricus Phalloides for Homoeopathic Preparations*
Ignatia for Homoeopathic Preparations*
Nux-vomica for Homoeopathic Preparations*
* denotes a monograph of the European Pharmacopoeia
2016 Introduction I-xxxiii

Blood-related Products
Normal Immunoglobulin for Subcutaneous Administration*
Immunological Products
Influenza Vaccine (Live, Nasal)*
Radiopharmaceutical Preparations
Fluoroethyl-L-tyrosine (18F) Injection*

Omissions The following monographs of the British Pharmacopoeia 2015 are not
included in the British Pharmacopoeia 2016.
Medicinal and Pharmaceutical Substances
Amitriptyline Embonate
Carbenoxolone Sodium
Chloroform
Fosfestrol Sodium
Halibut-liver Oil
Nandrolone Phenylpropionate
Formulated Preparations: Specific Monographs
Aminoglutethimide Tablets
Chloroform Spirit
Chloroform and Morphine Tincture
Double Strength Chloroform Water
Codeine Linctus1
Paediatric Codeine Linctus1
Codergocrine Tablets
Desipramine Tablets
Diethylstilbestrol Pessaries
Dydrogesterone Tablets
Ergometrine Tablets
Fosfestrol Injection
Fosfestrol Tablets
Fructose Infusion
Glucose Irrigation Solution
Halibut-liver Oil Capsules
Isoconazole Pessaries
Menadiol Phosphate Injection
Methylcellulose Granules
Morphine and Atropine Injection
Neonatal Naloxone Injection
Nandrolone Phenylpropionate Injection
Orciprenaline Oral Solution
Orciprenaline Tablets
Paraffin Ointment
Protriptyline Tablets
Tretinoin Solution

* denotes a monograph of the European Pharmacopoeia

• This formulated preparation is now controlled by the monograph for Codeine Phosphate
Oral Solution.
I-xxxiv Introduction 2016

Herbal Drugs, Herbal Drug Preparations and Herbal Medicinal

Products
Standardised Liquorice Ethanolic Liquid Extract2
Concentrated Peppermint Emulsion

Technical Changes The following monographs in the British Pharmacopoeia 2016 have been
technically amended since the publication of the British Pharmacopoeia
2015, or have had a significant editorial change. This list does not include
revised monographs of the European Pharmacopoeia. An indication of the
nature of the change or the section of the monograph that has been
changed is given in italic type in the right hand column.
Medicinal and Pharmaceutical Substances
Nicorandil Loss on drying —> Water; Assay
Phenelzine Sulfate Assay

Formulated Preparations: Specific Monographs

Abacavir Tablets Identification; Related substances
Adapalene Cream Related substances
Adapalene Gel Related substances
Alendronic Acid Tablets Dissolution; Related substances —>
Phosphate and Phosphite; Assay
Alfuzosin Tablets Identification; Related substances
Prolonged-release Alfuzosin Tablets Identification; Related substances
Aminophylline Injection Content of theophylline
Aminophylline Tablets Content of ethylenediamine
Prolonged-release Aminophylline Content of ethylenediamine; Labelling
Tablets
Carbimazole Tablets Identification (colour test deleted);
Thiamazole and other related
substances
Chloral Hydrate Oral Solution Definition
Clobazam Oral Suspension Removal of unlicensed status; Non­
interchangeability statement added;
Dissolution (deleted)
Clobazam Tablets Non-interchangeability statement
added
Clonazepam Tablets Non-interchangeability statement
added
Clonidine Tablets Assay
Clotrimazole Pessaries Definition; Identification test A
Codeine Phosphate Oral Solution Definition; Extemporaneous
Preparation (deleted); Identification;
Assay
Desmopressin Tablets Uniformity of content
Dexamethasone Sodium Phosphate Content of dexamethasone phosphate
Injection -* Content of dexamethasone;
Identification; Free dexamethasone -*■
Related substances; Assay; Labelling
Prolonged-release Dipyridamole Related substances
Capsules
Dipyridamole Infusion Related substances

2 Monograph suppressed by European Pharmacopoeia Commission on-1 April 2015.

2016 Introduction I-xxxv

Doxorubicin Injection Related substances

Econazole Pessaries Definition; Identification test A;
Related substances
Enoxaparin Sodium Injection Sodium
Ergocalciferol Injection Definition; Content of ergocalciferol
Ethinylestradiol Tablets Uniformity of content
Paediatric Ferrous Sulfate Oral Extemporaneous Preparation (deleted)
Solution
Glimepiride Tablets Dissolution
Goserelin Implants Drug release; Related substances tests
A , B and C
Heparin Injection Identification tests A and B; Assay
Hydroxychloroquine Tablets Disintegration; Related substances
Hydroxyzine Oral Solution Identification test A; Related
substances; Impurities
Kaolin Mixture Extemporaneous Preparation (deleted)
Kaolin and Morphine Mixture Definition; Extemporaneous
Preparation (deleted)
Lamotrigine Tablets Non-interchangeability statement
added
Dispersible Lamotrigine Tablets Non-interchangeability statement
added
Levonorgestrel Tablets Identification test A
Lisinopril Oral Solution Related substances
Lymecycline Capsules Light-absorbing impurities (deleted);
Related substances; Water
Aromatic Magnesium Carbonate Extemporaneous Preparation (deleted)
Mixture
Magnesium Hydroxide Mixture Extemporaneous Preparation (deleted)
Magnesium Sulfate Mixture Extemporaneous Preparation (deleted)
Magnesium Trisilicate Mixture Extemporaneous Preparation (deleted)
Melphalan Injection Related substances test B
Methotrexate Injection Related substances '
Methotrexate Oral Solution Identification
Morphine Sulfate Injection Assay
Naloxone Injection Identification test A; Related
substances
Omeprazole Oral Suspension Alkalinity (deleted); Dissolution
Oxymetholone Tablets Reference to unlicensed status added
Liquid Paraffin Oral Emulsion Definition; Extemporaneous
Preparation (deleted)
Liquid Paraffin and Magnesium Definition; Extemporaneous
Hydroxide Oral Emulsion Preparation (deleted)
Paroxetine Tablets Related substances; Impurities
Phénobarbital Elixir Non-interchangeability statement
added
Paediatric Phénobarbital Oral Patient monitoring statement added
Solution
Phénobarbital Tablets Non-interchangeability statement
added
Phénobarbital Sodium Tablets Non-interchangeabUity statement
added
Phentolâmine Injection Related substances
I-xxxvi Introduction 2016

Phenytoin Capsules Non-interchangeability statement

added; Related substances
Phenytoin Oral Suspension Non-interchangeability statement
added
Phenytoin Tablets Non-interchangeability statement
added
Potassium Citrate Mixture Extemporaneous Preparation (deleted)
Prednisolone Oral Solution Related substances
Primidone Oral Suspension Non-interchangeability statement
added
Primidone Tablets Non-interchangeability statement
added
Ramipril Capsules Related substances
Ramipril Tablets Related substances
Simvastatin Oral Suspension Acidity or alkalinity (deleted)
Sodium Bicarbonate Oral Solution Labelling
Compound Sodium Chloride Extemporaneous Preparation (deleted)
Mouthwash
Sodium Chloride Oral Solution Definition; Labelling
Sodium Valproate Oral Solution Non-interchangeabUity statement
added; Identification; Related
substances
Sodium Valproate Tablets Non-interchangeability statement
added; Related substances
Gastro-resistant Sodium Valproate Non-interchangeability statement
Tablets added; Dissolution; Related substances
Sumatriptan Injection Content of sumatriptan succinate ->
Content of sumatriptan;
Identification; Impurities A and H;
Related substances; Assay
Sumatriptan Nasal Spray Impurities A and H; Related
substances; Assay
Sumatriptan Tablets Content of sumatriptan succinate —►
Content of sumatriptan;
Identification; Dissolution; Impurities
A and H; Related substances; Assay
Vinblastine Injection Labelling
Vincristine Injection Requirements for powder for re­
constitution replaced by requirements
for ready-to-use solution; Labelling
Zidovudine Capsules Related substances
Zidovudine Tablets Related substances
Zidovudine and Lamivudine Tablets Related substances
Zuclopenthixol Decanoate Injection Identification

Herbal Drugs, Herbal Drug Preparations and Herbal Medicinal

Products
Acid Gentian Mixture Extemporaneous Preparation (deleted)
Alkaline Gentian Mixture Extemporaneous Preparation (deleted)
2016 Introduction I-xxxvii

Changes in Tide The following list gives the alterations in the titles of monographs of the
British Pharmacopoeia 2015 that have been retained in the British
Pharmacopoeia 2016.

BRITISH PH A RM A CO PO EIA BRITISH PHARM A CO PO EIA

2015 2016

Medicinal and Pharm aceutical Substances

Noscapine Hydrochloride Noscapine Hydrochloride Hydrate
Sodium Alendronate Sodium Alendronate Trihydrate

Blood-related Products
Normal Immunoglobulin Normal Immunoglobulin for
Intramuscular Administration

Herbal Drugs, H erbal D rug Preparations and Herbal Medicinal

Products
Extracts Herbal Drug Extracts
2016 General Notices 1-1

General Notices
 1-2 General Notices 2016

CONTENTS OF THE GENERAL NOTICES

Part I Implementation of Pharmacopoeial Methods

Italic introduction Conventional Terms
European Pharmacopoeia Interchangeable Methods
References to Regulatory Documents
Part II
Italic introduction 1.2 Other Provisions Applying to General
Official Standards Chapters and Monographs
Definition of Terms Quantities
Expression of Standards Apparatus and Procedures
Temperature Water-bath
Weights and Measures Drying and Ignition to Constant Mass
Atomic Weights Reagents
Constant Weight Solvents
Expression of Concentrations Expression of Content
Water Bath Temperature
Reagents 1.3 General Chapters
Indicators Containers
Caution Statements 1.4 Monographs
Titles Tides
Chemical Formulae Relative Atomic and Molecular Masses
Definition Chemical Abstracts Service (CAS) Registry
Production Number
Manufacture of Formulated Preparations Definition
Freshly and Recently Prepared Limits of Content
Methods of Sterilisation Herbal Drugs
Water Production
Excipients Choice of Vaccine Strain^ Choice of
Colouring Agents Vaccine Composition
Antimicrobial Preservatives Potential Adulteration
Characteristics Characters
Solubility Solubility
Identification Identification
Reference Spectra Scope
Assays and Tests First and Second Identifications
Biological Assays and Tests Powdered Herbal Drugs
Reference Substances and Reference Preparations Tests and Assays
Chemical Reference Substances Scope
Biological Reference Preparations Calculation
Storage Limits
Labelling Indication of Permitted Limit of Impurities
Action and Use Herbal Drugs
Crude Drugs; Traditional Herbal and Equivalents
Complementary Medicines Culture Media
Monograph Tide Storage
Definition Labelling
Characteristics Warnings
Control Methods Impurities
Homoeopathic Medicines Functionality-related Characteristics of
Unlicensed Medicines Excipients
Reference Standards
Part HI
Italic introduction 1.5 Abbreviations and Symbols
General Notices of the European Pharmacopoeia Abbreviations used in the Monographs on
1.1 General Statements Immunoglobulins, Immunosera and
Quality Systems Vaccines
Alternative Methods Collections of Micro-organisms
Demonstration of Compliance with the 1.6 Units of the International System (SI) used
Pharmacopoeia in the Pharmacopoeia and Equivalence
Grade of Materials with other Units
General Monographs International System of Units (SI)
Validation of Pharmacopoeial Methods Notes
2016 General Notices 1-3

General Notices

Part I

The British Pharmacopoeia comprises the entire text within this publication. The
word ‘official3is used in the Pharmacopoeia to signify (of the Pharmacopoeia\ It
applies to any title, substance, preparation} method or statement included in the
general notices, monographs and appendices of the Pharmacopoeia. The
abbreviation for British Pharmacopoeia is BP.

European Monographs of the European Pharmacopoeia are reproduced in this edition

Pharmacopoeia of the British Pharmacopoeia by incorporation of the text published under
the direction of the Council of Europe (Partial Agreement) in accordance
with the Convention on the Elaboration of a European Pharmacopoeia
(Treaty Series No. 32 (1974) CMND 5763) as amended by the Protocol to
the Convention (Treaty Series No. MISC16 (1990) CMND 1133). They
are included for the convenience of users of the British Pharmacopoeia. In
cases of doubt or dispute reference should be made to the Council of
Europe text.
Monographs of the European Pharmacopoeia are distinguished by a
V * chaplet of stars against the title and by reference to the European
* Pharmacopoeia monograph number included immediately below the
title in italics. The beginning and end of text from the European
Pharmacopoeia are denoted by means of horizontal lines with the symbol
(Ph Eur* ranged leftand right, respectively.
The general provisions of the European Pharmacopoeia relating to
different types of dosage form are included in the appropriate general
monograph in that section of the British Pharmacopoeia entided
Monographs: Formulated Preparations. These general provisions apply to
all dosage forms of the type defined, whether or not an individual
monograph is included in the British Pharmacopoeia. In addition, the
provisions of the European Pharmacopoeia General Monograph for
Pharmaceutical Preparations apply to all dosage forms, whether or not an
individual monograph is included in the British Pharmacopoeia.
Texts of the European Pharmacopoeia are governed by the General
Notices of the European Pharmacopoeia. These are reproduced as Part HE
of these notices.
1-4 General Notices 2016

Part II

The following général notices apply to the statements made in the monographs of
the British Pharmacopoeia other than those reproduced from the European
Pharmacopoeia and to the statements made in the Appendices of the British
Pharmacopoeia other than when a method, test or other matter described in an
appendix is invoked in a monograph reproduced from the European
Pharmacopoeia.

Official Standards The requirements stated in the monographs of the Pharmacopoeia apply to
articles that are intended for medicinal use but not necessarily to articles
that may be sold under the same name for other purposes. An article
intended for medicinal use that is described by means of an official title
must comply with the requirements of the relevant monograph. A
formulated preparation must comply throughout its assigned shelf-life
(period of validity). The subject of any other monograph must comply
throughout its period of use.
A monograph is to be construed in accordance with any general
monograph or notice or any appendix, note or other explanatory material
that is contained in this edition and that is applicable to that monograph.
All statements contained in the monographs, except where a specific general
notice indicates otherwise and with the exceptions given below, constitute
standards for the official articles. An article is not of pharmacopoeial quality
unless it complies with all of the requirements stated. This does not imply
that a manufacturer is obliged to perform all the tests in a monograph in
order to assess compliance with the Pharmacopoeia before release of a
product. The manufacturer may assure himself that a product is of
pharmacopoeial quality by other means, for example, from data derived
from validation studies of the manufacturing process, from in-process
controls or from a combination of the two. Parametric release in
appropriate circumstances is thus not precluded by the need to comply with
the Pharmacopoeia. The general notice on Assays and Tests indicates that
analytical methods other than those described in the Pharmacopoeia may be
employed for routine purposes.
Requirements in monographs have been framed to provide appropriate
limitation of potential impurities rather than to provide against all possible
impurities. Material found to contain an impurity not detectable by means
of the prescribed tests is not of pharmacopoeial quality if the nature or
amount of the impurity found is incompatible with good pharmaceutical
practice.
The status of any statement given under the headings Definition,
Production, Characteristics, Storage, Labelling or Action and use is defined
within the general notice relating to the relevant heading. In addition to any
exceptions indicated by one of the general notices referred to above, the
following parts of a monograph do not constitute standards: (a) a graphic or
molecular formula given at the beginning of a monograph; (b) a molecular
weight; (c) a Chemical Abstracts Service Registry Number; (d) any
information given at the end of a monograph concerning impurities known
to be limited by that monograph; (e) information in any annex to a
2016 General Notices 1-5

monograph. Any statement containing the word ‘should5 constitutes

non-mandatory advice or recommendation.
The expression ‘unless otherwise justified and authorised’ means that the
requirement in question has to be met, unless a competent authority
authorises a modification or exemption where justified in a particular case.
The term ‘competent authority5 means the national, supranational or
international body or organisation vested with the authority for making
decisions concerning the issue in question. It may, for example, be a
licensing authority or an official control laboratory. For a formulated
preparation that is the subject of monograph in the British Pharmacopoeia
any justified and authorised modification to, or exemption from, the
requirements of the relevant general monograph of the European
Pharmacopoeia is stated in the individual monograph. For example, the
general monograph for Tablets requires that Uncoated Tablets, except for
chewable tablets, disintegrate within 15 minutes; for Calcium Lactate
Tablets a time of 30 minutes is permitted.
Many of the general monographs for formulated preparations include
statements and requirements additional to those of the European
Pharmacopoeia that are applicable to the individual monographs of the
British Pharmacopoeia. Such statements and requirements apply to all
monographs for that dosage form included in the Pharmacopoeia unless
otherwise indicated in the individual monograph.
Where a monograph on a biological substance or preparation refers to a
strain, a test, a method, a substance, etc., using the qualifications ‘suitable’
or ‘appropriate’ without further definition in the text, the choice of such
strain, test, method, substance, etc., is made in accordance with any
international agreements or national regulations affecting the subject
concerned.

Definition of Terms Where the term ‘about’ is included in a monograph or test it should be
taken to mean approximately (fairly correct or accurate; near to the actual
value).
Where the term ‘corresponds’ is included in a monograph or test it
should be taken to mean similar or equivalent in character or quantity.
Where the term ‘similar’ is included in a monograph or test it should be
taken to mean alike though not necessarily identical.
Further qualifiers (such as numerical acceptance criteria) for the above
terms are not included in the BP. The acceptance criteria for any individual
case is set based on the range of results obtained from known reference
samples, the level of precision of the equipment or apparatus used and the
level of accuracy required for the particular application. The user should
determine the variability seen in his/her own laboratory and set in-house
acceptance criteria that he/she judges to be appropriate based on the local
operating conditions.

Expression of Where the standard for the content of a substance described in a

Standards monograph is expressed in terms of the chemical formula for that substance
an upper limit exceeding 100% may be stated. Such an upper limit applies
to the result of the assay calculated in terms of the equivalent content of the
specified chemical formula. For example, the statement ‘contains not less
than 99.0% and not more than 101.0% of C 20H 24N 2O2JHCI’ implies that
the result of the assay is not less than 99.0% and not more than 101.0%,
calculated in terms of the equivalent content of C20H 24N 2O2JHCI.
1-6 General Notices 2016

Where the result of an assay or test is reqmred to be calculated with

. .. . reference to die dried, anhydrous or ignited substance, the substance free
from a specified solvent or to the peptide content, thệ determination of loss
on drying, water content, loss on ignition, content of the specified solvent
or peptide content is carried Gut by the method prescribed in the relevant
test in the monograph.

Temperature The Celsius thermometric scale is used in expressing temperatures.

Weights and The metric system of weights and measures is employed; SI Units have
Measures generally been adopted. Metric measures are required to have been
graduated at 20 ° and all measurements involved in the analytical operations
of die Pharmacopoeia are intended, unless otherwise stated, to be made at
that temperature. Graduated glass apparatus used in analytical operations
should comply with Class A requirements of the appropriate International
Standard issued by the International Organization for Standardization. The
abbreviation for litre is ‘L’ throughout die Pharmacopoeia. In line with
European Dừeetive 80/181/EEC, the abbreviation T is also permitted for
use.

Atomic Weights The atomic weights adopted are the values given in the Table of Relative
Atomic Weights 2001 published by the International Union of Pure and
Applied Chemistry (Appendix XXV).

Constant Weight The term ‘constant weight’, used in relation to the process of drying or the
process of ignition, means that two consecutive weighings do not differ by
more than 0.5 mg, the second weighing being made after an additional
period of drying or ignition under the specified conditions appropriate to
die nature and quantity of the residue (1 hour is usually suitable).

Expression of The term ‘per cent’ or more usually the symbol .*.%■is used with one of four
Concentrations different meanings in the expression of concenưations according to
cừcumstances. In order that the meaning to be attached to the expression
in each instance is clear, the following notation is used:
Per cent w/w (% w/w) (percentage weight in weight) expresses the
number of grams of solute in 100 g of product.
Per cent w/v (% w/v) (percentage weight in volume) expresses the
number of grams of solute in 100 mL of product.
Per cent v/v (% v/v) (percentage volume in volume) expresses the
number of millilitres of solute in 100 mL of product.
Per cent v/w (% v/w) (percentage volume in weight) expresses the
number OĨ millilitres of solute in 100 g of product.
Usually the sưength of solutions of solids in liquids is expressed as
percentage weight in volume, of liquids in liquids as percentage volume in
volume and of gases in liquids as percentage weight in weight.
When the concentration of a solution is expressed as parts per million
(ppm), it means weight in weight, unless otherwise specified.
When the concentration of a solution is expressed as parts of dissolved
substance in parts of the solution, it means parts by weight (g) of a solid in
parts by volume (mL) of the final solution; or parts by volume (mL) of a
liquid in pans by volume (mL) of the final solution; or parts by weight (g)
of a gas in parts by weight (g) of the final solution.
2016 General Notices 1-7

When the concentration of a solution is expressed in molarity designated

by the symbol m preceded by a number, it denotes the number of moles of
the stated solute contained in sufficient Purified Water (unless otherwise
stated) to produce 1 litre of solution.

Water Bath The term ‘water bath’ means a bath of boiling water, unless water at some
other temperature is indicated in the text. An alternative form of heating
may be employed providing that the required temperature is approximately
maintained but not exceeded.

Reagents The reagents required for the assays and tests of the Pharmacopoeia are
defined in appendices. The descriptions set out in the appendices do not
imply that the materials are suitable for use in medicine.

Indicators Indicators, the colours of which change over approximately the same range
of pH, may be substituted for one another but in the event of doubt or
dispute as to the equivalence of indicators for a particular purpose, the
indicator specified in the text is alone authoritative.
The quantity of an indicator solution appropriate for use in acid-base
titrations described in assays or tests is 0.1 mL unless otherwise stated in
the text.
Any solvent required in an assay or test in which an indicator is specified
is previously neutralised to the indicator, unless a blank test is prescribed.

Caution Statements A number of materials described in the monographs and some of the
reagents specified for use in the assays and tests of the Pharmacopoeia may
be injurious to health unless adequate precautions are taken. The principles
of good laboratory practice and the provisions of any appropriate
regulations such as those issued in the United Kingdom in accordance with
the Health and Safety at Work etc. Act 1974 should be observed at all times
in carrying out the assays and tests of the Pharmacopoeia.
Attention is drawn to particular hazards in certain monographs by means
of an italicised statement; the absence of such a statement should not
however be taken to mean that no hazard exists.

Titles Subsidiary tides, where included, have the same significance as the main
tides. An abbreviated title constructed in accordance with the directions
given in Appendix XXI A has the same significance as the main title.
Titles that are derived by the suitable inversion of words of a main or
subsidiary tide, with the addition of a preposition if appropriate, are also
official titles. Thus, the following are all official tides: Aspirin Tablets,
Tablets of Aspirin; Atropine Injection, Injection of Atropine.
A title of a formulated preparation that includes the full nonproprietary
name of the active ingredient or ingredients, where this is not included in ^
the title of the monograph, is also an official tide. For example, the title
Promethazine Hydrochloride Oral Solution has the same significance as
Promethazine Oral Solution and the title Brompheniramine Maleate Tablets
has the same significance as Brompheniramine Tablets.
Where the English tide at the head of a monograph in the European
Pharmacopoeia is different from that at the head of the text incorporated
into the British Pharmacopoeia, an Approved Synonym has been created on
the recommendation of the British Pharmacopoeia Commission. Approved
Synonyms have the same significance as the main title and are thus official
1-8 General Notices 2016

titles. A cumulative list of such Approved Synonyms is provided in

Appendix XXI B.
Where the names of pharmacopoeial substances, preparations and other
materials occur in the text they are printed with capital initial letters and
this indicates that materials of Pharmacopoeial quality must be used. Words
in the text that name a reagent or other material, a physical characteristic or
a process that is described or defined in an appendix are printed in italic
type, for example, methanol, absorbance, gas chromatography, and these imply
compliance with the requirements specified in the appropriate appendix.

Chemical Formulae When the chemical composition of an official substance is known or

generally accepted, the graphic and molecular formulae, the molecular
weight and the Chemical Abstracts Service Registry Number are normally
given at the beginning of the monograph for information. This information
refers to the chemically pure substance and is not to be regarded as an
indication of the purity of the official material. Elsewhere, in statements of
standards of purity and strength and in descriptions of processes of assay, it
is evident from the context that the formulae denote the chemically pure
substances.
Where the absolute stereochemical configuration is specified, the
International Union of Pure and Applied Chemistry (IUPAC) R/S and E/Z
systems of designation have been used. If the substance is an enantiomer of
unknown absolute stereochemistry the sign of the optical rotation, as
determined in the solvent and under the conditions specified in the
monograph, has been attached to the systematic name. An indication of
sign of rotation has also been given where this is incorporated in a trivial
name that appears on an IUPAC preferred list.
All amino acids, except glycine, have the L-configuration unless otherwise
indicated. The three-letter and one-letter symbols used for amino acids in
peptide and protein sequences are those recommended by the Joint
Commission on Biochemical Nomenclature of the International Union of
Pure and Applied Chemistry and the International Union of Biochemistry
and Molecular Biology.
In the graphic formulae the following abbreviations are used:

Me -C H 3 Bu* -CH(CH 3)CH 2CH 3

Et - c h 2c h 3 Bu" - c h 2c h 2c h 2c h 3
Pr! -CH(CH 3)2 Bur -C(CH 3)3
Pr” - c h 2c h 2c h 3 Ph - c 6h 5
Bul -C H 2CH(CH 3)2 Ac -c o c h 3

Definition Statements given under the heading Definition constitute an official

definition of the substance, preparation or other article that is the subject of
the monograph. They constitute instructions or requirements and are
mandatory in nature.
Certain medicinal or pharmaceutical substances and other articles are
defined by reference to a particular method of manufacture. A statement
that a substance or article is prepared or obtained by a certain method
constitutes part of the official definition and implies that other methods are
not permitted. A statement that a substance may be prepared or obtained by
a certain method, however, indicates that this is one possible method and
does not imply that other methods are proscribed.
2016 General Notices 1-9

Additional statements concerning the definition of formulated

preparations are given in the general notice on Manufacture of Formulated
Preparations.

Production Statements given under the heading Production draw attention to particular
aspects of the manufacturing process but are not necessarily comprehensive.
They constitute mandatory instructions to manufacturers. They may relate,
for example, to source materials, to the manufacturing process itself and its
validation and control, to in-process testing or to testing that is to be
carried out by the manufacturer on the final product (bulk material or
dosage form) either on selected batches or on each batch prior to release.
These statements cannot necessarily be verified on a sample of the final
product by an independent analyst. The competent authority may establish
that the instructions have been followed, for example, by examination of
data received from the manufacturer, by inspection or by testing
appropriate samples.
The absence of a section on Production does not imply that attention to
features such as those referred to above is not required. A substance,
preparation or article described in a monograph of the Pharmacopoeia is to
be manufactured in accordance with the principles of good manufacturing
practice and in accordance with relevant international agreements and
supranational and national regulations governing medicinal products.
Where in the section under the heading Production a monograph on a
vaccine defines the characteristics of the vaccine strain to be used, any test
methods given for confirming these characteristics are provided as examples
of suitable methods. The use of these methods is not mandatory.
Additional statements concerning the production of formulated
preparations are given in the general notice on Manufacture of Formulated
Preparations.

Manufacture of Attention is drawn to the need to observe adequate hygienic precautions in

Formulated the preparation and dispensing of pharmaceutical formulations. The
Preparations principles of good pharmaceutical manufacturing practice should be
observed.
The Definition in certain monographs for pharmaceutical preparations is
given in terms of the principal ingredients only. Any ingredient, other than
those included in the Definition, must comply with the general notice on
Excipients and the product must conform with the Pharmacopoeial
requirements.
The Definition in other monographs for pharmaceutical preparations is
presented as a full formula. No deviation from the stated formula is
permitted except those allowed by the general notices on Colouring Agents
and Antimicrobial Preservatives. Where additionally directions are given
under the heading Extemporaneous Preparation these are intended for the
extemporaneous preparation of relatively small quantities for short-term
supply and use. When so prepared, no deviation from the stated directions
is permitted. If, however, such a pharmaceutical preparation is
manufactured on a larger scale with the intention that it may be stored,
deviations from the stated directions are permitted provided that the final
product meets the following criteria:
I-10 General Notices 2016

( 1) compliance with all of the requirements stated in the monograph;

(2) retention of the essential characteristics of the preparation made strictly
in accordance with the directions of the Pharmacopoeia.
Monographs for yet other pharmaceutical preparations include both a
Definition in terms of the principal ingredients and, under the side-heading
Extemporaneous Preparation, a full formula together with, in some cases,
directions for their preparation. Such full formulae and directions are
intended for the extemporaneous preparation of relatively small quantities
for short-term supply and use. When so prepared, no deviation from the
stated formula and directions is permitted. If, however, such a
pharmaceutical preparation is manufactured on a larger scale with the
intention that it may be stored, deviations from the formula and directions
stated under the heading Extemporaneous Preparation are permitted
provided that any ingredient, other than those included in the Definition,
complies with the general notice on Excipients and that the final product
meets the following criteria:
( 1) accordance with the Definition stated in the monograph;
(2) compliance with all of the requirements stated in the monograph;
(3) retention of the essential characteristics of the preparation made strictly
in accordance with the formula and directions of the Pharmacopoeia.
In the manufacture of any official preparation on a large scale with the
intention that it should be stored, in addition to following any instruction
under the heading Production, it is necessary to ascertain that the product
is satisfactory with respect to its physical and chemical stability and its state
of preservation over the claimed shelf-life. This applies irrespective of
whether the formula of the Pharmacopoeia and any instructions given under
the heading Extemporaneous Preparation are followed precisely or
modified. Provided that the preparation has been shown to be stable in
other respects, deterioration due to microbial contamination may be
inhibited by the incorporation of a suitable antimicrobial preservative. In
such circumstances the label states appropriate storage conditions, the date
after which the product should not be used and the identity and
concentration of the antimicrobial preservative.

Freshly and The direction, given under the heading Extemporaneous Preparation, that a
Recently Prepared preparation must be freshly prepared indicates that it must be made not
more than 24 hours before it is issued for use. The direction that a
preparation should be recendy prepared indicates that deterioration is likely
if the preparation is stored for longer than about 4 weeks at 15° to 25°.

Methods of The methods of sterilisation used in preparing the sterile materials

Sterilisation described in the Pharmacopoeia are given in Appendix XVIIL For aqueous
preparations, steam sterilisation (heating in an autoclave) is the method of
choice wherever it is known to be suitable. Any method of sterilisation must
be validated with respect to both the assurance of sterility and the integrity
of the product and to ensure that the final product complies with the
requirements of the monograph.

Water The term water used without qualification in formulae for formulated
preparations means either potable water freshly drawn direct from the
public supply and suitable for drinking or freshly boiled and cooled Purified
2016 General Notices 1-11

Water. The latter should be used if the public supply is from a local storage
tank or if the potable water is unsuitable for a particular preparation.

Excipients Where an excipient for which there is a pharmacopoeial monograph is used

in preparing an official preparation it shall comply with that monograph.
Any substance added in preparing an official preparation shall be
innocuous, shall have no adverse influence on the therapeutic efficacy of the
active ingredients and shall not interfere with the assays and tests of the
Pharmacopoeia. Particular care should be taken to ensure that such
substances are free from harmful organisms.

Colouring Agents If in a monograph for a formulated preparation defined by means of a full

formula a specific colouring agent or agents is prescribed, suitable
alternatives approved in the country concerned may be substituted.

Antimicrobial When the term ‘suitable antimicrobial preservative’ is used it is implied that
Preservatives the preparation concerned will be effectively preserved according to the
appropriate criteria applied and interpreted as described in the test for
efficacy of antimicrobial preservation (Appendix XVI C). In certain
monographs for formulated preparations defined by means of a full formula,
a specific antimicrobial agent or agents may be prescribed; suitable
alternatives may be substituted provided that their identity and
concentration are stated on the label.

Characteristics Statements given under the heading Characteristics are not to be

interpreted in a strict sense and are not to be regarded as official
requirements. Statements on taste are provided only in cases where this
property is a guide to the acceptability of the material (for example, a
material used primarily for flavouring). The status of statements on
solubility is given in the general notice on Solubility.
Solubility Statements on solubility given under the heading
Characteristics are intended as information on the approximate solubility at
a temperature between 15° and 25°, unless otherwise stated, and are not to
be considered as official requirements.
Statements given under headings such as Solubility in ethanol express
exact requirements and constitute part of the standards for the substances
under which they occur.
The following table indicates the meanings of the terms used in
statements of approximate solubilities.

Descriptive term Approximate volume of

solvent in millilitres per
gram of solute
very soluble less than 1
freely soluble from 1 to 10
soluble from 10 to 30
sparingly soluble from 30 to 100
slightly soluble from 100 to 1000
very slightly soluble from 1000 to 10,000
practically insoluble more than 10,000

The term ‘partly soluble’ is used to describe a mixture of which only

some of the components dissolve.
1-12 General Notices 2016

Identification The tests described or referred to under the heading Identification are not
necessarily sufficient to establish absolute proof of identity. They provide a
means of verifying that the identity of the material being examined is in
accordance with the label on the container.
Unless otherwise prescribed, identification tests are carried out at a
temperature between 15° and 25°.
Reference spectra Where a monograph refers to an infrared reference
spectrum, this spectrum is provided in a separate section of the
Pharmacopoeia. A sample spectrum is considered to be concordant with a
reference spectrum if the transmission minima (absorption maxima) of the
principal bands in the sample correspond in position, relative intensities and
shape to those of the reference. Instrumentation software may be used to
calculate concordance with a previously recorded reference spectrum.
When tests for infrared absorption are applied to material extracted from
formulated preparations, strict concordance with the specified reference
spectrum may not always be possible, but nevertheless a close resemblance
between the spectrum of the extracted material and the specified reference
spectrum should be achieved.

Assays an d Tests The assays and tests described are the official methods upon which the
standards of the Pharmacopoeia depend. The analyst is not precluded from
employing alternative methods, including jnethods of micro-analysis, in any
assay or test if it is known that the method used will give a result of
equivalent accuracy. Local reference materials may be used for routine
analysis, provided that these are calibrated against the official reference
materials. In the event of doubt or dispute, the methods of analysis, the
reference materials and the reference spectra of the Pharmacopoeia are
alone authoritative.
Where the solvent used for a solution is not named, the solvent is
Purified Water.
Unless otherwise prescribed, the assays and tests are carried out at a
temperature between 15° and 25°.
A temperature in a test for Loss on drying, where no temperature range
is given, implies a range of ± 2 ° about the stated value.
Visual com parative tests, unless otherwise prescribed, are carried out
using identical tubes of colourless, transparent, neutral glass with a flat
base. The volumes of liquid prescribed are for use with tubes 16 mm in
internal diameter; tubes with a larger internal diameter may be used but the
volume of liquid examined must be increased so that the depth of liquid in
the tubes is not less than that obtained when the prescribed volume of
liquid and tubes 16 mm in internal diameter are used. Equal volumes of the
liquids to be compared are examined down the vertical axis of the tubes
against a white background or, if necessary, against a black background.
The examination is carried out in diffuse light.
Where a direction is given that an analytical operation is to be carried out
‘in subdued light’, precautions should be taken to avoid exposure to direct
sunlight or other strong light. Where a direction is given that an analytical
operation is to be carried out ‘protected from light’, precautions should be
taken to exclude actinic light by the use of low-actinic glassware, working in
a dark room or similar procedures.
For preparations other than those of fixed strength, the quantity to be
taken for an assay or test is usually expressed in terms of the active
ingredient. This means that the quantity of the active ingredient expected to
2016 General Notices 1-13

be present and the quantity of the preparation to be taken are calculated

from the strength stated on the label.
In assays the approximate quantity to be taken for examination is
indicated but the quantity actually used must not deviate by more than
10% from that stated. The quantity taken is accurately weighed or
measured and the result of the assay is calculated from this exact quantity.
Reagents are measured and the procedures are carried out with an accuracy
commensurate with the degree of precision implied by the standard stated
for the assay.
In tests the stated quantity to be taken for examination must be used
unless any divergence can be taken into account in conducting the test and
calculating the result. The quantity taken is accurately weighed or measured
with the degree of precision implied by the standard or, where the standard
is not stated numerically (for example, in tests for Clarity and colour of
solution), with the degree of precision implied by the number of significant
figures stated. Reagents are measured and the procedures are carried out
with an accuracy commensurate with this degree of precision.
The limits stated in monographs are based on data obtained in normal
analytical practice; they take account of normal analytical errors, of
acceptable variations in manufacture and of deterioration to an extent
considered acceptable. No further tolerances are to be applied to the limits
prescribed to determine whether the article being examined complies with
the requirements of the monograph.
In determining compliance with a numerical limit, the calculated result of
a test or assay is first rounded to the number of significant figures stated,
unless otherwise prescribed. The last figure is increased by 1 when the part
rejected is equal to or exceeds one half-unit, whereas it is not modified
when the pan rejected is less than a half-unit.
In certain tests, the concentration of impurity is given in parentheses
either as a percentage or in parts per million by weight (ppm). In
chromatographic tests such concentrations are stated as a percentage
irrespective of the limit. In other tests they are usually stated in ppm unless
the limit exceeds 500 ppm. In those chromatographic tests in which a
secondary spot or peak in a chromatogram obtained with a solution of the
substance being examined is described as corresponding to a named
impurity and is compared with a spot or peak in a chromatogram obtained
with a reference solution of the same impurity, the percentage given in
parentheses indicates the limit for that impurity. In those chromatographic
tests in which a spot or peak in a chromatogram obtained with a solution of
the substance being examined is described in terms other than as
corresponding to a named impurity (commonly, for example, as any (other)
secondary spot or peak) but is compared with a spot or peak in a
chromatogram obtained with a reference solution of a named impurity, the
percentage given in parentheses indicates an impurity limit expressed in
terms of a nominal concentration of the named impurity. In
chromatographic tests in which a comparison is made between spots or
peaks in chromatograms obtained with solutions of different concentrations
of the substance being examined, the percentage given in parentheses
indicates an impurity limit expressed in terms of a nominal concentration of
the medicinal substance itself. In some monographs, in particular those for
certain formulated preparations, the impurity limit is expressed in terms of a
nominal concentration of the active moiety rather than of the medicinal
 I

1-14 General Notices 2016

substance itself. Where necessary for clarification the terms in which the
limit is expressed are stated within the monograph.
In all cases where an impurity limit is given in parentheses, the figures
given are approximations for information only; conformity with the
requirements is determined on the basis of compliance or otherwise with
the stated test.
The use of a proprietary designation to identify a material used in an
assay or test does not imply that another equally suitable material may not
be used.

Biological Assays Methods of assay described as Suggested methods are not obligatory, but
and Tests when another method is used its precision must be not less than that
required for the Suggested method.
For those antibiotics for which the monograph specifies a microbiological
assay the potency requirement is expressed in the monograph in
International Units (IU) per milligram. The material is not of
pharmacopoeial quality if the upper fiducial limit of error is less than the
stated potency. For such antibiotics the required precision of the assay is
stated in the monograph in terms of the fiducial limits of error about the
estimated potency.
For other substances and preparations for which the monograph specifies
a biological assay, unless otherwise stated, the precision of the assay is such
that the fiducial limits of error, expressed as a percentage of the estimated
potency, are within a range not wider than that obtained by multiplying by
a factor of 10 the square roots of the limits given in the monograph for the
fiducial limits of error about the stated potency.
In all cases fiducial limits of error are based on a probability of 95%
(P = 0.95).
Where the biological assay is being used to ascertain the purity of the
material, the stated potency means the potency stated on the label in terms
of International Units (IU) or other Units per gram, per milligram or per
millilitre. When no such statement appears on the label, the stated potency
means the fixed or minimum potency required in the monograph. This
interpretation of stated potency applies in all cases except where the
m onograph specifically directs otherwise.
Where the biological assay is being used to determine the total activity in
the container, the stated potency means the total number of International
Units (IU) or other Units stated on the label or, if no such statement
appears, the total activity calculated in accordance with the instructions in
the monograph.
Wherever possible the primary standard used in an assay or test is the
respective International Standard or Reference Preparation established by
the World Health Organization for international use and the biological
activity is expressed in International Units (IU).
In other cases, where Units are referred to in an assay or test, the Unit
for a particular substance or preparation is, for the United Kingdom, the
specific biological activity contained in such an amount of the respective
primary standard as the appropriate international or national organisation
indicates. The necessary information is provided with the primary standard.
Unless otherwise directed, animals used in an assay or a test are healthy
animals, drawn from a uniform stock, that have not previously been treated
with any material that will interfere with the assay or test. Unless otherwise
stated, guinea-pigs weigh not less than 250 g or, when used in systemic
 General Notices 1-15

toxicity tests, not less than 350 g. When used in skin tests they are white or
light coloured. Unless otherwise stated, mice weigh not less than 17 g and
not more than 22 g.
Certain of the biological assays and tests of the Pharmacopoeia are such
that in the United Kingdom they may be carried out only in accordance
with the Animals (Scientific Procedures) Act 1986. Instructions included in
such assays and tests in the Pharmacopoeia, with respect to the handling of
animals, are therefore confined to those concerned with the accuracy and
reproducibility of the assay or test.

Reference Certain monographs require the use of a reference substance, a reference

Substances and preparation or a reference spectrum. These are chosen with regard to their
Reference intended use as prescribed in the monographs of the Pharmacopoeia and
Preparations are not necessarily suitable in other circumstances.
Any information necessary for proper use of the reference substance or
reference preparation is given on the label or in the accompanying leaflet or
brochure. Where no drying conditions are stated in the leaflet or on the
label, the substance is to be used as received. No certificate of analysis or
other data not relevant to the prescribed use of the product are provided.
The products are guaranteed to be suitable for use for a period of three
months from dispatch when stored under the appropriate conditions. The
stability of the contents of opened containers cannot be guaranteed. The
current lot is listed in the BP Laboratory website catalogue. Additional
information is provided in Supplementary Chapter III E.
Chemical Reference Substances The abbreviation BPCRS indicates a
Chemical Reference Substance established by the British Pharmacopoeia
Commission. The abbreviation CRS or EPCRS indicates a Chemical
Reference Substance established by the European Pharmacopoeia
Commission. Some Chemical Reference Substances are used for the
microbiological assay of antibiotics and their activity is stated, in
International Units, on the label or on the accompanying leaflet and defined
in the same manner as for Biological Reference Preparations.
Biological Reference Preparations The majority of the primary
biological reference preparations referred to are the appropriate
International Standards and Reference Preparations established by the
World Health Organisation. Because these reference materials are usually
available only in limited quantities, the European Pharmacopoeia has
established Biological Reference Preparations (indicated by the
abbreviation BRP or EPBRP) where appropriate. Where applicable, the
potency of the Biological Reference Preparations is expressed in
International Units. For some Biological Reference Preparations, where an
international standard or reference preparation does not exist, the potency is
expressed in European Pharmacopoeia Units.

Storage Statements under the side-heading Storage constitute non-mandatory

advice. The substances and preparations described in the Pharmacopoeia
are to be stored under conditions that prevent contamination and, as far as
possible, deterioration. Unless otherwise stated in the monograph, the
substances and preparations described in the Pharmacopoeia are kept in
well-closed containers and stored at a temperature not exceeding 25°.
Precautions that should be taken in relation to the effects of the
atmosphere, moisture, heat and light are indicated, where appropriate, in
1-16 General Notices 2016

the monographs. Further precautions may be necessary when some

materials are stored in tropical climates or under other severe conditions.
The expression ‘protected from moisture’ means that the product is to be
stored in an airtight container. Care is to be taken when the container is
opened in a damp atmosphere. A low moisture content may be maintained,
if necessary, by the use of a desiccant in the container provided that direct
contact with the product is avoided.
The expression ‘protected from light’ means that the product is to be
stored either in a container made of a material that absorbs actinic light
sufficiently to protect the contents from change induced by such light or in
a container enclosed in an outer cover that provides such protection or
stored in a place from which all such light is excluded.
The expression ‘tamper-evident container3 means a closed container fitted
with a device that reveals irreversibly whether the container has been
opened, whereas, the expression ‘tamper-proof container’ means a closed
container in which access to the contents is prevented under normal
conditions of use. The two terms are considered to be synonymous by the
European Pharmacopoeia Commission.

Labelling The labelling requirements of the Pharmacopoeia are not comprehensive,

and the provisions of regulations issued in accordance with the
requirements of the territory in which the medicinal product is to be used
should be met.
Licensed medicines intended for use within the United Kingdom must
comply with the requirements of The Human Medicines Regulations 2012
and European Directive 2001/83/EC, Title V (as amended) in respect of
their labelling and package leaflets, together with those regulations for the
labelling of hazardous materials.
Best practice guidance on the labelling and packaging of medicines for
use in the United Kingdom advises that certain items of information are
deemed critical for the safe use of the medicine (see “Best Practice
Guidance on the Labelling and Packaging of Medicines” issued by the
MHRA, 2012) . Further information and guidance on the labelling of
medicinal products can be found in Supplementary Chapter I G.
Such matters as the exact form of wording to be used and whether a
particular item of information should appear on the primary label and
additionally, or alternatively, on the package or exceptionally in a leaflet are,
in general, outside the scope of the Pharmacopoeia. When the term ‘label’
is used in Labelling statements of the Pharmacopoeia, decisions as to where
the particular statement should appear should therefore be made in
accordance with relevant legislation.
The label of every official formulated preparation other than those of
fixed strength also states the content of the active ingredient or ingredients
expressed in the terms required by the monograph. Where the content of
active ingredient is required to be expressed in terms other than the weight
of the official medicinal substance used in making the formulation, this is
specifically stated under the heading Labelling. Unless otherwise stated in
the monograph, the content of the active ingredient is expressed in terms of
the official medicinal substance used in making the formulation.
These requirements do not necessarily apply to unlicensed preparations
supplied in accordance with a prescription. For requirements for unlicensed
medicines see the general monograph on Unlicensed Medicines.
2016 General Notices 1-17

Action an d Use The statements given under this heading in monographs are intended only
as information on the principal pharmacological actions or the uses of the
materials in medicine or pharmacy. It should not be assumed that the
substance has no other action or use. The statements are not intended to be
binding on prescribers or to limit their discretion.

C rude D rugs; Herbal and complementary medicines are classed as medicines under European
T raditional H erbal Directive 2001/83/EC as amended. It is emphasised that, although requirements
and C om plem entary for the quality of the material are provided in the monograph to assist the
M edicines registration scheme by the UK Licensing Authority, the British Pharmacopoeia
Commission has not assessed the safety or efficacy of the material in traditional
use.
Monograph Title For traditional herbal medicines, the monograph title
is a combination of the binomial name together with a description of use.
Monographs for the material that has not been processed (the herbal drug)
and the processed material (the herbal drug preparation) are published
where possible. To distinguish between the two, the word ‘Processed’ is
included in the relevant monograph title.
Definition Under the heading Definition, the botanical name together
with any synonym is given. Where appropriate, for material that has not
been processed, information on the collection/harvesting and/or treatment/
drying of the whole herbal drug may be given. For processed materials, the
method of processing, where appropriate, will normally be given in a
separate section.
Characteristics References to odour are included only where this is
highly characteristic. References to taste are not included.
Control methods Where applicable, the control methods to be used in
monographs are:
(a) macroscopical and microscopical descriptions and chemical/
chromatographic tests for identification
(b) tests for absence of any related species
(c) microbial test to assure microbial quality
(d) tests for inorganic impurities and non-specific purity tests, including
extractive tests, Sulfated ash and Heavy metals where appropriate
(e) test for Loss on drying or Water
(f) wherever possible, a method for assaying the active constituent(s) or
suitable marker constituent (s).
The macroscopical characteristics include those features that can be seen
by the unaided eye or by the use of a hand lens. When two species/
subspecies of the same plant are included in the Definition, individual
differences between the two are indicated where possible.
The description of the microscopical characteristics of the powdered drug
includes information on the dominant or the most specific characters.
Where it is considered to be an aid to identification, illustrations of the
powdered drug may be provided.
The following aspects are controlled by the general monograph for
Herbal Drugs: they are required to be free from moulds, insects, decay,
animal matter and animal excreta. Unless otherwise prescribed the amount
of foreign matter is not more than 2 % w/w. Microbial contamination should
be minimal.
1-18 General Notices 2016

In determining the content of the active constituents or the suitable

marker substances measurements are made with reference to the dried or
anhydrous herbal drug. In the tests for Acid-insoluble ash. Ash, Extractive
soluble in ethanol. Loss on drying, Sulfated ash, Water, Water-soluble ash
and Water-soluble extractive of herbal drugs, the calculations are made with
reference to the herbal drug that has not been specifically dried unless
otherwise prescribed in the monograph.

Homoeopathic Homoeopathic medicines are classed as medicines under European Directive

Medicines 2001/83IEC as amended. It is emphasised that, although requirements for the
quality of the material are provided in the relevant monograph in order to assist
the simplified registration scheme by the UK Licensing Authority, the British
Pharmacopoeia Commission has not assessed the safety or efficacy of the material
in use.
All materials used for the production of homoeopathic medicines,
including excipients, must comply with European Pharmacopoeia or British
Pharmacopoeia monographs for those materials. Where such European
Pharmacopoeia or British Pharmacopoeia monographs do not exist, each
material used for the production of homoeopathic medicines must comply
with an official national pharmacopoeia of a Member State.
British Pharmacopoeia monographs for homoeopathic medicines apply to
homoeopathic stocks and mother tinctures only, but may be prefaced by a
section which details the quality requirements applicable to the principle
component where there is no European Pharmacopoeia or British
Pharmacopoeia monograph for the material. These monographs also
include either general statements on the methods of preparation or refer to
specific methods of preparation given in the European Pharmacopoeia.
Homoeopathic stocks and mother tinctures undergo the further process
referred to as potentisation. Potentisation is a term specific to homoeopathic
medicine and is a process of dilution of stocks and mother tinctures to
produce the final product.
Identification tests are established for the components in homoeopathic
stocks and usually relate to those applied to the materials used in the
production of the homoeopathic stocks. An assay is included for the
principal component(s) where possible. For mother tinctures, an
identification test, usually chromatographic, is established and, where
applicable, an assay for the principle component(s); where appropriate,
other tests, related to the solvent, dry matter or known adulterants, are
included.
Specifications have not been set for final homoeopathic products due to
the high dilution used in their preparation and the subsequent difficulty in
applying analytical methodology.
Statements under Crude Drugs; Traditional Herbal and Complementary
Medicines also apply to homoeopathic stocks and mother tinctures, when
appropriate.

Unlicensed The General Monograph for Unlicensed Medicines applies to those

Medicines formulations used in human medicine that are prepared under a
Manufacturer’s ‘Specials’ Licence or prepared extemporaneously under the
supervision of a pharmacist, whether or not there is a published monograph
for the specific dosage form.
An article intended for medicinal use that is described by means of an
official title must comply with the requirements of the relevant monograph.
2016 General Notices 1-19

A formulated preparation must comply throughout its assigned shelf-life

(period of validity). The subject of any other monograph must comply
throughout its period of use.
Unlicensed medicines that are prepared under a Manufacturer’s
‘Specials’ Licence comply with the requirements of the General Monograph
for Pharmaceutical Preparations, the requirements of the General
Monograph for Unlicensed Medicines and, where applicable, the
requirements of the individual monograph for the specific dosage form.
Unlicensed medicines prepared extemporaneously under the supervision
of a pharmacist comply with the requirements of the General Monograph
for Pharmaceutical Preparations, the requirements of the General
Monograph for Unlicensed Medicines and, where applicable, the
requirements of the individual monograph for the specific dosage form.
While it is expected that extemporaneous preparations will demonstrate
pharmacopoeial compliance when tested, it is recognised that it might not
be practicable to carry out the pharmacopoeial tests routinely on such
formulations. In the event of doubt or dispute, the methods of analysis, the
reference materials and the reference spectra of the Pharmacopoeia are
alone authoritative.
 I

1-20 General Notices 2016

Part III

Monographs and other texts of the European Pharmacopoeia that are incorporated
in thừ edition of the British Pharmacopoeia are governed by the general notices of
the European Pharmacopoeia; these are reproduced below.
GENERAL NOTICES OF THE EUROPEAN
PHARMACOPOEIA
1.1. GENERAL STATEMENTS
The General Notices apply to all monographs and other texts of the
European Pharmacopoeia.
The official texts of the European Pharmacopoeia are published in
English and French. Translations in other languages may be prepared by
die signatory States of die European Pharmacopoeia Convention. In case of
doubt or dispute, die English and French versions are alone authoritative.
In the texts of the European Pharmacopoeia, the word ‘Pharmacopoeia5
without qualification means the European Pharmacopoeia. The official
abbreviation Ph. Eur. may be used to indicate the European
Pharmacopoeia.
The use of the title or the subtitle of a monograph implies that the article
complies with die requirements of die relevant monograph. Such references
to monographs in die texts of the Pharmacopoeia are shown using die
monograph title and reference number in italics.
A preparation must comply throughout its period of validity; a distinct
period of validity and/or specifications for opened or broached containers
may be decided by the competent authority. The subject of any other
monograph must comply throughout its period of use. The period of
validity that is assigned to any given article and the time from which that
period is to be calculated are decided by the competent authority in light of
experimental results of stability studies.
Unless otherwise indicated in the General Notices or in the monographs,
statements in monographs constitute mandatory reqmrements. General
chapters become mandatory when referred to in a monograph, unless such
reference is made in a way that indicates that it is not die intention to make
the text referred to mandatory but rather to cite it for information.
The active substances, excipients, pharmaceutical preparations and other
articles described in the monographs are intended for human and veterinary
use (unless explicitly restricted to one of these uses).

Quality systems The quality standards represented by monographs are valid only where the
articles in question are produced within the framework of a suitable quality
system. The quality system must assure that the articles consistently meet
die requirements of die Pharmacopoeia.

Alternative methods The tests and assays described are the official methods upon which the
standards of die Pharmacopoeia are based. With the agreement of die
competent authority, alternative methods of analysis may be used for
conưol purposes, provided that the methods used enable an unequivocal
decision to be made as to whether compliance with the standards of the
2016 General Notices 1-21

monographs would be achieved if the official methods were used. In the

event of doubt or dispute, the methods of analysis of the Pharmacopoeia are
alone authoritative.

Demonstration of (1) An article is not of Pharmacopoeia quality unless it complies with all
compliance with the the requirements stated in the monograph. This does not imply that
Pharmacopoeia performance of all the tests in a monograph is necessarily a prerequisite
for a manufacturer in assessing compliance with the Pharmacopoeia
before release of a product. The manufacturer may obtain assurance
that a product is of Pharmacopoeia quality on the basis of its design,
together with its control strategy and data derived, for example, from
validation studies of the manufacturing process.
(2) An enhanced approach to quality control could utilise process analytical
technology (PAT) and/or real-time release testing (including parametric
release) strategies as alternatives to end-product testing alone. Real-time
release testing in circumstances deemed appropriate by the competent
authority is thus not precluded by the need to comply with the
Pharmacopoeia.
(3) Reduction of animal testing: the European Pharmacopoeia is dedicated
to phasing out the use of animals for test purposes, in accordance with
the 3Rs (Replacement, Reduction, Refinement) set out in the European
Convention for the Protection of Vertebrate Animals used for
Experimental and Other Scientific Purposes. In demonstrating
compliance with the Pharmacopoeia as indicated above ( 1),
manufacturers may consider establishing additional systems to monitor
consistency of production. With the agreement of the competent
authority, the choice of tests performed to assess compliance with the
Pharmacopoeia when animal tests are prescribed is established in such
a way that animal usage is minimised as much as possible.

Grade of materials Certain materials that are the subject of a pharmacopoeial monograph may
exist in different grades suitable for different purposes. Unless otherwise
indicated in the monograph, the requirements apply to all grades of the
material. In some monographs, particularly those on excipients, a list of
functionality-related characteristics that are relevant to the use of the
substance may be appended to the monograph for information. Test
methods for determination of one or m ore of these characteristics may be
given, also for information.

General Substances and preparations that are the subject of an individual

monographs monograph are also required to comply with relevant, applicable general
monographs. Cross-references to applicable general monographs are not
normally given in individual monographs.
General monographs apply to all substances and preparations within the
scope of the Definition section of the general monograph, except where a
preamble limits the application, for example to substances and preparations
that are the subject of a monograph of the Pharmacopoeia.
General monographs on dosage forms apply to all preparations of the
type defined. The requirements are not necessarily comprehensive for a
given specific preparation and requirements additional to those prescribed
in the general monograph may be imposed by the competent authority.
1-22 General Notices 2016

General monographs and individual monographs are complementary. If

the provisions of a general monograph do not apply to a particular product,
this is expressly stated in the individual monograph.

Validation of The test methods given in monographs and general chapters have been
pharmac opoeial validated in accordance with accepted scientific practice and current
methods recommendations on analytical validation. Unless otherwise stated in the
monograph or general chapter, validation of the test methods by the analyst
is not required.

Implementation o f When implementing a pharmacopoeial method, the user must assess

pharmac opoeial whether and to what extent the suitability of the method under the actual
methods conditions of use needs to be demonstrated according to relevant
monographs, general chapters and quality systems.

Conventional terms The term ‘competent authority’ means the national, supranational or
international body or organisation vested with the authority for making
decisions concerning the issue in question. It may, for example, be a
national pharmacopoeia authority, a licensing authority or an official control
laboratory.
The expression ‘unless otherwise justified and authorised’ means that the
requirements have to be met, unless the competent authority authorises a
modification or an exemption where justified in a particular case.
Statements containing the word ‘should’ are informative or advisory.
In certain monographs or other texts, the terms ‘suitable’ and
‘appropriate’ are used to describe a reagent, micro-organism, test method
etc.; if criteria for suitability are not described in the monograph, suitability
is demonstrated to the satisfaction of the competent authority.
Medicinal product (a) Any substance or combination of substances
presented as having properties for treating or preventing disease in human
beings and/or animals; or (b) any substance or combination of substances
that may be used in or administered to human beings and/or animals with a
view either to restoring, correcting or modifying physiological functions by
exerting a pharmacological, immunological or metabolic action, or to
making a medical diagnosis.
Herbal m edicinal product Any medicinal product, exclusively
containing as active ingredients one or more herbal drugs or one or more
herbal drug preparations, or one or more such herbal drugs in combination
with one or more such herbal drug preparations.
Active substance Any substance intended to be used in the manufacture
of a medicinal product and that, when so used, becomes an active
ingredient of the medicinal product. Such substances are intended to
furnish a pharmacological activity or other direct effect in the diagnosis,
cure, mitigation, treatment or prevention of disease, or to affect the
structure and function of the body.
Excipient (auxiliary substance). Any constituent of a medicinal product
that is not an active substance. Adjuvants, stabilisers, antimicrobial
preservatives, diluents, antioxidants, for example, are excipients.

Interchangeable Certain general chapters contain a statement that the text in question is
methods harmonised with the corresponding text of the Japanese Pharmacopoeia
and/or the United States Pharmacopeia and that these texts are
interchangeable. This implies that if a substance or preparation is found to
2016 General Notices 1-23

comply with a requirement using an interchangeable method from one of

these pharmacopoeias it complies with the requirements of the European
Pharmacopoeia. In the event of doubt or dispute, the text of the European
Pharmacopoeia is alone authoritative.

References to Monographs and general chapters may contain references to documents

regulatory issued by regulatory authorities for medicines, for example directives and
documents notes for guidance of the European Union. These references are provided
for information for users for the Pharmacopoeia. Inclusion of such a
reference does not modify the status of the documents referred to, which
may be mandatory or for guidance.
1.2. OTHER PROVISIONS APPLYING TO GENERAL
CHAPTERS AND MONOGRAPHS
Quantities In tests with numerical limits and assays, the quantity stated to be taken for
examination is approximate. The amount actually used, which may deviate
by not more than 10 per cent from that stated, is accurately weighed or
measured and the result is calculated from this exact quantity. In tests
where the limit is not numerical, but usually depends upon comparison
with the behaviour of a reference substance in the same conditions, the
stated quantity is taken for examination. Reagents are used in the
prescribed amounts.
Quantities are weighed or measured with an accuracy commensurate with
the indicated degree of precision. For weighings, the precision corresponds
to plus or minus 5 units after the last figure stated (for example, 0.25 g is to
be interpreted as 0.245 g to 0.255 g). For the measurement of volumes, if
the figure after the decimal point is a zero or ends in a zero (for example,
10.0 mL or 0.50 mL), the volume is measured using a pipette, a volumetric
flask or a burette, as appropriate; otherwise, a graduated measuring cylinder
or a graduated pipette may be used. Volumes stated in microlitres are
measured using a micropipette or micro syringe.
It is recognised, however, that in certain cases the precision with which
quantities are stated does not correspond to the number of significant
figures stated in a specified numerical limit. The weighings and
measurements are then carried out with a sufficiently improved accuracy.

Apparatus and Volumetric glassware complies with Class A requirements of the appropriate
procedures International Standard issued by the International Organisation for
Standardisation.
Unless otherwise prescribed, analytical procedures are carried out at a
temperature between 15 °C and 25 °C.
Unless otherwise prescribed, comparative tests are carried out using
identical tubes of colourless, transparent, neutral glass with a flat base; the
volumes of liquid prescribed are for use with tubes having an internal
diameter of 16 mm, but tubes with a larger internal diameter may be used
provided the volume of liquid used is adjusted (2.1.5). Equal volumes of
the liquids to be compared are examined down the vertical axis of the tubes
against a white background, or if necessary against a black background. The
exam ination is carried out in diffuse light.
Any solvent required in a test or assay in which an indicator is to be used
is previously neutralised to the indicator, unless a blank test is prescribed.
1-24 General Notices 2016

Water-bath The term ‘water-bath’ means a bath of boiling water unless water at
another temperature is indicated. Other methods of heating may be
substituted provided the temperature is near to but not higher than 100 °C
or the indicated temperature.

Drying and ignition The terms ‘dried to constant mass’ and ‘ignited to constant mass’ mean
to constant mass that 2 consecutive weighings do not differ by more than 0.5 mg, the 2nd
weighing following an additional period of drying or of ignition respectively
appropriate to the nature and quantity of the residue.
Where drying is prescribed using one of the expressions ‘in a desiccator’
or ‘in vacuo’, it is carried out using the conditions described in chapter
2.2.32. Loss on drying.

Reagents The proper conduct of the analytical procedures described in the

Pharmacopoeia and the reliability of the results depend, in part, upon the
quality of the reagents used. The reagents are described in general chapter
4. It is assumed that reagents of analytical grade are used; for some
reagents, tests to determine suitability are included in the specifications.

Solvents Where the name of the solvent is not stated, the term ‘solution’ implies a
solution in water.
Where the use of water is specified or implied in the analytical
procedures described in the Pharmacopoeia or for the preparation of
reagents, water complying with the requirements of the monograph Purified
water (0008) is used, except that for many purposes the requirements for
bacterial endotoxins (Purified water in bulk) and microbial contamination
(Purified water in containers) are not relevant. The term ‘distilled water’
indicates purified water prepared by distillation.
The term ‘ethanol’ without qualification means anhydrous ethanol. The
term ‘alcohol’ without qualification means ethanol (96 per cent). Other
dilutions of ethanol are indicated by the term ‘ethanol’ or ‘alcohol’ followed
by a statement of the percentage by volume of ethanol (C2H 60 ) required.

Expression of In defining content, the expression ‘per cent’ is used according to

content circumstances with one of 2 meanings:
— per cent m/m (percentage, mass in mass) expresses the number of
grams of substance in 100 g of final product;
— per cent V/V (percentage, volume in volume) expresses the number of
millilitres of substance in 100 mL of final product.
The expression ‘parts per million’ (or ppm) refers to mass in mass, unless
otherwise specified.

Temperature Where an analytical procedure describes temperature without a figure, the

general terms used have the following meaning:
— in a deep-freeze: below -1 5 °C;
— in a refrigerator: 2 °C to 8 °C;
— cold or cool: 8 °C to 15 °C;
— room temperature: 15 °C to 25 °C.
2016 General Notices 1-25

1.3. GENERAL CHAPTERS

Containers Materials used for containers are described in general chapter 3.1. General
names used for materials, particularly plastic materials, each cover a range
of products varying not only in the properties of the principal constituent
but also in the additives used. The test methods and limits for materials
depend on the formulation and are therefore applicable only for materials
whose formulation is covered by the preamble to the specification. The use
of materials with different formulations, and the test methods and limits
applied to them, are subject to agreement by the competent authority.
The specifications for containers in general chapter 3.2 have been
developed for general application to containers of the stated category, but in
view of the wide variety of containers available and possible new
developments, the publication of a specification does not exclude the use, in
justified circumstances, of containers that comply with other specifications,
subject to agreement by the competent authority.
Reference may be made within the monographs of the Pharmacopoeia to
the definitions and specifications for containers provided in chapter 3.2.
Containers. The general monographs for pharmaceutical dosage forms may,
under the heading Definition/Production, require the use of certain types of
container; certain other monographs may, under the heading Storage,
indicate the type of container that is recommended for use.
1.4. MONOGRAPHS
Titles Monograph titles are in English and French in the respective versions and
there is a Latin subtitle.

Relative Atomic and The relative atomic mass (A r) or the relative molecular mass (Mr) is shown,
Molecular Masses as and where appropriate, at the beginning of each monograph. The relative
atomic and molecular masses and the molecular and graphic formulae do
not constitute analytical standards for the substances described.

Chemical Abstracts CAS registry numbers are included for information in monographs, where
Service (CAS) applicable, to provide convenient access to useful information for users.
Registry Number CAS Registry Number^ is a registered trademark of the American
Chemical Society.

Definition Statements under the heading Definition constitute an official definition of

the substance, preparation or other article that is the subject of the
monograph.
Limits of content Where limits of content are prescribed, they are those
determined by the method described under Assay.
Herbal drugs In monographs on herbal drugs, the definition indicates
whether the subject of the monograph is, for example, the whole drug or
the drug in powdered form. Where a monograph applies to the drug in
several states, for example both to the whole drug and the drug in
powdered form, the definition states this.

Production Statements under the heading Production draw attention to particular

aspects of the manufacturing process but are not necessarily comprehensive.
They constitute mandatory requirements for manufacturers, unless
otherwise stated. They may relate, for example, to source materials; to the
manufacturing process itself and its validation and control; to in-process
1-26 General Notices 2016

testing] or to testing that is to be carried out by the manufacturer on the

final article, either on selected batches or on each batch prior to release.
These statements cannot necessarily be verified on a sample of the final
article by an independent analyst. The competent authority may establish
that the instructions have been followed, for example, by examination of
data received from the manufacturer, by inspection of manufacture or by
testing appropriate samples.
The absence of a Production section does not imply that attention to
features such as those referred to above is not required.
Choice of vaccine strain, Choice of vaccine composition The
Production section of a monograph may define the characteristics of a
vaccine strain or vaccine composition. Unless otherwise stated, test methods
given for verification of these characteristics are provided for information as
examples of suitable methods. Subject to approval by the competent
authority, other test methods may be used without validation against the
method shown in the monograph.

Potential Due to the increasing number of fraudulent activities and cases of

Adulteration adulteration, information may be made available to Ph. Eur. users to help
detect adulterated materials (i.e. active substances, excipients, intermediate
products, bulk products and finished products).
To this purpose, a method for the detection of potential adulterants and
relevant limits, together with a reminder that all stages of production and
sourcing are subjected to a suitable quality system, may be included in this
section of monographs on substances for which an incident has occurred or
that present a risk of deliberate contamination. The frequency of testing by
manufacturers or by users (e.g. manufacturers of intermediate products,
bulk products and finished products, where relevant) depends on a risk
assessment, taking into account the level of knowledge of the whole supply
chain and national requirements.
This section constitutes requirements for the whole supply chain, from
manufacturers to users (e.g. manufacturers of intermediate products, bulk
products and finished products, where relevant). The absence of this section
does not imply that attention to features such as those referred to above is
not required.

Characters The statements under the heading Characters are not to be interpreted in a
strict sense and are not requirements.
Solubility In statements of solubility in the Characters section, the terms
used have the following significance, referred to a temperature between
15 °C and 25 °C.

Descriptive terra Approximate volume of solvent in millilitres

per gram of solute
Very soluble less than 1

Freely soluble from 1 to 10

Soluble from 10 to 30

Sparingly soluble from 30 to 100

Slightly soluble from 100 to 1000

Very slightly soluble from 1000 to 10 000

Practically insoluble more than 10 000

2016 General Notices 1-27

The term ‘partly soluble’ is used to describe a mixture where only some
of the components dissolve. The term ‘miscible’ is used to describe a liquid
that is miscible in all proportions with the stated solvent.

Identification Scope The tests given in the Identification section are not designed to give
a full confirmation of the chemical structure or composition of the product;
they are intended to give confirmation, with an acceptable degree of
assurance, that the article conforms to the description on the label.
First and second identifications Certain monographs have
subdivisions entitled ‘First identification’ and ‘Second identification’. The
test or tests that constitute the ‘First identification’ may be used in all
circumstances. The test or tests that constitute the ‘Second identification’
may be used in pharmacies provided it can be demonstrated that the
substance or preparation is fully traceable to a batch certified to comply
with all the other requirements of the monograph.
Certain monographs give two or more sets of tests for the purpose of the
first identification, which are equivalent and may be used independently.
One or more of these sets usually contain a cross-reference to a test
prescribed in the Tests section of the monograph. It may be used to
simplify the work of the analyst carrying out the identification and the
prescribed tests. For example, one identification set cross-refers to a test for
enantiomeric purity while the other set gives a test for specific optical
rotation: the intended purpose of the two is the same, that is, verification
that the correct enantiomer is present.
Powdered herbal drugs Monographs on herbal drugs may contain
schematic drawings of the powdered drug. These drawings complement the
description given in the relevant identification test.

Tests and Assays Scope The requirements are not framed to take account of all possible
impurities. It is not to be presumed, for example, that an impurity that is
not detectable by means of the prescribed tests is tolerated if common sense
and good pharmaceutical practice require that it be absent. See also below
under Impurities.
Calculation Where the result of a test or assay is required to be
calculated with reference to the dried or anhydrous substance or on some
other specified basis, the determination of loss on drying, water content or
other property is carried out by the method prescribed in the relevant test
in the monograph. The words ‘dried substance’ or ‘anhydrous substance’
etc. appear in parentheses after the result.
Where a quantitative determination of a residual solvent is carried out
and a test for loss on drying is not carried out, the content of residual
solvent is taken into account for the calculation of the assay content of the
substance, the specific optical rotation and the specific absorbance. No
further indication is given in the specific monograph.
Limits The limits prescribed are based on data obtained in normal
analytical practice; they take account of normal analytical errors, of
acceptable variations in manufacture and compounding and of deterioration
to an extent considered acceptable. No further tolerances are to be applied
to the limits prescribed to determine whether the article being examined
complies with the requirements of the monograph.
In determining compliance with a numerical limit, the calculated result of
a test or assay is first rounded to the number of significant figures stated,
unless otherwise prescribed. The limits, regardless of whether the values are
1-28 General Notices 2016

expressed as percentages or as absolute values, are considered significant to

the last digit shown (for example 140 indicates 3 significant figures). The
last figure of the result is increased by one when the part rejected is equal to
or exceeds one half-unit, whereas it is not modified when the part rejected
is less than a half-unit.
Indication of permitted lim it of impurities The acceptance criteria
for related substances are expressed in monographs either in terms of
comparison of peak areas (comparative tests) or as numerical values. For
comparative tests, the approximate content of impurity tolerated, or the
sum of impurities, may be indicated in brackets for information only.
Acceptance or rejection is determined on the basis of compliance or non-
compliance with the stated test. If the use of a reference substance for the
named impurity is not prescribed, this content may be expressed as a
nominal concentration of the substance used to prepare the reference
solution specified in the monograph, unless otherwise described.
Herbal drugs For herbal drugs, the sulfated ash, total ash, water-soluble
matter, alcohol-soluble matter, water content, content of essential oil and
content of active principle are calculated with reference to the drug that has
not been specially dried, unless otherwise prescribed in the monograph.
Equivalents Where an equivalent is given, for the purposes of the
Pharmacopoeia only the figures shown are to be used in applying the
requirements of the monograph.
Culture media The culture media described in monographs and general
chapters have been found to be satisfactory for the intended purpose.
However, the components of media, particularly those of biological origin,
are of variable quality, and it may be necessary for optimal performance to
modulate the concentration of some ingredients, notably:
— peptones and meat or yeast extracts, with respect to their nutritive
properties;
— buffering substances;
— bile salts, bile extract, deoxycholate, and colouring matter, depending
on their selective properties;
— antibiotics, with respect to their activity.

Storage The information and recommendations given under the heading Storage do
not constitute a pharmacopoeial requirement but the competent authority
may specify particular storage conditions that must be met.
The articles described in the Pharmacopoeia are stored in such a way as
to prevent contamination and, as far as possible, deterioration. Where
special conditions of storage are recommended, including the type of
container (see section 1.3. General chapters) and limits of temperature, they
are stated in the monograph.
The following expressions are used in monographs under Storage with
the meaning shown.
In an airtight container Means that the product is stored in an airtight
container (3.2). Care is to be taken when the container is opened in a damp
atmosphere. A low moisture content may be maintained, if necessary, by
the use of a desiccant in the container provided that direct contact with the
product is avoided.
Protected fr o m light Means that the product is stored either in a
container made of a material that absorbs actinic light sufficiently to protect
the contents from change induced by such light, or in a container enclosed
2016 General Notices 1-29

in an outer cover that provides such protection, or is stored in a place from

which all such light is excluded.

Labelling In general, labelling of medicines is subject to supranational and national

regulation and to international agreements. The statements under the
heading Labelling are not therefore comprehensive and, moreover, for the
purposes of the Pharmacopoeia only those statements that are necessary to
demonstrate compliance or non-compliance with the monograph are
mandatory. Any other labelling statements are included as
recommendations. When the term ‘label’ is used in the Pharmacopoeia, the
labelling statements may appear on the container, the package, a leaflet
accompanying the package, or a certificate of analysis accompanying the
article, as decided by the competent authority.

Warnings Materials described in monographs and reagents specified for use in the
Pharmacopoeia may be injurious to health unless adequate precautions are
taken. The principles of good quality control laboratory practice and the
provisions of any appropriate regulations are to be observed at all times.
Attention is drawn to particular hazards in certain monographs by means of
a warning statement; absence of such a statement is not to be taken to
mean that no hazard exists.

Impurities A list of all known and potential impurities that have been shown to be
detected by the tests in a monograph may be given. See also chapter 5.10.
Control of impurities in substances for pharmaceutical use. The impurities are
designated by a letter or letters of the alphabet. Where a letter appears to be
missing, the impurity designated by this letter has been deleted from the list
during monograph development prior to publication or during monograph
revision.

Functionality- Monographs on excipients may have a section on functionality-related

Related characteristics. The characteristics, any test methods for determination and
Characteristics of any tolerances are not mandatory requirements; they may nevertheless be
Excipients relevant for use of the excipient and are given for information (see also
section 1.1. General statements).

Reference Certain monographs require the use of reference standards (chemical

Standards reference substances, herbal reference standards, biological reference
preparations, reference spectra). See also chapter 5.12. Reference standards.
The European Pharmacopoeia Commission establishes the official reference
standards, which are alone authoritative in case of arbitration. These
reference standards are available from the European Directorate for the
Quality of Medicines & HealthCare (EDQM). Information on the available
reference standards and a batch validity statement can be obtained via the
EDQM website.
1-30 General Notices 2016

1.5. ABBREVIATIONS AND SYMBOLS

A Absorbance mp M elting point

*1 per cent Specific absorbance r,20 Refractive index
A \ cm
At Relative atom ic mass Ph. Eur. U. European Pharm acopoeia U nit

Md Specific optical rotation PPb Parts per billion (micrograms per kilogram)
bp Boiling point ppm Parts per million (milligrams p er kilogram)
BRP Biological Reference Preparation R Substance or solution defined under
CRS Chemical Reference Substance 4. Reagents
j20 Rf Retardation factor (see chapter 2.2.46)
“20 Relative density
X W avelength R:t U sed in chrom atography to indicate the ratio
o f the distance travelled by a substance to the
HRS H erbal reference standard distance travelled by a reference substance
IU International U nit RV Substance used as a prim ary standard in
M M olarity volumetric analysis (chapter 4.2.1)
Mr Relative m olecular mass

Abbreviations used in the monographs on imm unoglobulins, immunosera and vaccines

L D 50 T h e statistically determ ined quantity o f a Lo/10 dose T h e largest quantity of a toxin that, in the
substance that, when administered by the conditions of the test, when mixed with 0.1 IU
specified route, may be expected to cause the o f antitoxin and adm inistered by the specified
death o f 50 p er cent of the test animals within route, does not cause symptoms of toxicity in
a given period the test animals within a given period
M LD M inim um lethal dose L f dose T h e quantity of toxin or toxoid that flocculates
in the shortest time with 1 IU o f antitoxin
L + /1 0 dose T h e smallest quantity of a toxin that, in the
conditions of the test, when mixed with 0.1 IU CCIDso T h e statistically determ ined quantity o f virus
of antitoxin and administered by the specified th at may be expected to infect 50 p er cent of
route, causes th e death of the test anim als the cell cultures to which it is added
within a given period
EID50 T h e statistically determ ined quantity o f virus
L + dose T he smallest quantity o f a toxin that, in the th at may be expected to infect 50 per cent of
conditions of th e test, when mixed with 1 IU fertilised eggs into which it is inoculated
of antitoxin an d adm inistered by the specified ID 50 T h e statistically determ ined quantity of a virus
route, causes the death of the test animals that may be expected to infect 50 per cent of
within a given period the animals into which it is inoculated
lr/100 dose T he smallest quantity o f a toxin that, in the T h e statistically determ ined dose of a vaccine
P D 50
conditions o f the test, when mixed with that, in the conditions of the test, may be
0.01 IU of antitoxin and injected expected to protect 50 per cent o f the animals
intracutaneously causes a characteristic against a challenge dose of the micro­
reaction at the site o f injection within a organisms or toxins against which it is active
given period
EDsn T h e statistically determ ined dose of a vaccine
L p/10 dose T h e smallest quantity o f toxin that, in the that, in the conditions of the test, may be
conditions o f the test, when mixed w ith 0.1 IU expected to induce specific antibodies in
of antitoxin an d adm inistered by the specified 50 per cent o f the animals for the relevant
route, causes paralysis in the test anim als vaccine antigens
within a given period
PFU Pock-forming units or plaque-form ing units
SPF Specified-pathogen-free.
2016 General Notices 1-31

Collections of micro-organisms

A TCC American Type Culture Collection NCTC N ational Collection of Type Cultures
10801 University Boulevard C entral Public Health Laboratory
Manassas, Virginia 20110-2209, USA Colindale Avenue
London NW9 5HT, Great Britain
C.I.P. Collection de Bactéries de l’Institut Pasteur
B.P. 52, 25 rue du D octeur Roux NCYC N ational Collection of Yeast C ultures
75724 Paris Cedex 15, France A FRC Food Research Institute
Colney Lane
IM I International Mycological Institute
Norwich NR4 7UA, Great Britain
Bakeham Lane
Surrey T W 20 9TY, Great Britain N IT E Biological Resource Center
D epartm ent of Biotechnology
I.P . Collection Nationale de Culture de
N ational Institute o f Technology and
Microorganismes (C.N .C.M .)
Evaluation
Institut Pasteur
2-5-8 Kazusakamatari, Kisarazu-shi, Chiba,
25, rue du D octeur Roux
292-0818
75724 Paris Cedex 15, France
Japan
N C IM B N ational Collection of Industrial and M arine
S.S.I. Statens Serum Institut
Bacteria Ltd
80 Amager Boulevard, Copenhagen, D enm ark
23 St Machar Drive
Aberdeen AB2 1RY, Great Britain
N CPF N ational Collection of Pathogenic Fungi
London School of Hygiene and Tropical
Medicine
Keppel Street
London W C1E 7 H T , Great Britain

1.6. UNITS OF THE INTERNATIONAL SYSTEM (SI) USED IN

THE PHARMACOPOEIA AND EQUIVALENCE WITH OTHER
UNITS
International The International System of Units comprises 3 classes of units, namely base
System O f Units units, derived units and supplementary units1. The base units and their
(SI) definitions are set out in Table 1.6-1.
The derived units may be formed by combining the base units according
to the algebraic relationships linking the corresponding quantities. Some of
these derived units have special names and symbols. The SI units used in
the Pharmacopoeia are shown in Table 1.6-2.
Some important and widely used units outside the International System
are shown in Table 1.6-3.
The prefixes shown in Table 1.6-4 are used to form the names and
symbols of the decimal multiples and submultiples of SI units.

1 The definitions of the units used in the International System are given in the booklet "Le Systeme International d’Unites (SI)” published by

the Bureau International des Poids et Mesures, Pavillion de Breteuil, F-92310 Sevres.
1-32 General Notices 2016

Notes 1. In the Pharmacopoeia, the Celsius temperature is used (symbol r).

This is defined by the following equation:

t = T-To

where T0 = 213.15 K by definition. The Celsius or centigrade

temperature is expressed in degree Celsius (symbol °C). The unit
‘degree Celsius’ is equal to the unit ‘kelvin’.
2. The practical expressions of concentrations used in the Pharmacopoeia
are defined in the General Notices.
3. The radian is the plane angle between two radii of a circle that cut off
on the circumference an arc equal in length to the radius.
4. In the Pharmacopoeia, conditions of centrifugation are defined by
reference to the acceleration due to gravity (g):

g = 9.806 65 m • s ~ 2

5. Certain quantities without dimensions are used in the Pharmacopoeia:

relative density (2.2.5), absorbance (2.2.25), specific absorbance
(2.2.25) and refractive index (2.2.6).
6. The microkatal is defined as the enzymic activity that, under defined
conditions, produces the transformation (e.g. hydrolysis) of 1 micromole
of the substrate per second.

Table 1.6.-1. - S I base units

Quantity Unit Definition
Name Symbol Name Symbol

Length I metre m The metre is the length of the path travelled by light in a vacuum during a time
interval of 1/299 792 458 of a second.
Mass m kilogram kg The kilogram is equal to the mass of the international prototype of the kilogram.

The second is the duration of 9 192 631 770 periods of the radiation corresponding
Time t second s to the transition between the two hyperfine levels of the ground state of the
caesium-133 atom.
Electric current I ampere A The ampere is that constant current which, maintained in two straight parallel
conductors of infinite length, of negligible circular cross-section and placed 1 metre
apart in vacuum would produce between these conductors a force equal to 2 x 10'7
newton per metre of length.
Thermodynamic T kelvin K The kelvin is the fraction 1/273.16 of the thermodynamic temperature of the triple
temperature point of water.
Amount of substance n mole mol The mole is the amount of substance of a system containing as many elementary
entities as there are atoms in 0.012 kilogram of carbon-12*.
Luminous intensity I, candela cd The candela is the luminous intensity in a given direction of a source emitting
monochromatic radiation with a frequency of 540 x 10IJ hertz and whose energy
intensity in that direction is 1/683 watt per steradian.
* When the mole is used, the elementary entities must be specified and may be atoms, molecules, ions, electrons, other particles or specified groups of
such particles.
2016 General Notices 1-33

Table 1.6.-2. - SI units used in the European Pharmacopoeia and equivalence with other units
Quantity Unit

Name Name Symbol Expression b SI Expression in other Conversion of other units into SI units
Symbol
base units SI units
Wave number V one per metre 1/m m '1

pm

3° 3"
Wavelength X micrometre

o o
nanometre nm

Area A, S square metre m2 mJ

Volume V cubic metre m3 m3 1 mL = 1 cm3 = 10'6 m3

Frequency V hertz Hz s '1

Density P kilogram per cubic kg/m3 kg-m"3 1 g/mL = 1 g/cm3 =■ 103 kg-m'3
metre
Velocity V metre per second m/s m-s'1

Force F newton N m-kg-s"5 1 dyne = 1 g-cm-s'3 = 10*s N

1 kp = 9.806 65 N
Pressure P pascal Pa m‘ Lkg-s'2 N-m'5 1 dyne/cm2 = 10*1 Pa = 10'1N-m"2
1 atm = 101 325 Pa =* 101.325 kPa
1 bar - 10s Pa = 0.1 MPa
1 mm Hg =* 133.322 387 Pa
lT o rr= 133.322 368 Pa
1 psi = 6.894 757 kPa
Dynamic V pascal second Pa-s m'^kg-s'1 N-s-m'2 1 P - 10'1Pa-s - 10'1N-s-m'1
viscosity 1 cP =* 1 mPa-s
Kinematic V square metre per m2/ s m2-s*‘ Pa-s-n^-kg-1 1 St = 1 cm2-s'1* 10'* m2-s'’
viscosity second N-m-s-kg"1
Energy W joule J m2-kg-s"2 N-m 1 erg - 1 cm2-g-s‘2 = 1 dyne-cm = 10'7J
l e a l» 4.1868 J
Power P watt W m2-kg-s'3 N-m-s"1 1 e r g / s - 1 dyne-cm-s'1=
Radiant flux J-s"1 10'7 W = 10'7 N-m-s'1* 10-7J-s'1
Absorbed dose D gray Gy m^s"2 /•kg-1 1 rad » lO'2 Gy
(of radiant
energy)
Electric U volt V m2- kg-s'3-A*‘ W-A*1
potential,
electromotive
force
Electric R ohm Q m2- kg-s'3-A'2 V-A-*
resistance
Quantity of Q coulomb c A-s
electricity
Activity of a A becquerel Bq s '1 1 Ci - 37-109 Bq = 37-109 s '1
radionuclide
Concentration c mole per cubic mol/m3 mol-m'3 1 m o l/L * 1 M * 1 mol/dm5 - 103 mol-m'3
(of amount of metre
substance),
molar
concentration
Mass P kilogram per cubic kg/m3 kg-m'3 1 g/L = 1 g/dm3» 1 kg-m'3
concentration metre
1-34 General Notices 2016

Table 1.6.-3. - Units used with the International System

Quantity Unit Value in SI units

Name Symbol

Time minute min 1 min = 60 s

hour h 1 h = 60 mũi = 3600 s

day d 1 d = 24 h - 86 400 s

Plane angle degree » 1° = (n/180) rad

Volume liừe L 1 L “ 1 dm1 ” 10"3 m3

Mass tonne t 1 t » 103 kg

Rotational revolution r/m in 1 r/min =* (1/60) s*1

frequency per minute

Table 1.6.4. - Decimal multiples and sub-multiples o f units

Factor Prefix Symbol Factor Prefix Symbol

1018 exa E 10-1 deci d

10>5 peta p 10-* centi c

10“ tera T 10-3 milli m

109 giga G 10-* micro ụ

106 mega M 10-9 nano n

103 kilo k 10-« pico p

102 hecto h 10-“ fern to f

10' deca da 10-18 atto a

Monographs

Medicinal and Pharmaceutical

Substances A to I
2016 General Monographs 1-37

T h e manufacture o f active substances m ust take place under

MEDICINAL AND PHARMACEUTICAL conditions o f good manufacturing practice.
SUBSTANCES T h e provisions of general chapter 5.10 apply to the control of
impurities in substances for pharmaceutical use.
W hether or no t it is specifically stated in the individual
monograph th at the substance for pharmaceutical me:
Substances ★ ★ — is a recom binant protein or another substance obtained as
★ ★ a direct gene product based on genetic modification,
for Pharmaceutical Use ***** where applicable, the substance also complies with the
(Ph. Eur. monograph 2034) requirements o f the general monograph Products of
recombinant DNA technology (0784)’,
PhEur------ -----------------------------------------------------------------------------—
— is obtained from animals susceptible to transmissible
D E F IN IT IO N spongiform encephalopathies other than by experimental
Substances for pharmaceutical use are any organic or challenge, where applicable, the substance also complies
inorganic substances that are used as active substances or with the requirements of the general monograph Products
excipients for the production of medicinal products for with risk of transmitting agents of animal spongiform
hum an or veterinary use. They may be obtained from natural encephalopathies (1483)',
sources or produced by extraction from raw materials, — is a substance derived from a fermentation process,
fermentation or synthesis. whether or n o t the micro-organisms involved are modified
T his general m onograph does not apply to herbal drugs, by traditional procedures or recom binant D N A (rDNA)
herbal drugs for homoeopathic preparations, herbal drug technology, where applicable, the substance also complies
preparations, extracts, or m other tinctures for homoeopathic with the requirements o f the general monograph Products
preparations, which are the subject of separate general offermentation (1468).
monographs (Herbal drugs (1433), Herbal drugs for I f solvents are used during production, they are of suitable
homoeopathic preparations (2045), Herbal drug quality. In addition, their toxidty and their residual level are
preparations (1434), Extracts (0765), Mother tinctures for taken into consideration (5.4). If water is used during
homoeopathic preparations (2029)). It does not apply to raw production, it is o f suitable quality.
materials for homoeopathic preparations, except where there I f substances are produced or processed to yield a certain
is an individual m onograph for the substance in the non- form or grade, that specific form or grade of the substance
homoeopathic p art of the Pharmacopoeia. complies with the requirements of the monograph. Certain
Where a substance for pharmaceutical use not described in functionality-related tests may be described to control
an individual monograph o f the Pharmacopoeia is used in a properties that may influence the suitability of the substance
medicinal product prepared for the special needs of and subsequently the properties o f dosage forms prepared
individual patients, the need for compliance with the present from it.
general monograph is decided in the light o f a risk Powdered substances May be processed to obtain a certain
assessment that takes account o f the available quality o f the degree of fineness (2.9.35).
substance and its intended use.
Compacted substances Are processed to increase the particle
W here medicinal products are manufactured using size or to obtain particles o f a specific form and/or to obtain
substances for pharmaceutical use of hum an or animal origin, a substance with a higher bulk density.
the requirements o f chapter 5.1.7. Viral safety apply.
Coated active substances Consist o f particles of the active
Substances for pharmaceutical use may be used as such or as substance coated with one or more suitable excipients.
starting materials for subsequent formulation to prepare
Granulated active substances Are particles of a specified size
medicinal products. Depending on the formulation, certain
and/or form produced from the active substance by
substances may be used either as active substances or as
granulation directly or with one or more suitable excipients.
excipients. Solid substances may be compacted, coated,
granulated, pow dered to a certain fineness, or processed in I f substances are processed with excipients, these excipients
other ways. A m onograph is applicable to a substance comply with the requirements of the relevant monograph or,
processed with an excipient only where such processing is where no such monograph exists, the approved specification.
mentioned in the definition section of the monograph. W here active substances have been processed with excipients
Substance for pharmaceutical use of special grade Unless to produce, for example, coated or granulated substances, the
otherwise indicated or restricted in the individual processing is carried out under conditions of good
monographs, a substance for pharmaceutical use is intended manufacturing practice and the processed substances are
for hum an and veterinary use, and is of appropriate quality regarded as intermediates in the manufacture of a medicinal
for the m anufacture o f all dosage forms in which it can be p ro d u ct
used. CHARACTERS
Polymorphism Individual monographs do not usually specify T h e statements under the heading Characters
crystalline or am orphous forms, unless bioavailability is (e.g. statements about the solubility or a decomposition
affected. All forms of a substance for pharmaceutical use point) are n o t to be interpreted in a strict sense and are not
comply with the requirements of the monograph, unless requirements. T hey are given for information.
otherwise indicated. W here a substance may show polymorphism, this may be
P R O D U C T IO N stated under Characters in order to draw this to the attention
Substances for pharm aceutical use are manufactured by o f the user who may have to take this characteristic into
procedures that are designed to ensure a consistent quality consideration during formulation o f a preparation.
and comply with the requirements of the individual
m onograph or approved specification.
 1-38 General Monographs 2016

ID E N T IF IC A T IO N T he requirem ents above do n o t apply to biological and

W here under Identification an individual monograph biotechnological products, oligonucleotides,
contains subdivisions entitled ‘First identification’ and radiopharmaceuticals, products of ferm entation and semi­
‘Second identification’, the test or tests th a t constitute the synthetic products derived therefrom, to crude products of
‘First identification’ may be used in all circumstances. animal or plant origin or herbal products.
T he test or tests that constitute the ‘Second identification’ F o r active substances in a new application for a medicinal
may be used in pharmacies provided it can be dem onstrated p roduct for hum an use, the requirem ents o f the guideline on
that the substance or preparation is fully traceable to a batch the limits o f genotoxic impurities and the corresponding
certified to comply with all the other requirem ents o f the questions and answers docum ents published on the website
monograph. o f the European Medicines Agency (or similar evaluation
C ertain monographs give two o r m ore sets of tests for the principles for non-E uropean U nion m em ber states) m ust be
purpose of the first identification, which are equivalent and followed.
may be used independently. O ne o r m ore o f these sets R e sid u a l so lv en ts
usually contain a cross-reference to a test prescribed in the are limited according to the principles defined in chapter 5.4,
Tests section o f the m onograph. It may be used to simplify using general m ethod 2.4.24 or another suitable method.
the work of the analyst carrying out the identification and the W here a quantitative determ ination o f a residual solvent is
prescribed tests. For example, one identification set cross- carried out and a test for loss on drying is n o t carried out,
refers to a test for enantiomeric purity while the other set the content o f residual solvent is taken into account for
gives a test for specific optical rotation: the intended purpose calculation o f the assay content o f the substance, the specific
of the two is the same, that is, verification that the correct optical rotation and the specific absorbance.
enantiom er is present.
M ic ro b io lo g ic a l q u a lity
TESTS Individual monographs give accqptance criteria for
P o ly m o rp h is m (5.9) microbiological quality wherever such control is necessary.
If the nature o f a crystalline or am orphous form imposes T able 5.1.4.-2. - Acceptance criteria for microbiological quality of
restrictions on its use in preparations, the nature o f the rum-sterile substances for pharmaceutical use in chapter 5.1.4.
specific crystalline or am orphous form is identified, its Microbiological quality of non-sterUe pharmaceutical preparations
morphology is adequately controlled and its identity is stated and substances for pharmaceutical use gives recom mendations
on the label. on microbiological quality th a t are o f general relevance for
R e la te d su b s ta n c e s substances subject to microbial contamination. D epending on
Unless otherwise prescribed or justified and authorised, the nature of the substance and its intended use, different
organic impurities in active substances are to be reported, acceptance criteria may be justified.
identified wherever possible, and qualified as indicated in S te rility (2. 6 .I)
T able 2034.-1 or in Table 2034.-2 for peptides obtained by I f intended for use in the m anufacture o f sterile dosage forms
chemical synthesis. w ithout a further appropriate sterilisation procedure, o r if
offered as sterile grade, the substance for pharmaceutical use
Table 2034.-1. - Reporting, identification and qualification of complies with the test for sterility.
organic impurities in active substances B a c te ria l e n d o to x in s ('2.6.14)
Use Maximum Report­ Identification Qualification I f offered as bacterial endotoxin-free grade, the substance for
daily ing threshold threshold pharm aceutical use complies with the test for bacterial
dose threshold
endotoxins. T h e limit and test m ethod (if n o t gelation
Human £ 2 g/day > 0.05 per > 0.10 per > 0.15 per
use or cent cent or a cent or a m ethod A) are stated in the individual monograph. T h e limit
human daily intake daily intake is calculated in accordance with the recom mendations in
and of > 1.0 mg of > 1.0 mg general chapter 5.1.10. Guidelines for using the test for bacterial
veterinary (whichever is (whichever is
use the lower) the lower) endotoxins, unless a lower limit is justified from results from
Human > 2 g/day > 0.03 per > 0.05 per cent > 0.05 per production batches or is required by the com petent authority.
use or cent cent W here a test for bacterial endotoxins is prescribed, a test for
human
and pyrogens is n o t required.
veterinary
use
P y ro g e n s (2.6.8)
Veterinary Not > 0.10 per > 0.20 per cent > 0.50 per I f the test for pyrogens is justified rather than the test for
use only applicable cent cent bacterial endotoxins and if a pyrogen-free grade is offered,
the substance for pharm aceutical use complies with the test
for pyrogens. T h e limit and test m ethod are stated in die
Table 2034.-2. - Reporting, identification and qualification of individual m onograph or approved by the competent
organic impurities in peptides obtained by chemical synthesis authority. Based on appropriate test validation for bacterial
endotoxins and pyrogens, the test for bacterial endotoxins
Reporting Identification Qualification
may replace the test for pyrogens.
threshold threshold threshold
> 0.1 per cent > 0.5 per cent > 1.0 per cent A d d itio n a l p ro p e rtie s
Control o f additional properties (e.g. physical characteristics,
functionality-related characteristics) m ay be necessary for
Specific thresholds m ay be applied for impurities known to individual m anufacturing processes or formulations. Grades
be unusually potent or to produce toxic o r unexpected (such as sterile, endotoxin-free, pyrogen-free) may be
pharmacological effects. produced with a view to m anufacture o f preparations for
If the individual m onograph does n o t provide suitable control parenteral administration or other dosage forms and
for a new im purity, a suitable test for control must be
developed and included in the specification for the substance.
2016 Abacavir Sulfate 1-39

appropriate requirements may be specified in an individual Content

monograph. 99.0 per cent to 101.0 per cent (anhydrous substance).
ASSAY CHARACTERS
Unless justified and authorised, contents of substances for Appearance
pharmaceutical use are determined. Suitable methods are W hite or almost white powder.
used. Solubility
L A B E L L IN G Soluble in water, practically insoluble in ethanol
In general, labelling is subject to supranational and national (96 per cent) and in methylene chloride.
regulation and to international agreements. T h e statements IDENTIFICATION
under the heading Labelling therefore are n o t comprehensive A. Infrared absorption spectrophotometry (2.2.24).
and, moreover, for the purposes of the Pharmacopoeia only
Comparison abacavir sulfate CRS.
those statements that are necessary to demonstrate
compliance or non-compliance with the monograph are B. Enantiomeric purity (see Tests).
mandatory. Any other labelling statements are included as C. Solution S (see Tests) gives reaction (a) of sulfates
recommendations. W hen the term ‘label' is used in the (2.3.1).
Pharmacopoeia, the labelling statements may appear on the TESTS
container, the package, a leaflet accompanying the package or Solution S
a certificate of analysis accompanying the article, as decided Dissolve 0.250 g in water R and dilute to 25.0 m L with the
by the com petent authority. same solvent
Where appropriate, the label states that the substance is:
Enantiomeric purity
— intended for a specific use;
Liquid chromatography (2.2.29).
— of a distinct crystalline form;
— of a specific degree of fineness;
Solution A Mix 0.5 m L of trifluoroacenc acid R and 100 mT. of
— compacted;
methanol R.
— coated; Solution B Mix 30 volumes of methanol R, 30 volumes of 2-
— granulated; propanol R and 40 volumes of heptane R.
— sterile; Test solution Dissolve 40 mg of the substance to be examined
— free from bacterial endotoxins; in 30 m L of solution A. Sonicate until dissolution is
— free from pyrogens; complete. Add 30 m L of 2-propanol R and dilute to
— containing gliding agents. 100.0 m L with heptane R.
W here applicable, the label states: Reference solution (a) Dissolve 2 m g of abacavirfor system
— the degree o f hydration; suitability CRS (containing impurities A and D) in 1.5 m L of
— the nam e and concentration of any excipient. solution A. Sonicate until dissolution is complete.
__________________________________________________________ Ph Eur Add 1.5 m L of 2-propanol R and dilute to 5.0 m L with
heptane R.
Reference solution (b) Dilute 1.0 m L of the test solution to
100.0 m L with solution B. Dilute 1.0 m L of this solution to
10.0 m L with solution B.
Abacavir Sulfate Column:
*. * — size: I = 0.25 m , 0 = 4.6 mm;
(Ph Eur monograph 2589) *
— stationary phase: amylose derivative of silica gel for chiral
separation R (10 |im); .
— temperature: 30 °C.
Mobile phase:
— mobile phase A: diethylamine R, 2-propanol R, heptane R
(0.1:15:85 V/V/V);
— mobile phase B: heptane R, 2-propanol R (50:50 V!V)\

Time Mobile phase A Mobile phase B

(min) (per cent V/V) (per cent V/V)
C28H 38N 120 6S 671 188062-50-2 0 -2 5 100 0

A ctio n a n d u se 25 - 27 100 -»0 0 100

Nucleoside reverse transcriptase inhibitor; antiviral (HIV). 27 - 37 0 100
P re p a r a tio n s
Abacavir Oral Solution
Flow rate 1.0 mL/min.
Abacavir Tablets
Detection Spectrophotometer at 286 nm.
Abacavir, Zidovudine and Lamivudine Tablets
Injection 20 nL.
Abacavir and Lamivudine Tablets
Identification of impurities Use the chromatogram supplied
PhEir __________________________________________________________ with abacavir for system suitability CRS and the chromatogram
obtained with reference solution (a) to identify the peaks due
D E F IN IT IO N
to impurities A and D.
Bis[[(l5,4R)-4-[2-amino-6-(cyclopropylamiiio)-9i/-purin-9-
yl]cyclopent-2-enyl]methanol] sulfate.
 1-40 Abacavir Sulfate 2016

Relative retention W ith reference to abacavir (retention — total: n o t more than 5 times the area o f the principal peak
time = about 17 min): im purity D = about 0.8; in the chrom atogram obtained with reference solution (b)
im purity A = about 0.9. (0.5 per cent);
System suitability: reference solution (a): — disregard limit. 0.5 times the area o f the principal peak in
— resolution: minim um 1.5 betw een the peaks due to the chrom atogram obtained with reference solution (b)
impurities D and A; m inim um 1.5 between the peaks due (0.05 per cent).
to impurity A and abacavir. H eav y m e ta ls (2.4.8)
Limit. M aximum 20 ppm.
— impurity A: not m ore than 3 times the area o f the 12 m L of solution S complies with test A. Prepare the
principal peak in the chrom atogram obtained with reference solution using 2 m L of lead standard solution
reference solution (b) (0.3 p er cent). (1 ppm Pb) R.
Related substances W a te r (2.5.32)
Liquid chromatography (2.2.29). Prepare the solutions M aximum 0.5 p er cent, determined on 60.0 mg.
immediately before use and transfer them to low-adsorption, inert
S u lfa te d a s h (2.4.14)
glass vials.
M aximum 0.2 per cent, determined on 1.0 g.
Test solution Dissolve 25 mg o f the substance to be examined
in water R and dilute to 100.0 m l. with the same solvent. A SSAY
Sonicate until dissolution is complete. Dissolve 0.300 g in 50 m L of water R. T itrate with 0.1 M
Reference solution (a) Dissolve 2.5 mg o f abacavir for peak sodium hydroxide, determining the end-point
potentiometrically (2 . 2 . 2 0 ).
identification CRS (containing impurities B and D ) in
10.0 m L of water R. 1 m L of 0.1 M sodium hydroxide is equivalent to 33.54 mg of
Reference solution (b) D ilute 1.0 m L of the test solution to C 28H 38N 12O 6S.
100.0 m L with water R. D ilute 1.0 m L o f this solution to IM P U R IT IE S
10.0 m L w ith water R. Specified impurities: A , B.
Column: Other detectable impurities (the following substances would, if
— size. I = 0.15 m , 0 = 3.9 m m ; present at a sufficient level, be detected by one or other o f
— stationary phase: end-capped octadecylsQyl silica gelfor the tests in the monograph. They are limited by the general
chromatography R (5 pm); acceptance criterion for other/unspecified impurities and/or
— temperature: 30 °C. by the general m onograph Substances for pharmaceutical use
Mobile phase: (2034). It is therefore n o t necessary to identify these
— mobile phase A: dilute 0.5 m L of trifluoroacedc acid R in impurities for dem onstration o f compliance. See also 5.10.
1000 m L o f water R ; Control of impurities in substances for pharmaceutical use): C, D,
— mobile phase B: water R, methanol R (15:85 V!V)\ E, F.

Time Mobile phase A Mobile phase B

(min) (per cent V7V) (per cent V/V)
0 -5 95 5
5 - 25 95 + 70 5 + 30
25 -4 0 70->10 30 + 90

Flow rate 1.0 mL/min.

Detection Spectrophotom eter at 254 nm . A. [(li?,45)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-
yl] cydopent-2-enyl] methanol,
Irqection 20 pL.
Identification of impurities Use the chromatogram supplied
with abacavir for peak identification CRS and the
chrom atogram obtained with reference solution (a) to
identify the peaks due to impurities B and D.
Relative retention W ith reference to abacavir (retention
time = about 22 min): im purity D = about 1.04;
impurity B = about 1.3.
System suitability: reference solution (a):
— peak-to-vaEey ratio: m inim um 3.0, where Hp = height
above the baseline o f the peak due to impurity D and nh 2
Hv = height above the baseline o f the lowest point o f the
curve separating this peak from the peak due to abacavir. B. 6-(cyclopropylamino)-9-[(li?,4S)-4-[[(2,5-diam ino-6-
Limits: chloropyrimidin-4-yi)oxy]methyI] cyclopent-2-enyl] -9H-
— impurity B: not m ore than twice the area o f the principal purine-2-am ine,
peak in the chrom atogram obtained with reference
solution (b) (0.2 p er cent);
— unspecified impurities: for each impurity, not m ore than the
area of the principal peak in the chromatogram obtained
with reference solution (b) (0.10 per cent);
2016 Acacia 1-41

NH2 CHARACTERS
Acacia is almost completely b u t very slowly soluble, after

HjNr x >N about 2 h, in twice its mass of water leaving only a very small
residue of vegetable particles; the liquid obtained is colourless
or yellowish, dense, viscous, adhesive, translucent and weakly
a d d to blue litmus paper. Acada is practically insoluble in
ethanol (96 per cent).
C. [(15j4R )-4-(2,6-diarnino-9/f-purin-9-yl)cyclopent-2-
IDENTIFICATION
enyl] m ethanol,
A. A cada occurs as yellowish-white, yellow or pale amber,
sometimes with a pinkish tint, friable, opaque, spheroidal,
oval or reniform pieces (tears) o f a diameter from about
1-3 cm, frequently with a cracked surface, easily broken into
irregular, whitish or slightly yellowish angular fragments with
conchoidal fracture and a glassy and transparent appearance.
^ nI^ " >
h2na n In the centre o f an unbroken tear there is sometimes a small
cavity.
H
"Oc OH
B. Reduce to a powder (355) (2.9.12). T he pow der is white
or yellowish-white. Examine under a microscope using a
D. [(1 i?34i?)-4 -[2-amino-6-(cyclopropylainino)-9/f-purin-9- 50 per cent V/V solution of glycerol R. T h e pow der shows the
yl] cyclopent-2-enyl] methanol, following diagnostic characters: angular, irregular, colourless,
transparent fragments. Only traces of starch or vegetable
tissues are visible. N o stratified membrane, is ap p arent
^N H C. Examine the chromatograms obtained in the test for
glucose and fructose.
Results T h e chromatogram obtained with the test solution
h2n^ n^ n shows 3 zones due to galactose, arabinose and rhamnose.
H—r ^ \ ^ H N o other im portant zocies are visible, particularly in the
upper p art o f the chromatogram.
D . Dissolve 1 g o f the powdered herbal drug (355) (2.9.12)
E. [(li?,35)-3-[2-amino-6-(cyclopropylamino)-9/f-purin-9- in 2 m L of water R by stirring frequently for 2 h. Add 2 m L
yl] cyclopentyl] methanol, o f ethanol (96 per cent) R. After shaking, a white, gelatinous
mucilage is formed which becomes fluid on adding 10 m L of
water R.
NH TESTS
Solution S

H2NJ¿C> N N
Dissolve 3.0 g o f the powdered herbal drug (355) (2.9.12) in
25 m L o f water R by stirring for 30 min. Allow to stand for
30 min and dilute to 30 m L with water R.
"O<>x 0H
h í‘ h 3c ch3
Insoluble matter
M aximum 0.5 per cent.
T o 5.0 g o f the powdered herbal drug (355) (2.9.12) add
F. 6-(cyclopropylamino)-9- [(li?,45)-4- [ [(1,1 - 100 m L o f water R and 14 m L of dilute hydrochloric add R,
dimethylethyl)oxy] methyl] cyclopent-2-enyl] -9jFí-purine-2- boil gently for 15 min, shaking frequently and filter while hot
amine. through a tared sintered-glass filter (2.1.2). W ash with hot
PhEur water R and dry at 100-105 °C. The residue weighs a
maximum o f 25 mg.
Glucose and fructose
Thin-layer chromatography (2.2.27).

Acacia ★★+ ★★ Test solution T o 0.100 g of the powdered herbal drug (355)
★ ★ (2.9.12) in a thick-walled centrifuge tube add 2 m L of a
(Ph. Ever, monograph 0307) ***** 100 g/L solution of trifluoroacetic acid R, shake vigorously to
dissolve the forming gel, stopper the tube and heat the
Action and use mixture at 120 °C for 1 h. Centrifuge the hydrolysate,
Bulk-forming laxative; excipient transfer the clear supernatant carefully into a 50 m L flask,
W hen Powdered Acacia is prescribed or demanded, material add 10 m L of water R and evaporate the solution to dryness
complying with the requirements below with the exception of under reduced pressure. To the resulting clear film add
Identification test A shall be dispensed or supplied. 0.1 m L o f water R and 0.9 m L of methanol R. Centrifuge to
separate the amorphous predpitate. Dilute the supernatant, if
PhEur. necessary, to 1 m L with methanol R.
DEFINITION Reference solution Dissolve 10 mg of arabinose R, 10 mg of
Air-hardened, gummy exudate flowing naturally from or galactose R, 10 mg of glucose R, 10 mg o f rhamnose R and
obtained by incision of the trunk and branches o f Acacia 10 mg of xylose R in 1 m L of water R and dilute to 10 m L
Senegal L. Willd. (syn. Senegalia Senegal (L.) Britton), other with methanol R.
species of Acacia of African origin and Acacia seyal Delile.
 1-42 Acacia 2016

Plate TUG silica gel plate R. part of the monograph since they also represent mandatory quality
Mobile phase 16 g/L solution of sodium dihydrogen phosphate R, criteria. In such cases, a cross-reference to the tests described in the
butanol R , acetone R (10:40:50 VIV/V). mandatory part is included in the Functionality-related
Application 10 ^L as bands. characteristics section. Control of the characteristics can contribute
to the quality of a medicinal product by improving the consistency
Development A Over a path o f 10 cm. of the manufacturing process and the performance of the medicinal
Drying A In a current o f warm air for a few minutes. product during use. Where control methods are cited, they are
Development B Over a path o f 15 cm using the same mobile recognised as being suitable for the purpose, but other methods can
phase. also be used. Wherever results for a particular characteristic are
Drying B A t 110 °C for 10 m in. reported, the control method must be indicated.
Detection treat with amsaldehyde solution R and heat at 110 °C The following characteristic may be relevant for acacia used as a
for 10 min. viscosity-increasing agent andlor suspending agent in aqueous
Results T he chromatogram obtained with the reference preparations.
solution shows 5 clearly separated coloured zones due to Apparent viscosity
galactose (greyish-green or green), glucose (grey), arabinose D eterm ine the dynamic viscosity using a capillary viscometer
(yellowish-green), xylose (greenish-grey or yellowish-grey) (2.2.9) or a rotating viscometer (2.2.10) on a 100 g/L
and rham nose (yellowish-green), in order of increasing RF solution o f acacia (dried substance).
value. T he chrom atogram obtained with the test solution ___________________________________________________________ PhEur
shows no grey zone and no greyish-green zone between the
zones corresponding to galactose and arabinose in the
chrom atogram obtained with the reference solution.
S ta r c h , d e x trin a n d a g a r Spray-dried Acacia ******
T o 10 m L o f solution S previously boiled and cooled add
0.1 m L of 0.05 M iodine. N o blue or reddish-brown colour (Ph Eur monograph 0308) *
develops. PhEur___________________________________________________________

S te rc u lia g u m D E F IN IT IO N
A. Place 0.2 g of the powdered herbal drug (355) (2.9.12) in Spray-dried acacia is obtained from a solution of acacia.
a 10 m L ground-glass-stoppered cylinder graduated in-
CHARACTERS
0.1 m L . Add 10 m L o f ethanol (60 per cent VIV) R and
shake. Any gel form ed occupies a m aximum o f 1.5 mT- It dissolves completely and rapidly, after about 20 m in, in
twice its mass of water. T h e liquid obtained is colourless or
B. T o 1.0 g of the powdered herbal drug (355) (2.9.12) add
yellowish, dense, viscous, adhesive, translucent and weakly
100 m L of water R and shake. Add 0.1 m L of methyl red
a d d to blue litmus paper. Spray-dried acada is practically
solution R. N o t m ore than 5.0 m L o f 0.01 M sodium hydroxide
insoluble in ethanol (96 p er cent).
is required to change the colour o f the indicator.
ID E N T IF IC A T IO N
T a n n in s
T o 10 m L o f solution S add 0.1 m L o f ferric chloride A. Examined under a microscope, in ethanol (96 per cent) R,
solution R l. A gelatinous precipitate is formed, bu t neither the the powder is seen to consist predom inantly o f spheroidal
precipitate nor the liquid are dark blue. particles about 4-40 (jm in diameter, w ith a central cavity
containing 1 or several air-bubbles; a few m inute flat
T ra g a c a n th
fragments are p resen t Only traces o f starch granules are
Examine the chromatograms obtained in the test for glucose visible. N o vegetable tissue is seen.
and fructose.
B. Examine the chromatograms obtained in the test for
Results T he chrom atogram obtained with the test solution glucose and fructose.
shows no greenish-grey or yellowish-grey zone corresponding
to the zone of xylose in the chrom atogram obtained with the
Results T h e chrom atogram obtained with the test solution
shows 3 zones due to galactose, arabinose and rham nose.
reference solution.
N o other im portant zones are visible, particularly in the
L oss o n d ry in g (2.2.32) upper p art o f the chromatogram.
M axim um 15.0 p er cent, determ ined on 1.000 g o f the
C. Dissolve 1 g of the drug to be examined in 2 m L of
pow dered herbal drug (355) (2.9.12) by drying in an oven at
water R by stirring frequently for 20 min. A dd 2 m L of
105 °C.
ethanol (96 per cent) R. After shaking a white gelatinous
T o ta l a sh (2.4.16) m udlage is formed which becomes fluid on adding 10 m L of
M axim um 4.0 per cent. water R.
M ic ro b ia l c o n ta m in a tio n TESTS
T A M C : acceptance criterion 104 C FU /g (2.6.12).
S o lu tio n S
TY M C : acceptance criterion 104 C FU /g (2.6.12). Dissolve 3.0 g o f th e drug to be examined in 25 m L of
Absence o f Escherichia cod (2.6.13). water R by stirring for 10 min. Allow to stand for 20 m in and
Absence o f Salmonella (2.6.13). dilute to 30 m L with water R.

F U N C T IO N A L IT Y -R E L A T E D C H A R A C T E R IS T IC S G lu co se a n d fru c to se
Thin-layer chromatography (2.2.27).
This section provides information on characteristics that are
recognised as being relevant control parameters for one or more Test solution T o 0.100 g in a thick-walled centrifuge tube add
functions of the substance when used as an excipient (see chapter 2 m L o f a 100 g/L solution o f trifluoroacetic acid R, shake
5.15). Some of the characteristics described in the Functionality- vigorously to dissolve the forming gel, stopper the tube and
related characteristics section may also be present in the mandatory h eat the mixture at 120 °C for 1 h. Centrifuge the
hydrolysate, transfer the clear supernatant carefully, into a
2016 Acamprosate Calcium 1-43

50 m L flask, add 10 m L o f water R and evaporate to dryness Absence of Salmonella (2.6.13).

under reduced pressure. T o the resulting clear film add FU N C T IO N A L IT Y -R E L A T E D C H A R A C T E R IS T IC S
0.1 m L o f water R and 0.9 m L of methanol R. Centrifuge to
This section provides information on characteristics that are
separate the amorphous precipitate. Dilute the supernatant, if
recognised as being relevant control parameters for one or more
necessary, to 1 m L with methanol R.
functions of the substance when used as an excipient (see chapter
Reference solution Dissolve 10 mg o f arabinose R, 10 m g of 5.15). This section is a non-mandatory part of the monograph
galactose R, 10 m g o f glucose R, 10 m g of rhamnose R and and it is not necessary to verify the characteristics to demonstrate
10 mg o f xylose R in 1 m l. of water R and dilute to 10 m L compliance. Control of these characteristics can however contribute
with methanol R. to the quality of a medicinal product by improving the consistency
Plate TLC silica gel plate R. of the manufacturing process and the performance of the medicinal
Mobile phase 16 g/L solution o f sodium dihydrogen phosphate R, product during use. Where control methods are cited, they are
butanol R , acetone R (10:40:50 V/V/V). recognised as being suitable for the purpose, but other methods can
Application 10 pL as bands. also be used. Wherever results for-a particular characteristic are
reported, the control method must be indicated.
Development A Over a path o f 10 cm.
The following characteristic may be relevantfor spray-dried acacia
Drying A In a current of warm air for a few minutes. used as a viscosity-increasing agent and/or suspending agent in
Development B Over a path of 15 cm using the same mobile aqueous preparations.
phase.
A p p a re n t v isco sity
Drying B At 110 °C for 10 min. Determine the dynamic viscosity using a capillary viscometer
Detection Spray with ardsaldehyde solution R and heat at (2.2.9) or a rotating viscometer (2.2.10) on a 100 g/L
110 °C for 10 min. solution of spray-dried acacia (dried substance).
Results T h e chromatogram obtained with the reference PhEur
solution shows 5 clearly separated coloured zones due to
galactose (greyish-green or green), glucose (grey), arabinose
(yellowish-green), xylose (greenish-grey or yellowish-grey)

★★*★★
and rham nose (yellowish-green), in order of increasing
Rp value. T he chromatogram obtained with the test solution Acamprosate Calcium ★ ★
shows n o grey zone and no greyish-green zone between the *****
(Ph Eur monograph 1585)
zones corresponding to galactose and arabinose in the
chrom atogram obtained w ith the reference solution.
S ta r c h , d e x trin a n d a g a r
T o 10 m l. of solution S previously boiled and cooled add Ca2+
0.1 m L of 0.05 M iodine. N o blue or reddish-brown colour J2
develops.
Sterculia gum C io H ^ C a N ^ A ^ 400.5 77337-73-6
A. Place 0.2 g in a 10 m L ground-glass-stoppered cylinder
graduated in 0.1 mL. Add 10 m L of ethanol A ctio n a n d u se
(60 per cent VIV) R and shake. Any gel formed occupies not Treatm ent o f alcoholism.
more than 1.5 mL.
P re p a r a tio n
B. T o 1.0 g add 100 m L o f water R and shake. Add 0.1 m L Gastro-resistant Acamprosate Tablets
o f methyl red solution R. N o t more than 5.0 m L of
0.01 M sodium hydroxide is required to change the colour of PhEur__________________________________
the indicator. D E F IN IT IO N
T a n n in s Calcium bis[3-(acetylamino)propane-l-sulfonate].
T o 10 m L of solution S add 0.1 m L o f ferric chloride C o n te n t
solution R l. A gelatinous precipitate is formed, b u t neither the 98.0 per cent to 102.0 per cent (dried substance).
precipitate nor the liquid shows a dark blue colour.
CHARACTERS
Tragacanth
A p p e a ra n c e
Examine the chromatograms obtained in the test for Glucose
White or almost white powder.
and fructose.
Results T h e chromatogram obtained with the test solution Solubility
shows no greenish-grey o r yellowish-grey zone corresponding Freely soluble in water, practically insoluble in ethanol
to the zone of xylose in the chromatogram obtained with the (96 per cent) and in methylene chloride.
reference solution. ID E N T IF IC A T IO N
L oss o n d ry in g (2.2.32) A. Infrared absorption spectrophotometry (2.2.24).
M axim um 10.0 per cent, determined on 1.000 g by drying in Comparison Ph. Eur. reference spectrum of acamprosate calcium.
an oven at 105 °C. B. It gives reaction (a) o f calcium (2.3.1).
T o ta l a s h C2.4.16) TESTS
M axim um 4.0 per c e n t
S o lu tio n S
M ic ro b ia l c o n ta m in a tio n Dissolve 5.0 g in carbon dioxide-free water R and dilute to
T A M C : acceptance criterion 104 CFU/g (2.6.12). 100 m L with the same solvent.
T Y M C : acceptance criterion 102 C FU/g (2.6.12). A p p e a ra n c e o f so lu tio n
Absence of Escherichia colt (2.6.13). Solution S is clear (2.2.1) and colourless (2 .2 .2 , Method II).
 1-44 Acarbose 2016

p H (2.2.3) 0.1 M sodium hydroxide, determining the end-point

5.5 to 7.0 for solution S. potentiometrically (2.2.20).
Im p u rity A 1 m L of 0.1 M sodium hydroxide corresponds to 20.02 m g of
Liquid chromatography (2.2.29). CioH20CaN2O8S2.
Test solution Dissolve 0.40 g of the substance to be examined IM P U R IT IE S
in distilled water R and dilute to 20.0 m L with the same
solvent. D ilute 10.0 m L of this solution to 100.0 m L with H2N^ V / S O , H
borate buffer solution pH 10.4 R. Place 3.0 m L of the solution
obtained in a 25 m L ground-glass-stoppered tube. A. 3-aminopropane- 1-sulfonic a d d (homotaurine).
Add 0.15 m L of a freshly prepared 5 g/L solution o f
__________________________________________________________ PhEur
fluorescamine R in acetomtrUe R. Shake immediately and
vigorously for 30 s. Place in a water-bath at 50 °C for
30 min. Cool under a stream o f cold water. Centrifuge and
filter the supernatant through a suitable mem brane filter
(nominal pore size 0.45 pm), 25 mm in diameter. Acarbose *****
Reference solution Dissolve 50 m g o f acamprosate *. *
impurity A CRS in distilled water R and dilute to 200.0 m L (Ph. Eur. monograph 2089) *
with the same solvent. Dilute 0.4 m L o f the solution to
100.0 m L with borate buffer solution pH 10.4 R. T reat 3.0 m L
of this solution in the same way as the test solution
Column:
— size. I = 0.15 m , 0 = 4.6 mm;
— stationary phase, spherical octadecylsilyl silica gel for OH OH OH OH
chromatography R (5 pm) with a specific surface area of
170 m 2/g and a pore size o f 12 nm .
C25H43N018 646 56180-94-4
Mobile phase acetomtrUe R, methanol R, 0.1 M phosphate buffer
solution pH 6.5 R (10:10:80 VIVIV). A ctio n a n d u se
Flow rate 1 mL/min. Alpha-glucosidase inhibitor; treatm ent o f diabetes mellitus.
Detection Spectrophotom eter at 261 nm . PhEur__________________________________________________________
Irgection 20 |iL.
D E F IN IT IO N
Run time 6 times the retention time o f impurity A 0-4,6-Dideoxy-4- [ [(1 S,4R,5S, 6 S)-4,5,6-trihydroxy-3-
Retention times Fluorescamine = about 4 min; (hydroxymethyl) cyclohex-2-enyl] amino] -a-D-glucopyrano syl-
im purity A = about 8 min; acamprosate is not detected by (1-+ 4)-0-a-D-glucopyranosyl-( 1-*4)-D-glucopyranose, which
this system. is produced by certain strains of Actinopkmes utahensis.
Limits: C o n te n t
— impurity A: not m ore than the area of the corresponding 95.0 per cent to 102.0 per cent (anhydrous substance).
peak in the chromatogram obtained with the reference
solution (0.05 per cent). CHA RACTERS
A p p e a ra n c e
H eav y m e ta ls (2.4.8)
W hite or yellowish, hygroscopic, amorphous powder.
M aximum 10 ppm.
Dissolve 2.0 g in distilled water R and dilute to 20 m L with S o lubility
the same solvent. 12 m L o f the solution complies with test A. Very soluble in water, soluble in methanol, practically
insoluble in methylene chloride.
Prepare the reference solution using 10 m L of lead standard
solution (1 ppm Pb) R. ID E N T IF IC A T IO N
L oss o n d ry in g ('2.2.32) A. Infrared absorption spectrophotometry (2.2.24).
M aximum 0.4 per cent, determ ined on 1.000 g by drying in Comparison acarbose for identification CRS.
an oven at 105 °C. B. Examine the chromatograms obtained in the assay.
A SSAY Results T he prindpal peak in the chromatogram obtained
T o 4 g of cation-exchange resin R (75-150 pm) add 20 m L of with the test solution is similar in retention time and size to
disnUed water R and stir magnetically for 10 min. Introduce the prindpal peak in the chromatogram obtained with
this suspension into a glass column, 45 cm long and 2.2 cm reference solution (a).
in internal diameter, equipped with a polytetrafluoroethylene TESTS
flow cap covered by a glass-wool plug. Allow a few millilitres
S o lu tio n S
of this solution to flow, then place a plug o f glass wool over
Dissolve 1.00 g in carbon dioxide-free water R and dilute to
the resin. Pass 50 m L o f 1 M hydrochloric acid through the
20.0 m L with the same solvent.
column. T he p H of the eluate is close to 1. W ash with
3 quantities, each of 200 m L, o f distilled water R to obtain an p H (2.2.3)
eluate at p H 6. Dissolve 0.100 g o f the substance to be 5.5 to 7.5 for solution S.
examined in 15 m L of distilled water R. Pass through the S pecific o p tic a l ro ta tio n (2.2.7)
colum n and wash with 3 quantities, each o f 25 m l , o f + 168 to + 183 (anhydrous substance).
distilled water R, collecting the eluate. Allow to elute until an D ilute 2.0 m L o f solution S to 10.0 m L with water R.
eluate at p H 6 is obtained. Titrate the solution obtained with
A b so rb a n c e (2.2.25)
M aximum 0.15 at 425 nm for solution S.
2016 Acarbose 1-45

Related substances chromatogram obtained with reference solution (c)

Liquid chrom atography (2.2.29). (0.3 per cent);
Test solution Dissolve 0.200 g of the substance to be — impurity E: n o t more than 0.2 times the area of the
examined in water R and dilute to 10.0 m L with the same principal peak in the chromatogram obtained with
solvent. reference solution (c) (0.2 per cent);
— arty other impurity: for each impurity, n o t more than
Reference solution (a) Dissolve the contents of a vial of
0.2 times the area o f the principal peak in the
acarbose CRS in 5.0 m L of water R.
chromatogram obtained with reference solution (c)
Reference solution (b) Dissolve the contents of a vial of (0.2 p er cent);
acarbose for peak identification CRS (acarbose containing — total: n o t more than 3 times the area o f the principal peak
impurities A, B, C , D , E, F and G) in 1 m L of water R. in the chromatogram obtained with reference solution (c)
Reference solution (c) Dilute 1.0 m L of the test solution to (3.0 p er cent);
100.0 m L with water R. — disregard limit. 0.1 times the area of the prindpal peak in
Column: the chromatogram obtained with reference solution (c)
— size: I = 0.25 m , 0 = 4 mm; (0.1 per cent).
— stationary phase: aminopropylsUyl silica gel for H eav y m e ta ls (2.4.8)
chromatography R (5 pm); M aximum 20 ppm.
— temperature: 35 °C.
Dissolve 1.5 g in water R and dilute to 15 m L with the same
Mobile phase M ix 750 volumes of acetomtrUe R1 and solvent. If the solution is not clear, cany out prefiltration and
250 volumes of a solution containing 0.60 g/L of potassium use the filtrate. 10 m L complies with test E. Prepare the
dihydrogen phosphate R and 0.35 g/L o f disodium hydrogen reference solution using 20 m L o f lead standard solution
phosphate dihydrate R. (1 ppm Pb) R.
Flow rate 2.0 mL/min. W a te r (2.5.12)
Detection Spectrophotom eter at 210 nm. M aximum 4.0 per cent, determined on 0.300 g.
Injection 10 pL o f the test solution and reference solutions (b) S u lfa te d a s h (2.4.14)
and (c). M aximum 0.2 per cent, determined on 1.0 g.
Run time 2.5 times the retention time of acarbose.
A SSAY
Identification of impurities Use the chromatogram supplied Liquid chromatography (2.2.29) as described in the test for
with acarbose for peak identification CRS and the related substances with the following modification.
chromatogram obtained with reference solution (b) to
Injection T est solution and reference solution (a).
identify the peaks due to impurities A, B, C , D, E, F and G.
Calculate the percentage content o f C 25H 43N O i 8 taking into
Relative retention W ith reference to acarbose (retention
account the assigned content o f acarbose CRS.
time = about 16 min): impurity D = about 0.5;
impurity B = about 0.8; impurity A = about 0.9; STORA GE
impurity C = about 1.2; impurity E = about 1.7; In an airtight container.
impurity F = about 1.9; impurity G = about 2.2.
IM P U R IT IE S
System suitability, reference solution (b): Specified impurities A, B, C, D , E, F , G
— the chromatogram obtained is similar to the
chromatogram supplied with acarbose for peak
Other detectable impurities (the following substances would, if
present at a sufficient levd, be detected by one or other of
identification CRS;
— peak-to-vaRey ratio: minimum 1.2, where Hp — height the tests in the monograph. T hey are limited by the general
acceptance criterion for other/unspecified impurities. It is
above the baseline of the peak due to impurity A and
therefore n o t necessary to identify these impurities for
Hv = height above the baseline o f the lowest point o f the
demonstration of compliance. See also 5.10. Control of
curve separating this peak from the peak due to acarbose.
impurities in substances for pharmaceutical use): H.
Limits:
— correction factors: for the calculation of content, multiply
the peak areas o f the following impurities by the
corresponding correction factor impurity B = 0.63;
im purity D = 0.75; impurity E = 1.25;
impurity F = 1.25; impurity G = 1.25;
— impurity C: not m ore than 1.5 times the area o f the
principal peak in the chromatogram obtained with
reference solution (c) (1.5 per cent);
— impurity D: n o t m ore than the area of the principal peak
in the chromatogram obtained with reference solution (c) A. 0-4,6-dideoxy-4-[[(15,4i?,5«S,66)-4,5,6-trihydroxy-3-
(1.0 per cent); (hydroxymethyl) cyclohex-2 -enyl] amino] -a-r>-
— impurity A: n o t m ore than 0.6 times the area of the glucopyranosyl-(l -►4)-0-a-D-glucopyranosyl-( 1 -> 4 )-d -
principal peak in the chromatogram obtained with arafono-hex-2 -ulopyranose,
reference solution (c) (0.6 per cent);
— impurity B: not m ore than 0.5 times the area o f the
principal peak in the chromatogram obtained with
reference solution (c) (0.5 per cent);
— impurities F, G: for each impurity, not more than
0.3 times the area o f the principal peak in the
 1-46 Acebutolol Hydrochloride 2016

B. (li?,4i?,55,6iQ-4,5,6-trihydroxy-2-
(hydroxymethyl)cydohex-2-enyl 4-0-[4,6-dideoxy-4-
[ [(1 S,4uR,55, 65)-4,5,6-trihydroxy-3-
(hydroxymethyl)cyclohex-2-enyI] am ino]-a-D -
glucopyranosyl] -^-D -glucopyranoside,
G. a-D-glucopyranosyl 0 -4,6-dideoxy-4-[[(15,4ß ,55,65)-
4j5j6-trihydroxy-3-(hydroxymethyl)cyclohex-2-
en yl]am in o]-a-D -gIu cop yran osyl-(l -> 4 )-0 -a -D -
glucopyranosyl-(l ->4)-0 -a-D-glucopyranoside (a -D -
glucopyranosyl a-acarboside).

C. a-D-glucopyranosyl 4-0-[4,6-dideoxy-4-[[(15,4R,55,65)- H . 0 -4,6-dideoxy-4-[[(15,4i?,55,65)-4,5,6-trihydroxy-3-

4,5,6-trihydroxy-3-(hydroxymethyi)cydohex-2- (hydroxymethyl)cyclohex-2-enyl] amino] - a - D -
enyl] am ino]-a-D -glu cop yran osyl]-a-D -glucopyranosid e, glucopyranosyl-( 1-*■4)-0 -6-deoxy-a-D-glucopyranosyl-
(1 ->4)-D-glucopyranose.
ho ch HO PhEur

Acebutolol Hydrochloride ★★*★★

OH OH OH ★ ★
(Ph Eur monograph 0871) * * * * *
D . 4 -0 - [4,6-dideoxy-4- [[(15,4i?,55, 65)-4,5,6-trihydroxy-3-
(hydroxymethyl)cyclohex-2-enyl] amino] -a-D-
glucopyranosyl] -D -glucopyranose,

G )T 0H
lo \ — * OH
and enantiomer

Ci{1H29CIN2O4 3 72.9 34381-68-5

O X"—f Ö x —- f °
OH OH OH A ctio n a n d u se
Beta-adrenoceptor antagonist.
E. 0 -4 ,6-dideoxy-4- [[(15,4i?,55, 65)-4,5,6-trihydroxy-3- P re p a ra tio n s
(hydroxymethyi)cyclohex-2-enyI] amino] -a-D - Acebutolol Capsules
glucopyranosyl-(l -►4)-0-a-D-giucopyranosyl-(l -> 4 )-0 -a- Acebutolol Tablets
D-glucopyranosyl-(l ->4)-D-anzÄmo-hex-2-ulopyranose
PhEur.
(4-0-a-acarbosyl-D-fhictopyranose),
DEFINITION
HO HO. HO HO N- [3-Acetyl-4- [(2J?5)-2-hydroxy-3-
[(1 -methyletbyl) amino] propoxy] phenyl] butanamide
hydrochloride.
OH
C o n te n t
99.0 per cent to 101.0 per cent (dried substance).
OH OH OH OH OH
CHARACTERS
F. 0 -4,6-dideoxy-4- [[(15,4i?,55,65)-4,5,6-trihydroxy-3- A p p e a ra n c e
(hydroxymethyl) cyclohex-2-enyl] amino] -om> W hite or alm ost white, crystalline powder.
glucopyranosyl-(l -»4)-0 -a-D-glucopyranosyl-(l ->4)-0-a- S olu b ility
D -g lu co p y ra n o sy l-(l -*■4)-D-glucopyranose (4-0 -a- Freely soluble in w ater and in ethanol (96 per cent), very
acarb osyl-D -glu cop yran ose), slightly soluble in acetone and in-methylene chloride.
2016 Acebutolol Hydrochloride 1-47

mp mobile phase A Dilute 0.5 m L o f this solution to 50.0 m L

A bout 143 °C. with mobile phase A.
ID E N T IF IC A T IO N Reference solution (b) Dissolve the contents of a vial of
First identification B, D. acebutolol impurity I CRS in 1.0 m L of mobile phase A.
Second identification A , C, D. Reference solution (c) M ix 2.0 m L o f reference solution (a)
and 1.0 m L of reference solution (b) and dilute to 10.0 m L
A. Ultraviolet and visible absorption spectrophotometry
with mobile phase A.
(2.2.25).
Reference solution (d) Dissolve 5.0 m g o f acebutolol
Test solution Dissolve 20.0 mg in a 0.1 per cent V/V solution
impurity C CRS in 10 m L of acetomtrûe R and dilute to
of hydrochloric acid R and dilute to 100.0 m L with the same
25.0 m L with mobile phase A. D ilute 0.5 m L of this solution
acid solution. Dilute 5.0 m L of this solution to 100.0 m L
to 50.0 m L with mobile phase A.
with a 0.1 per cent V/V solution of hydrochloric add R.
Reference solution (e) Dissolve 5.0 m g o f acebutolol
Spectral range 220-350 nm.
impurity B CRS in 10.0 m L of acetonitrile R and dilute to
Absorption maxima At 233 nm and 322 nm. 25.0 m L with mobile phase A. D ilute 1.0 m L of this solution
Specific absorbance at the absorption maximum 555 to 605 at to 50.0 m L with mobile phase A.
233 nm . Column:
B. Infrared absorption spectrophotometry (2.2.24). — sizer. I = 0.125 m, 0 = 4 mm,
Preparation Discs. — stationary phase:, end-capped octadecylsüyl silica gel for
Comparison acebutold hydrochloride CRS. chromatography R (5 pm),
— temperature: 40 °C.
C. Thin-layer chromatography (2.2.27).
Mobile phase:
Test solution Dissolve 20 mg of die substance to be examined — mobile phase A: mix 2.0 m L of phosphoric add R, and
in methanol R and dilute to 20 m L with the same solvent. 3.0 m L of triethylamine R and dilute to 1000 m L with
Reference solution (a) Dissolve 20 mg of acebutolol water R;
hydrochloride CRS in methanol R and dilute to 20 m L with the — mobile phase B: mix equal volumes o f acetonitrile R and
same solvent. mobile phase A;
Reference solution (b) Dissolve 20 mg o f pindolol CRS in
methanol R and dilute to 20 m L with the same solvent. Time Mobile phase B
Mobile phase A
T o 1 m L o f this solution add 1 m L of reference solution (a). (min) (per cent V/V) (per cent V/V)
Plate TLC silica gel F 254 plate R. 0-2 98 2
Mobile phase perchloric add R, methanol R, water R 2 - 30.5 98-» 10 2 -> 90
(5:395:600 V/V/V).
30.5 - 41 10 90
Application 10 pL.
Development Over 3/4 of the plate.
Drying In air. Flow rate 1.2 mlVmin.
Detection Examine in ultraviolet light at 254 nm. Detection Spectrophotom eter at 240 nm .
System suitability T he chromatogram obtained with reference Injection 25 pL.
solution (b) shows 2 clearly separated principal spots. System suitability: reference solution (c):
Results T h e principal spot in the chromatogram obtained with — resolution: minim um 7.0 between the peaks due to
the test solution is similar in position and size to the principal im purity I and acebutolol.
spot in th e chromatogram obtained with reference Limits:
solution (a). — impurity B: n o t m ore than the area of the principal peak
D . It gives reaction (a) of chlorides (2.3.1). in the chromatogram obtained with reference solution (e)
(0.2 per cent);
TESTS — impurity C: not m ore than the area o f the principal peak
A p p e a ra n c e o f so lu tio n in the chromatogram obtained with reference solution (d)
T he solution is not more opalescent than reference (0.1 per cent);
suspension II (2.2.1) and not more intensely coloured than — impurity I: not m ore than twice the area of the principal
reference solution BY 5 (2.2.2, Method II). peak in the chromatogram obtained with reference
Dissolve 0.5 g in water R and dilute to 10 m L with the same solution (a) (0.2 p er cent);
solvent. — any other impurity: for each impurity, not more than the
p H (2.2.3) area of the principal peak in the chromatogram obtained
5.0 to 7.0. with reference solution (a) (0.1 per cent);
— total: no t more th an 5 times the area o f the principal peak
Dissolve 0.20 g in carbon dioxide-free water R and dilute to
in the chrom atogram obtained with reference solution (a)
20 m L w ith the same solvent.
(0.5 per cent);
R e la te d su b s ta n c e s — disregard limit. 0.5 times the area of the principal peak in
Liquid chromatography (2.2.29). the chromatogram obtained with reference solution (a)
Test solution Dissolve 0.100 g of die substance to be (0.05 per cent).
examined in mobile phase A and dilute to 50.0 m L with H eav y m e ta ls (2.4.8)
mobile phase A. M axim um 20 ppm.
Reference solution (a) Dissolve 20.0 m g of the substance to be Dissolve 0.50 g in 20.0 m L of water R. T h e solution
examined in mobile phase A and dilute to 100.0 m L with complies with test E. Prepare the .reference solution by
 1-48 Aceclofenac 2016

diluting 10.0 m L o f lead standard solution (1 ppm Pb) R to

20.0 m L w ith water R.
and enantiomer
L oss o n d ry in g (2.2.32)
M aximum 0.5 per cent, determined on 1.000 g by drying in
an oven at 105 °C for 3 h.
S u lfated a s h (2.4.14) F. R = OH: N- [3-acetyl-4- [(2&S)-2,3-
M aximum 0.1 per cent, determined on 1.0 g. dihydroxypropoxy]phenyl]butanamide,
A SSAY I. R = NH-CH2-CH3: N - [3-acetyl-4- [(2J?S)-3-(ethylamino)-
Dissolve 0.300 g in 50 m L o f ethanol (96 per cent) R and add 2-hydroxypropoxy]phenyl]butanamide,
1 m L of 0.1 M hydrochloric acid. Carry out a potentiometric
titration (2.2.20), using 0.1 M sodium hydroxide. Read the H3C C H 3 H3C
OH T OH
volume added between the 2 points o f inflexion.
1 m L of 0.1 M sodium hydroxide is equivalent to 37.29 m g of
C 18H 29CIN 2O 4.
STORAGE
Protected from light.
IM P U R IT IE S G . NyN'-[[( 1-methylethyl)imino]bis[(2-hydroxypropane-l,3-
Specified impurities A, B, C, D , E, F, G , H , I, J, K. diyl) oxy(3-acetyl-1,4-phenylene)]] dibutanam ide (biamine),

O and enantiomer

A N - [3-acetyl-4- [(2i?3)-oxiran-2-
ylmethoxy]phenyl]butanamide, H . NyN'-[(2-hydroxypropane-l,3-diyl)bis[oxy(3-acetyl-1,4-
phenylene)]]dibutanamide.
R2 H OH PhEur
° ^ X ^ N CH3
j and enantiomer
R1. CH3

Aceclofenac ★ ★
★ ★
B. R I = R2 = C O -C H 3: N - [3-acetyl-4-[(2i?5)-2-hydroxy-3-
[( 1-methylethyl) amino] propoxy]phenyl] acetamide (Ph Eicr monograph 1281) *****
(diacetolol),
o co 2h
D . R I = H , R2 = CO-CH3: 1- [5-amino-2-[(2&S)-2-
hydroxy-3-[( 1-methyiethyl)amino]propoxy]phenyI] ethanone,
E. R I = C O -C H 2-C H 2-C H 33 R2 = H : N-[4-[(2i?5)-2-
o a
hydroxy-3-
[( 1-methylethyl) amino] propoxy]phenyI] butanamide,
J. R I = CO-CH2-CH3, R2 = CO-CH3: Ar-[3-acetyl-4-
[(2i?5)-2-hydroxy-3- C16H13a 2N04 354.2 89796-99-6
[( 1-methylethyl) amino] propoxy] phenyl] propanamide,
K R I = R2 = C O -C H 2-C H 2-C H 3: N-[3-butanoyl-4- A ctio n a n d u se
[(2i?5)-2-hydroxy-3- Cyclo-oxygenase inhibitor; analgesic; anti-inflammatory.
[( 1-methylethyl) amino] propoxy]phenyI]butanamide,
PhEur_______________________________________________________

DEFINITION
[[[2-[(2,6-Dichlorophenyl)amino]phenyl]acetyl]oxy]acetic
ad d .
C o n te n t
99.0 per cent to 101.0 p er cent (dried substance).
CHARACTERS
C. N-(3-acetyl-4-hydroxyphenyI)butanamide, A p p e a ra n c e
W hite or almost white, crystalline powder.
S o lu b ility
Practically insoluble in water, freely soluble in acetone,
soluble in ethanol (96 p er cent).
ID E N T IF IC A T IO N
First identification B.
2016 Aceclofenac 1-49

Second identification A, C. Time Mobile phase A Mobile phase B

(min)____________ (per cent V/V)________ (per cent V/V)
A. Ultraviolet and visible absorption spectrophotometry
0 - 25 70 -» 50 30 -» 50
(2.2.25).
Test solution Dissolve 50.0 mg in methanol R and dilute to 25 - 30 50 -» 20 50 -» 80
100.0 m L with the same solvent. Dilute 2.0 m L o f the 30 - 50 20 80
solution to 50.0 m L with methanol R.
Spectral range 220-370 nm. Flow rate 1.0 mL/min.
Absorption maximum At 275 nm. Detection Spectrophotom eter at 275 nm.
Specific absorbance at the absorption maximum 320 to 350. Injection 10 jiL o f the test solution and reference
solutions (c), (e), (f) and (g).
B. Infrared absorption spectrophotometry (2.2.24).
Identification of impurities Use the chromatogram supplied
Comparison: Ph. Eur. reference spectrum of aceclofenac.
with aceclofenac for peak identification CRS and the
C . Dissolve about 10 mg in 10 m L of ethanol (96 per cent) R. chromatogram obtained witinreference solution (g) to
T o 1 m L o f the solution, add 0.2 m L of a mixture, prepared identify the peaks due to impurities B, C, D , E and G.
immediately before use, o f equal volumes o f a 6 g/L solution
Relative retention W ith reference to aceclofenac (retention
o f potassium ferricyanide R and a 9 g/L solution of ferric
time = about 11 min): impurity A = about 0.8;
chloride R. Allow to stand protected from light for 5 min.
impurity G = about 1.3; impurity H = about 1.5;
A dd 3 m l, of a 10.0 g/L solution of hydrochloric acid R. Allow
impurity I = about 2.3; impurity D = about 3.1;
to stand protected from light for 15 min. A blue colour
impurity B = about 3.2; impurity E = about 3.3;
develops and a precipitate is formed.
impurity C = about 3.5; impurity F = about 3.7.
TESTS System suitability: reference solution (c):
Related substances — resolution: minim um 5.0 between the peaks due to
Liquid chromatography (2.2.29). Prepare the solutions impurity A and aceclofenac.
immediately before use. Limits:
Solvent mixture M obile phase A, mobile phase B (30:70 V/V). — impurity A: n o t more than the area o f the corresponding
Test solution Dissolve 50.0 mg o f the substance to be peak in the chromatogram obtained with reference
examined in the solvent mixture and dilute to 25.0 m L with solution (c) (0.2 per cent);
the solvent mixture. — impurities B, C, D, E, G: for each impurity, no t more than
Reference solution (a) Dissolve 21.6 mg of diclofenac the area of the peak due to aceclofenac in the
sodium CRS (impurity A) in the solvent mixture and dilute to chrom atogram obtained with reference solution (e)
50.0 m L with the solvent mixture. (0.2 per cent);
— impurity F: n o t more than the area o f the corresponding
Reference solution (b) Dilute 2.0 m L of the test solution to
peak in the chromatogram obtained with reference
10.0 m L with the solvent mixture.
solution (e) (0.2 per cent);
Reference solution (c) Mix 1.0 m L o f reference solution (a) — impurity H: n o t more than the area o f the corresponding
and 1.0 m L o f reference solution (b) and dilute to 100.0 m L peak in the chromatogram obtained with reference
w ith the solvent mixture. solution (e) (0.1 per cent);
Reference solution (d) Dissolve 4.0 mg of aceclofenac — impurity I: no t more than the area of the corresponding
impurity F CRS and 2.0 mg of aceclofenac impurity H CRS in peak in the chromatogram obtained with reference
the solvent mixture, then dilute to 10.0 m L with the solvent solution (f) (0.1 per cent);
mixture. — unspecified impurities: not more than 0.5 times die area of
Reference solution (e) Mix 1.0 m L of reference solution (b) the peak due to aceclofenac in the chromatogram
and 1.0 m L o f reference solution (d) and dilute to 100.0 m L obtained with reference solution (e) (0.10 per cent);
with the solvent mixture. — total: n o t m ore than 0.7 per cent;
Reference solution (j) Dissolve the contents of a vial of — disregard Ixmitr. 0.1 times the area of the peak due to
diclofenac impurity A CRS (aceclofenac impurity I) in 1.0 m L aceclofenac in the chromatogram obtained with reference
o f the solvent mixture, add 1.5 m L of the solvent mixture solution (e) (0.02 per cent).
and mix, Heavy m etals ( 2.4.8)
Reference solution (g) Dissolve 4 mg of aceclofenac for peak M aximum 10 ppm .
identification CRS (containing impurities B, C, D , E and G) T o 2.0 g in a silica crucible, add 2 m L of sulfuric acid R to
in 2.0 m L o f the solvent mixture. wet die substance. H eat progressively to ignition and
Column: continue heating until an almost white or at m ost a greyish
— size: I = 0.25 m , 0 = 4.6 mm; residue is obtained. Carry out the ignition at a tem perature
— stationary phase: spherical end-capped octadecylsüyl silica gel not exceeding 800 °C. Allow to cool. Add 3 m L of
for chromatography R (5 pm) with a pore size o f 10 n m hydrochloric acid R and 1 m L o f nitric acid R. H eat and
and a carbon loading of 19 per cent; evaporate slowly to dryness. Cool and add 1 m L of a 100 g/L
— temperature'. 40 °C. solution o f hydrochloric acid R and 10.0 m L o f cUsaHed
Mobile phase: water R. N eutralise with a 1.0 g/L solution of ammonia R
— mobile phase A: 1.12 g/L solution of phosphoric acid R using 0.1 m L o f phenolphthalein solution R as indicator.
adjusted to p H 7.0 with a 42 g/L solution of sodium Add 2.0 m L of a 60 g/L solution of anhydrous acetic acid R
hydroxide R, and dilute to 20 m L with distilled water R. 12 m L of the
— mobile phase B: water R, acetonitrüe R (10:90 ViV)\ solution complies with test A. Prepare the reference solution
using lead standard solution (1 ppm Pb) R.
 1-50 Aceclofenac 2016

L oss o n d ry in g (2.2.32)
o
M aximum 0.5 per cent, determ ined on 1.000 g by drying in
an oven at 105 °C.
S u lfa te d a s h (2.4.14)
M aximum 0.1 per cent, determ ined on 1.0 g.
A SSA Y
Dissolve 0.300 g in 40 m L o f methanol R. T itrate with 0.1 M
sodium hydroxide, determining the end-point E. ethyl [[[2-[(2,6-
potentiometrically (2 . 2 . 2 0 ). dichlorophenyl)amino]phenyl] acetyl] oxy] acetate (ethyl ester
1 m L of 0.1 M sodium hydroxide is equivalent to 35.42 mg o f o f aceclofenac),
C 16H 13CI2N O 4.
STORAGE o
Protected from light.
IM P U R IT IE S
Specified impurities A, B, C, D , E, F , G, H , I

F. benzyl [[[2-[(2,6-
dichlorophenyl)amino]phenyI]acetyl]oxy]acetate (benzyl ester
o f aceclofenac),

O
A [2-[(2,6-dichlorophenyi)amino]phenyI] acetic acid
(diclofenac).

G. [[[[[2-[(2,6-
dichlorophenyl)amino]phenyl]acetyI] oxy] acetyl] oxy]acetic
acid (acetic aceclofenac),
B. methyl [2-[(2j6-dichlorophenyl)amino]phenyl]acetate
(methyl ester of diclofenac),
o
^ Y ^ r 0' ^ o / v 0 ^ ' c ° 2H

H . [ [[ [[[[2-[(2,6-dichlorophenyl)amino]phenyl] acetyl] oxy]

C. ethyl [2-[(2,6-dichlorophenyl)amino]phenyl]acetate (ethyl
acetyl]oxy]acetyl]oxy]acetic a d d (diacetic acedofenac),
ester o f diclofenac),

• OCT^
1. 1-(2 ,6-dichlorophenyl)-1,3-dihydro-2/f-indol-2-one.
PhEur
D . methyl [[[2-[(2,6-
dichlorophenyl)amino]phenyl]acetyl]oxy]acetate (methyl ester
of aceclofenac),
2016 Acemetacin 1-51

★ ★ — temperature: 40 °C.
Acemetacin ★ ★ Mobile phase:
(Ph Eitr monograph 1686) ***** — mobile phase A: dissolve 1.0 g of potassium dihydrogen
phosphate R in 900 m L of water R, adjust to p H 6.5 with
1 M sodium hydroxide and dilute to 1000 m L with
water R,
— mobile phase B: acetonitrUefor chromatography R]

Time Mobile phase A Mobile phase B

(min) (percent V/V) (per cent V/V)
0-5 95 5

C jxH wCINO î 415.8 53164-05-9 5 -9 95->65 5 -»35

9 - 16 65 35
A c tio n a n d u se
16-28 65 -»20 35 -»80
Cyclo-oxygenase inhibitor; analgesic; anti-inflammatory.
28 - 34 20 80
PhEur_______________________________ ___________________________

DEFINITION
[ [[ l-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3- Flow rate 1.0 mTVmin.
yl] acetyl] oxy] acetic ad d . Detection Spectrophotom eter at 235 nm.
C o n te n t Injection 20 pL.
99.0 per cent to 101.0 per cent (dried substance). Identification of impurities:
CHARACTERS — use the chromatogram supplied with acemetacin
A p p e a ra n c e impurity mixture CRS and the chromatogram obtained
Yellow or greenish-yellow, crystalline powder. with reference solution (e) to identify the peaks due to
impurities C, D , E and F;
Solubility — use the chromatogram obtained with reference
Practically insoluble in water, soluble in acetone, slightly solution (b) to identify the peak due to impurity B.
soluble in anhydrous ethanol.
Relative retention W ith reference to acemetacin (retention
It shows polymorphism (5.9). tim e = about 15 min): impurity A = about 0.7;
IDENTIFICATION impurity B = about 0.9; impurity F = about 1.2;
Infrared absorption spectrophotometry (2.2.24). impurity C = about 1.3; impurity D = about 1.5;
Comparison acemetacin CRS. impurity E = about 2.2.
I f the spectra obtained in the solid state show differences, System suitability Reference solution (d):
dissolve the substance to be examined and the reference — peak-to-valley ratio: minimum 15, where Hp = htight
substance separately in acetone R, evaporate to dryness and above the baseline of the peak due to impurity B and
record new spectra using the residues. Hv = height above the baseline of the lowest point o f the
curve separating this peak from the peak due to
TESTS acemetacin.
R e la te d su b sta n c e s Limits:
Liquid chromatography (2.2.29). — correction factors: for the calculation of content, multiply
Test sohaion Dissolve 0.100 g of the substance to be the peak areas o f the following impurities by the
examined in acetonitrUe for chromatography R and dilute to corresponding correction factor: impurity C = 1.3;
20.0 m L with the same solvent. impurity D = 1.4; impurity F = 1.3;
Reference solution (a) Dilute 5.0 mT. of the test solution to — impurity E: no t more than 3 times the area of the
50.0 m L with acetonitrUe for chromatography R. Dilute 1.0 m L p rindpal peak in the chromatogram obtained with
o f this solution to 100.0 m L with acetonitrUe for reference solution (a) (0.3 per cent);
chromatography R. — impurity B: not more than the area of the mi-responding
Reference sohaion (b) Dissolve 5.0 mg of acemetacin peak in the chromatogram obtained with reference
impurity A CRS and 10.0 m g o f indometacin CRS solution (c) (0.2 per cent);
(impurity B) in acetonitrUe for chromatography R, and dilute to — impurity A: not more than the area of the corresponding
50.0 m L with the same solvent. peak in the chromatogram obtained with reference
solution (c) (0.1 per cent);
Reference solution (c) Dilute 1.0 m L o f reference solution (b)
— impurities C, D, F: for each impurity, not more than the
to 20.0 m L with acetomtrUe for chromatography R.
area o f the prindpal peak in th e chromatogram obtained
Reference sohaion (d) T o 1 m L of reference solution (b), add with reference solution (a) (0.1 per cent);
10 m L o f the test solution and dilute to 20 m L with — unspecified impurities: for each impurity, not more than the
acetomtrUefor chromatography R. area o f the prindpal peak in the chromatogram obtained
Reference sohaion (e) Dissolve the contents o f a vial of with reference solution (a) (0.10 per cent);
acemetacin impurity mixture CRS (containing impurities C, D , — total: n o t more than 4 times the area of the principal peak
E and F) in 1.0 m L o f the test solution. in the chromatogram obtained with reference solution (a)
Column: (0.4 per cent);
— size: I = 0.25 m , 0 = 4 mm; — disregard limit. 0.5 times the area of the prindpal peak in
— stationary phase: spherical end-capped octadecylsUyl siHca gel the chromatogram obtained w ith reference solution (a)
for chromatography R (5 pm); (0.05 p er cent).
 1-52 Acenocoumarol 2016

H eav y m e ta ls (2.4.8)
M aximum 20 ppm.
Acenocoumarol
Solvent mixture methanol R, acetone R (10:90 V!V).
0.250 g complies with test H . Prepare the reference solution
using 0.5 m L o f lead standard sohaion (10 ppm Pb) R.
L oss o n d ry in g (2.2.32)
M aximum 0.5 per cent, determ ined on 1.000 g by drying in
an oven at 105 °C.
S u lfa te d a s h (2.4.14)
M aximum 0.1 per cent, determined on 1.0 g. and enantiomer

A SSAY
Dissolve 0.350 g in 20 m L o f acetone R and add 10 m L of c 19h 15n o 6 353.3 152-72-7
water R. T itrate with 0.1 M sodium hydroxide, determining the
end-point potentiometrically (2 . 2 . 2 0 ). A ctio n a n d use
Vitamin K epoxide reductase inhibitor; oral anticoagulant.
1 m L o f 0.1 M sodium hydroxide is equivalent to 41.58 mg
o f C 2iH i8C 1N 06. P re p a r a tio n
Acenocoumarol Tablets
STORA GE
Protected from light. D E F IN IT IO N
IM P U R IT IE S Acenocoumarol is (R.S)-4-hydroxy-3-( 1-p-nitrophenyl-3-
Specified impurities A, B, C, D , E, F oxobutyl)coumarin. It contains not less than 98.5% and not
more than 100.5% o f C 19H15N 0 6, calculated with reference
COjH to the dried substance.
C H A R A C T E R IS T IC S
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.
A. 4-chlorobenzoic acid,
ID E N T IF IC A T IO N
T he infrared absorption spectrum, Appendix II A, is concordant
with the reference spectrum of acenocoumarol (RS 001). If the
R3
spectra are n o t concordant, dissolve 0.1 g o f the substance
being examined in 10 m L o f acetone and add water drop wise
until the solution becomes turbid. H eat on a w ater bath until
the solution is clear and allow to stand. Filter, wash the
crystals with a mixture o f equal volumes of acetone and water
and dry at 100° at a pressure o f 2 kPa for 30 minutes.
B. R1 = R2 = R3 = H : [l-(4-chlorobenzoyl)-5-methoxy-2- Prepare a new spectrum o f the residue.
methylindol-3-yl]acetic acid (indom etadn),
TESTS
C. R1 = Cl, R2 = H , R3 = C H 2 -C 0 2H: [[[l-(3,4-
C la rity a n d c o lo u r o f so lu tio n
dichlorobenzoyl)-5-methoxy-2-methyl-l.H'-indol-3- A. A 2.0% w/v solution in acetone is clear, Appendix IV A.
yl] acetyl] oxy] acetic acid,
B. T he absorbance of a 4-cm layer of a 2.0% w/v solution in
D. R1 = H , R2 = C (C H 3)3, R3 = C H 2 -C 0 2H : [[[1-
acetone at 460 nm is not more than 0.12, Appendix II B.
(4-chlorobenzoyl)-6-( 1,1 -dimethylethyl)-5-methoxy-2-methyl-
1H-indol-3-yl] acetyl] oxy] acetic ad d , C. A 2.0% w/v solution in 0.1m sodium hydroxide is dear,
Appendix IV A, and yellow.
E. R1 = R2 = H , R3 = C H 2 -C 0 -0 -C (C H 3) 3:
1,1 -dimethylethyl [[[1 -(4-chlorobenzoyl)-5-methoxy-2- L ig h t a b so rp tio n
methyl- l/i-indol-3-yl] acetyl] oxy]acetate, Absorbance of a 0.001% w/v solution in a mixture of
1 volume o f 1m hydrochloric add and 9 volumes o f methanol at
F. R1 = R2 = H , R3 = C H 2- C 0 -0 - C H 2- C 0 2H: [[[[[1-
the maximum at 306 nm, 0.50 to 0.54, calculated with
(4-chlorobenzoyl)-5-methoxy-2-methjd-l/i-indol-3-
reference to the dried substance, Appendix II B.
yl] acetyl] oxy] acetyl] oxy] acetic ad d .
R e la te d su b sta n c e s
__________________________________________________________ PhEur
Carry out the m ethod for thin-layer chromatography,
Appendix HI A, using the following solutions in acetone.
(1) 2.0% w/v of the substance being examined.
(2) 0.0020% w/v o f the substance being examined.
CHROMATOGRAPHIC CONDITIONS
(a) U se as the coating silica gd GF2 s4 -
(b) Use die mobile phase as described below.
(c) Apply 20 nL of each solution.
(d) Develop the plate to 15 cm.
2016 Acesulfame Potassium 1-53

(e) After removal o f the plate, allow it to dry in air and Reference solution (a) Dissolve 5 mg o f acesulfame
im m ediately examine under ultraviolet light (254 nm). potassium CRS in water R and dilute to 5 m L with the same
solvent.
M O BILE PHASE
20 volumes of glacial acetic acid, 50 volumes of cychhexane Reference solution (b) Dissolve 5 mg o f acesulfame
and 50 volumes o f cHchloromethane.
potassium CRS and 5 m g o f saccharin sodium R in water R and
dilute to 5 m L 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 acetate R
solution (1) is n o t more intense than the spot in the
(10:60:60 V/V/V).
chrom atogram obtained with solution (2) (0.1%).
Application 5 pL as bands.
L oss o n d ry in g
W hen dried to constant weight at 105°, loses not more than Development Twice over 2/3 of the plate.
0.5% o f its weight. U se 1 g. Drying In a current o f warm air.
S u lfa te d ash Detection Examine in ultraviolet light at 254 nm.
N o t m ore than 0.1% , Appendix IX A. System suitability: reference solution (b):
— the chromatogram shows 2 clearly separated zones.
A SSA Y
Dissolve 0 .6 g in 5 0 m L of acetone and titrate with
Results T he principal zone in the chromatogram obtained
with the test solution is similar in position and size to the
0 .1 m sodium hydroxide VS using bromothymol blue solution R3
prindpal zone in the chromatogram obtained with reference
as indicator. Repeat the operation without the substance
solution (a).
being examined. T h e difference between the titrations
represents the am ount of sodium hydroxide required. Each C. 0.5 m L o f solution S (see Tests) gives reaction (b) of
mT. o f 0 .1 m sodium hydroxide KS is equivalent to 3 5 .3 3 m g of potassium (2.3.1).
C 19H 15N CV TESTS
S o lu tio n S
Dissolve 10.0 g in carbon dioxide-free water R and dilute to
50 m L with the same solvent.
Acesulfame Potassium ****** A p p e a ra n c e o f so lu tio n
*+ +* Solution S is clear (2.2.1) and colourless (2.2.2, Method IT).
(Ph Eur monograph 1282) *
A cid ity o r alk a lin ity
T o 20 m L of solution S add 0.1 m L of bromothymol blue
solution R l. N o t m ore than 0.2 m L o f 0.01 M hydrochloric
acid or 0.01 M sodium hydroxide is required to change the
colour of the indicator.
Im p u rity A
Thin-layer chrom atography (2.2.27).
Test solution Dissolve 0.80 g of the substance to be examined
C4H4KNO4S 201.2 55589-62-3
in water R and dilute to 10 m L with the same solvent
A c tio n a n d use Reference solution (a) Dissolve 50 m g o f acetylacetamide R
Sweetening agent. (impurity A) in water R and dilute to 25 m L with the same
solvent. T o 5 m L o f the solution add 45 m L of water R and
PhEur________________________________________________________ __ dilute to 100 m L with methanol R.
D E F IN IT IO N Reference solution (b) T o 10 m L of reference solution (a) add
Potassium 6-methyl-1,2,3-oxathiazin-4-olate 2,2-dioxide. 1 m L o f the test solution and dilute to 20 m L with
C o n te n t 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
(2:15:74 V/VIV).
A p p e a ra n c e
W hite or almost white, crystalline powder or colourless Application 5 piL.
crystals. Development Over 2/3 o f the plate.
S o lu b ility Drying In air until the solvents are completely removed.
Soluble in water, very slightly soluble in acetone and in Detection Spray with phosphoric vanillin solution R and heat at
ethanol (96 per cent). 120 °C for about 10 min; examine in daylight
ID E N T IF IC A T IO N System suitability T h e chromatogram obtained with reference
First identification A , C. solution (a) shows a clearly visible spot and the
chromatogram obtained with reference solution (b) shows
Second identification B, C.
2 clearly separated spots.
A. Infrared absorption spectrophotometry (2.2.24).
Limit.
Comparison acesulfame potassium CRS. — impurity A: any spot due to im purity A is not more
B. Thin-layer chromatography (2.2.27). intense than the spot in the chromatogram obtained with
Test solution Dissolve 5 mg of the substance to be examined reference solution (a) (0.125 per cent).
in water R and dilute to 5 m L with the same solvent. I m p u rity B
Liquid chromatography (2.2.29).
 1-54 Acetazolamide 2016

Test solution Dissolve 0.100 g o f die substance to be IMPURITIES

examined in water R and dilute to 10.0 m L with the same Specified impurities: A , B.
solvent.
Reference solution (a) Dissolve 4.0 m g o f acesulfame potassium H3C\ ^ 0
impurity B CRS in water R and dilute to 100.0 m L with the
same solvent. Dilute 1.0 m L o f the solution to 200.0 m L
with water R.
Reference solution (b) Dissolve 0.100 g o f the substance to be
A. 3-oxobutanamide (acetylacetamide),
examined in reference solution (a) and dilute to 10.0 m L
with the same solution.
h 3c . .0 ^ »
Column: V s=o
— sizer. I = 0.25 m, 0 = 4.6 mm; ,NH
— stationary phase: octadecylsQyl silica gel for chromatography R ° A r ’
(3 nm).
Mobile phase M ix 40 volumes o f acetomtrUe R and 60 volumes B. 5-chloro-6-methyi-1,2,3-oxathiazm -4(3/f)-one 2,2-dioxide.
of a 3.3 g/L solution o f tetrabuiylammomwn hydrogen sulfate R.
__________________________________________________________ PhEur
Flow rate 1 mL/min.
Detection Spectrophotom eter at 234 nm .
Injection 20 nL.
Run time Twice the retention time o f acesulfame. Acetazolamide ★★* ★★
★ ★
Relative retention W ith reference to acesulfame (retention * * * * *
time = about 5.3 min): im purity B = about 1.6.
(Ph Eur monograph 0454)
System suitability: o\w/o H
— signal-to-noise ratio: minimum 10 for the peak due to ,s. N^CHs
impurity B in the chromatogram obtained with reference HjN' 'Y Y T
N -N
solution (a);
— peak-to-vaUey ratio: minimum 1.2, where Hp = height
above the baseline o f the peak due to impurity B and 222.2 59-66-5
Hv = height above the baseline o f the lowest point o f the Action and use
curve separating this peak from the peak due to Carbonic anhydrase inhibitor; diuretic; treatm ent of
acesulfame, in the chromatogram obtained with reference glaucoma and ocular hypertension; treatm ent o f m o untain
solution (b). sickness.
Limit: Preparation
— impurity B: not m ore than the area o f the principal peak Acetazolamide Tablets
in the chrom atogram obtained with reference solution (a)
PhEur__________________________________________________________
(20 ppm ).
D E F IN IT IO N
Fluorides
Maximum 3 ppm. 2V-(5-Sulfamoyi-1,3,4-thiadiazol-2-yI) acetamide.

Potentiom etry (2.2.36, Method I). Content

98.5 per cent to 101.0 per cent (dried substance).
Test solution Dissolve 3.000 g o f the substance to be
examined in distilled water R, add 15.0 m l. of total-ionic- CHARACTERS
strength-adjustment buffer R 1 and dilute to 50.0 m L with Appearance
distilled water R. White or almost white, crystalline powder.
Reference solutions T o 0.5 mT, 1.0 mT, 1.5 m L and 3.0 m L Solubility
of fluoride standard solution (10 ppm F) R add 15.0 m L of Very slighdy soluble in water, slighdy soluble in ethanol
total-ionic-strerigth-adjustment buffer R1 and dilute to 50.0 mL (96 per cent). It dissolves in dilute solutions o f alkali
with distilled water R. hydroxides.
Indicator electrode Fluoride-selective. It shows polymorphism (5.9).
Reference electrode Silver-silver chloride. IDENTIFICATION
H eav y m e ta ls (2.4.8) First identification A, B
M aximum 5 ppm . Second identification A , C, D
12 m L of solution S complies with test A. Prepare die A Ultraviolet and visible absoiption spectrophotometry
reference solution using lead standard solution (1 ppm Pb) R (2.2.25).
L oss o n d ry in g (2.2.32) Solution A Dissolve 30.0 m g in 0.01 M sodium hydroxide and
M aximum 1.0 per cent, determined on 1.000 g by drying in dilute to 100.0 m L with die same solvent. D ilute 10.0 m L of
an oven at 105 °C for 3 h. the solution to 100.0 m L with 0.01 M sodium hydroxide.
A SSA Y Solution B Dilute 25.0 m L o f solution A to 100.0 m L with
Dissolve 0.150 g in 50 m L o f anhydrous acetic add R. Titrate 0.01 M sodium hydroxide.
with 0.1 M perchloric add, determining the end-point Spectral range 230-260 nm for solution A; 260-350 n m for
potentiometrically (2 .2 . 2 0 ). solution B.
1 m L o f 0.1 M perchloric add is equivalent to 20.12 mg Absorption maximum A t 240 nm for solution A; at 292 nm for
Of C4H4KNO4S. solution B.
2016 Acetazolamide 1-55

Specific absorbance at the absorption maximum 162 to 176 for — impurities A , B, C, D, E, F: for each impurity, not more
solution A; 570 to 620 for solution B. than 1.5 times the area of the principal peak in the
B. Infrared absorption spectrophotometry (2.2.24). chromatogram obtained with reference solution (a)
(0.15 per cent);
Comparison acetazolamide CRS.
— unspecified impurities: for each impurity, not more than the
If the spectra obtained in the solid state show differences, area o f the principal peak in th e 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
— total: not m ore than 6 times the area o f the principal peak
dryness and record new spectra using the residues. in the chromatogram obtained with reference solution (a)
C. Introduce about 20 mg into a test-tube and add 4 m L of (0.6 per cent);
dilute hydrochloric add R and 0.2 g o f zinc powder R — disregard limit. 0.5 times the area of the principal peak in
Im mediately place a piece of lead acetate paper R over the the chromatogram obtained w ith reference solution (a)
m outh o f the tube. T he paper shows a brownish-black (0.05 per cent).
colour.
S u lfates (2.413)
D . Dissolve about 25 mg in a mixture o f 0.1 m L o f dilute M axim um 500 ppm.
sodium hydroxide sdurion R and 5 m L of water R Add 0.1 m L
T o 0.4 g add 20 m L o f distilled water R and dissolve by
o f copper sulfate solution R. A greenish-blue precipitate is
heating to boiling. Allow to cool w ith frequent shaking and
formed.
filter.
TESTS H eav y m e ta ls (2.48)
Appearance of solution M aximum 20 ppm.
T he solution is no t more opalescent than reference
1.0 g complies with test C. Prepare the reference solution
suspension II (2.2.1) and not more intensely coloured than
using 2 m L o f lead standard solution (10 ppm Pb) R.
reference solution Y 5 or BY5 (2.2.2, Method II).
L oss o n d ry in g (2.2.32)
Dissolve 1.0 g in 10 m L of 1 M sodium hydroxide.
M aximum 0.5 per cent, determined on 1.000 g by drying in
Related substances an oven at 105 °C.
Liquid chromatography (2.2.29).
S u lfa te d a sh (2.414)
Test solution Dissolve 40 mg o f die substance to be examined M aximum 0.1 per cent, determined on 1.0 g.
in the mobile phase and dilute to 100.0 m L with the mobile
phase. A SSAY
Reference solution (a) Dilute 1.0 m L o f the test solution to Dissolve 0.200 g in 25 m L of dimethylforrnamide R. T itrate
100.0 m L with the mobile phase. Dilute 1.0 m L o f this with 0.1 M ethanolic sodium hydroxide, determining the
end-point potentiometrically (2 . 2 . 2 0 ).
solution to 10.0 m L with the mobile phase.
Reference solution (b) Dissolve the contents o f a vial o f 1 m L of 0.1 M ethanolic sodium hydroxide is equivalent to
acetazolamide for system suitability CRS (containing 22.22 m g of C 4H 6N 4O 3S2.
impurities A, B, C , D , E and F) in 1.0 m L o f the mobile IM P U R IT IE S
phase. Specified impurities A, B, C, D , E, F
Cdumn: Other detectable impurities (the following substances would, if
— size: I = 0.15 m , 0 = 4.6 mm; present at a sufficient level, be detected by one o r other of
— stationary phase: end-capped propoxybenzene silica gel for the tests in the monograph. They are limited by the general
chromatography R (4 (im). acceptance criterion for other/unspecified impurities and/or
Mobile phase acetonitrüe for chromatography R, 6.8 g/L solution by the general monograph Substances for pharmaceutical use
of potassium dihydrogen phosphate R (10:90 V!V). (2034). It is therefore not necessary to identify these
Flow rate 1.0 mL/min. impurities for demonstration of compliance. See also 5.10.
Detection Spectrophotom eter at 265 nm. Control of impurities in substancesfor pharmaceutical use): G.
Injection 25 jil. H
Run time 3.5 times the retention time of acetazolamide.
Identification of impurities Use the chromatogram supplied
\
N -N
Y R1
with acetazolamide for system suitability CRS and the
chromatogram obtained with reference solution (b) to A. R1 = C O -C H 3, R2 = Cl: N -(5-chloro-l,3,4-thiadiazol-2-
identify the peaks due to impurities A, B, C, D , E and F. yl)acetamide,
Relative retention W ith reference to acetazolamide (retention B. R1 = C O -C H 3, R2 = H: 2V-(l,3,4-thiadiazol-2-
time = about 8 min): impurity E = about 0.3; yl)acetamide,
impurity D = about 0.4; impurity B = about 0.6;
C. R1 = C O -C H 3, R2 = SH: N-(5-sulfanyl-1,3,4-thiadiazol-
impurity C = about 1.4; impurity A = about 2.1;
2-yl)acetamide,
impurity F = about 2.6.
D . R 1 = H , R2 = S 0 2-N H 2: 5-am ino-1,3,4-thiadiazole-2-
System suitability: reference solution (b):
sulfonamide,
— resolution: minim um 2.0 between the peaks due to
impurities E and D . E. R1 = C O -C H 3, R2 = S 0 2-0 H : 5-acetamido-1,3,4-
thiadiazole-2-sulfonic ad d ,
Limits:
— correction factory, for the calculation o f content, multiply
the peak areas o f the following impurities by the
corresponding correction factor impurity B = 2.3;
impurity C = 2 . 6; impurity D = 1.6;
 1-56 Acetic Acid 2016

Dissolve the residue obtained in the test for residue on

"’V V rY x V V "
O N -N N -N 0
evaporation by h eating with 2 quantities, each of 15 m L, of
water R and dilute to 50.0 m L with water R (solution A).
12 m l. o f solution A complies with test A. Prepare the
F. N- [5- [(5-acetam ido-l 33,4-thiadiazol-2- reference solution using lead standard solution (2 ppm Pb) R.
yl) sulfonyl] sulfamoyl-1,3,4-thiadiazol-2-yI] acetamide, R esid u e o n e v a p o ra tio n
M aximum 0.01 per cent.
H
Sx sy N
H
N -N
2 Evaporate 20 g to dryness on a water-bath and dry at
100-105 °C. T h e residue weighs a maximum of 2.0 mg.

G. 5-am ino-1,3,4-thiadiazole-2-thioL A SSA Y

Weigh accurately a conical flask with a ground-glass stopper
PhEur
containing 25 m l, o f water R A dd 1.0 m L o f the substance
to be examined and weigh again accurately. A dd 0.5 m L of
phenolpkthalein solution R and titrate with 1 M sodium
***** hydroxide.
Glacial Acetic Acid ★ ★ 1 m i, o f 1 M sodium hydroxide is equivalent to 60.1 mg
(Ph Eitr monograph 0590) ***** o f C 2H 4O 2.
STO R A G E
0
In an airtight container.
I
H3C OH PhEur

C2H 4O 2 60.1 64-19-7

PhEur__________________________________________________________

D E F IN IT IO N Acetic Acid (6 per cent)

C o n te n t Dilute Acetic A d d
99.0 per cent mlm to 100.5 per cent mlm.
D E F IN IT IO N
CHARACTERS Acetic A d d (6 per cent) contains not less than 5.7% and not
A p p e a ra n c e more than 6.3% w/w of acetic ad d , C 2H 4O 2. It may be
Crystalline mass or clear, colourless, volatile liquid. prepared by mnring 182 g of Acetic A d d (3 3 per cent) with
S o lu b ility 818 g of Purified Water.
Miscible with water, with ethanol (96 per cent) and with IDENTIFICATION
methylene chloride.
A. Strongly addic.
ID E N T IF IC A T IO N B. W hen neutralised, yields the reactions characteristic of
A. A 100 g/L solution is strongly acid (2.2.4). acetates, Appendix VI.
B. T o 0.03 m L add 3 m L o f water R and neutralise with
TESTS
dilute sodium hydroxide solution R T he solution gives W eig h t p e r m L
reaction (b) of acetates (2.3.1).
About 1.005 g, Appendix V G.
TESTS H eav y m e ta ls
S o lu tio n S Evaporate 20.0 m l, to dryness and add 20 m L of water.
Dilute 20 m L to 100 m L with distilled water R 12 m L o f the resulting solution complies with limit test A for
A p p e a ra n c e heavy metals, Appendix VII. Use lead standard solution
T he substance to be examined is clear (2.2.1) and colourless ( 1 ppm Pb) to prepare the standard (1 ppm).

(2.2.2, Method II). C h lo rid e

F re e z in g p o in t (2.2.18) Dilute 5.0 m l, with suffident water to produce 100 m L
M inim um 14.8 °C. 15 m L o f the resulting solution complies with the limit test for
R e d u c in g su b s ta n c e s chlorides, Appendix VII (70 ppm).
Dilute 2.0 m L to 10.0 m L with water R Add 0.1 m L of S u lfate
0.02 M potassium permanganate. H eat on a water-bath for 12.5 m L o f the solution used in the test for Chloride, diluted
1 min, the colour remains pink. to 15 m l . with water, complies with the limit test for sulfates,
C h lo rid e s (2.4.4) Appendix VII (240 ppm ).
Maximum 25 mg/L. A ldehydes
Dilute 10 m L of solution S to 15 m L with water R. Distil 7 5 m l^ T o the first 5 m L of the distillate add 10 m L
o f a 5 % w/v solution o f mercuTy(n) chloride, make alkaline
S u lfates (2.4.13)
with 5m sodium hydroxide, allow to stand for 5 minutes and
M aximum 50 mg/L, determined on solution S.
addify with 1m sulfuric acid. T h e solution shows not more
Iro n (2.4.9) than a faint turbidity.
M aximum 5 ppm.
F o rm ic a c id a n d o x id isab le im p u ritie s
Dilute 5.0 m L of solution A obtained in the test for heavy Mix 5 m l . with 6 m l , of sulfuric acid and cool to 20°.
metals to 10.0 m L with water R. Add 0 .4 m L o f 0 .0 1 6 7 m potassium dichromate VS, allow to
H eav y m e ta ls (2.4.8) stand for 1 minute, add 25 m l, o f water and 1 m L o f freshly
M aximum 5 ppm. prepared dilute potassium iodide solution and titrate the
2016 Acetone 1-57

liberated iodine with 0.1m sodium tkiosidfate F 5 using starch mucilage as indicator. N ot less than 1.0 m L of 0.1m sodium
mucilage as indicator. N ot less than 0 .2 m L o f 0.1m sodium tkiosidfate VS is required.
thiosulfate F 5 is required. R ead ily o x id isa b le im p u ritie s
R ead ily o x id isab le im p u ritie s T o 5 .0 m L add 2 0 m L o f water and 0 .2 m L of
T o 2 5 m L add 0 .2 m L of 0.02m potassium permanganate FiS 0.02m potassium permanganate KS and allow to stand for
and allow to stand for 1 minute. T he pink colour is not 1 minute. T h e pink colour is not entirely discharged.
entirely discharged. N o n -v o la tile m a t te r
N o n -v o latile m a t te r W hen evaporated to dryness and dried at 105°, leaves not
W hen evaporated to dryness and dried at 105°, leaves not more th an 0 .01 % w/w of residue.
more than 0 .01 % w/w o f residue.
ASSAY
A SSAY Weigh 5 g into a stopper flask containing 5 0 m L o f water and
A dd 30 m L of water to 20 g in a stopper flask and titrate titrate w ith 1m sodium hydroxide F 5 using phenolphthalein
with 1m sodium hydroxide F 5 using phenolphthalein solution R1 solution R1 as indicator. Each mL o f 1m sodium hydroxide F S
as indicator. E ach m L of 1m sodium hydroxide F 5 is is equivalent to 6 0 .0 5 mg o f C2H 4 0 2.
equivalent to 60.05 mg of C 2H 4O 2.

Acetone ★ ★
★ ★
Acetic Acid (33 per cent) *****
(Ph Eur monograph 0872)
Acetic A d d
P r e p a r a tio n 0
Acetic A d d (6 p er cent) X , CH;
H3C
D E F IN IT IO N
Acetic A d d (33 p er cent) contains not less than 32.5% and
n o t m ore than 33.5% w/w of acetic a d d , C 2H 4O 2. CjHeO 58.08 67-64-1
PhEir___
C H A R A C T E R IS T IC S
A clear, colourless liquid. D E F IN IT IO N
M isdble with water, with ethanol (96%) and with glycerol. Propanone.

ID E N T IF IC A T IO N CHARACTERS
A. Strongly addic, even when diluted fredy. A p p e a ra n c e
Volatile, clear, colourless liquid.
B. W hen neutralised, yields the reactions characteristic of
acetates, A ppendix VI. S o lu b ility
M isdble w ith w ater and with ethanol (96 per cent).
TESTS
W eig h t p e r m l . T h e vapour is flammable.
1.040 to 1.042 g, Appendix V G. ID E N T IF IC A T IO N
H eav y m e ta ls A. Relative density (see Tests).
Evaporate 10.0 m L to dryness and add 20 m L o f water. B. T o 1 m L, add 3 m L o f dilute sodium hydroxide solution R
12 m L of the resulting solution complies with limit test A for and 0.3 m L of a 25 g/L solution o f sodium nitroprusside R.
heavy metals, Appendix VII. Use lead standard solution An intense red colour is produced which becomes violet with
(1 ppm Pb) to prepare the standard (2 ppm). the addition of 3.5 m L o f acetic acid R.
Chloride C. T o 10 m L of a 0.1 per cent VIV solution o f the substance
Dilute 5.0 m L w ith suffident water to produce 100 m L to be examined in ethanol (50 per cent VIV) R, add 1 m L o f a
15 m L of the resulting solution complies with the limit test for 10 g/L solution o f nitrobenzaldehyde R in ethanol
chlorides, Appendix VII (70 ppm). (50 per cent VIV) R and 0.5 mL o f strong sodium hydroxide
S u lfate solution R. Allow to stand for about 2 min and addify with
12.5 m L of the solution used in the test for Chloride, diluted
acetic acid R. A greenish-blue colour is produced.
to 15 m L with water, complies with the limit test for sulfates, TESTS
Appendix VII (240 ppm). A p p e a ra n c e o f so lu tio n
A ldehydes T o 10 m L add 10 m L o f water R. T he solution is clear
Distil 15 m L T o the first 5 m L of the distillate add 10 m L (2.2.1) and colourless (2.2.2, Method II).
of a 5% w/v solution of mercury(n) chloride, make alkaline A c id ity o r a lk a lin ity
w ith 5m sodium hydroxide, allow to stand for 5 minutes and T o 5 m L add 5 m L of carbon dioxide-free water R, 0.15 m L of
make addic w ith 1m sulfuric acid. The solution shows not phenolphthalein solution R and 0.5 m L of 0.01 M sodium
m ore than a faint turbidity. hydroxide. T h e solution is pink. A dd 0.7 m L o f 0.01 M
F o rm ic a c id a n d o x id isab le im p u ritie s hydrochloric acid and 0.05 m L of methyl red solution R.
M ix 5 m L with 6 m L o f sulfuric acid and cool to 20°. T h e solution is red or orange.
A dd 2 m L of 0 .0 1 6 7 m potassium dickromate F5, allow to R e lativ e d e n sity (2.2.5)
stand for 1 m inute, add 25 m L of water and 1 m L of freshly 0.790 to 0.793.
prepared dilute potassium iodide solution and titrate the
liberated iodine w ith 0.1m sodium thiosulfate VS using starch
 1-58 Acetylcholine Chloride 2016

R e d u c in g su b sta n c e s R esid u e o n e v a p o ra tio n

T o 30 m l. add 0.1 m l. o f 0.02 M potassium permanganate M aximum 50 ppm.
and allow to stand in the dark for 2 h. T he mixture is not Evaporate 20.0 g to dryness on a w ater-bath and dry at
completely decolourised 100-105 °C. T h e residue weighs a maximum o f 1 mg.
R e la te d su b sta n c e s W a te r (.2.5.12)
G as chromatography (2.2.28). Maximum 3 g/L, determined on 10.0 m L. U se 20 m L of
Test solution T he substance to be examined. anhydrous pyridine R as solvent.
Reference solution (a) T o 0.5 m L o f methanol R add 0.5 m L of STORAGE
2-propanol R and dilute to 100.0 m L with the test solution. Protected from light.
D ilute 1.0 m L of this solution to 10.0 m L with the test
IM P U R IT IE S
solution.
Specified impurities: A, B, C.
Reference solution (b) Dilute 100 pL o f benzene R to 100.0 m L
w ith the test solution. Dilute 0.20 m L o f this solution to A. C H 3-OH: m ethanol,
100.0 m L with the test solution. B. C H 3-C H O H -C H 3: propan-2-ol (isopropanol),
Column: C. O H ^ : benzene.
— material: fused silica, PhEur
— size. I = 50 m, 0 = 0.3 mm,
— stationary phase: macrogol 20 000 R (film thickness 1 (im).
Carrier gas helium for chromatography R.
Linear velocity 21 cm/s. Acetylcholine Chloride ★★*★*
★ ★
Split ratio 1:50. *****
(Ph Ever monograph 1485)
Temperature:
H3C + CH3
Time Temperature .N^ Cl
CH3
(min) (°C)
Column 0 -1 1 45 + 100
C7H16o n o 2 181.7 60-31-1
11-20 100

Injection port 150 A c tio n a n d u se

Cholinoceptor agonist.
Detector 250
PhEur__________________

Detection Flam e ionisation. D E F IN IT IO N

2-(Acetyloxy)-iV>W,N-trimethylethanaminium chloride.
Injection 1 pL.
Retention time Impurity C = about 7.5 min. C o n te n t
98.5 per cent to 101.5 per cent (dried substance).
System suitability:
— resolution: minimum 5.0 between the peak due to CHARACTERS
impurity A (2nd peak) and the peak due to impurity B A p p e a ra n c e
(3rd peak) in the chromatogram obtained with reference W hite or almost white crystalline powder or colourless
solution (a), crystals, very hygroscopic.
— signal-to-notse ratio: minim um 5 for the peak due to S o lu b ility
impurity C in the chromatogram obtained with reference Very soluble in water, freely soluble in alcohol, slightly
solution (b). soluble in methylene chloride.
Limits:
ID E N T IF IC A T IO N
— impurities A , B: for each impurity, n o t m ore than the
difference between the areas of the corresponding peaks First identification B, E.
in the chromatogram obtained with reference solution (a) Second identification A , C, D, E.
and the areas o f the corresponding peaks in the A. Melting point (2.2.14): 149 °C to 152 °C.
chromatogram obtained with the test solution Introduce the substance to be examined into a capillary tube.
(0.05 per cent VIV), D ry in an oven at 100-105 °C for 3 h. Seal the tube and
— impurity C: not more than die difference between the area determine the melting point.
o f die peak due to impurity C in the chromatogram
B. Infrared absorption spectrophotometry (2.2.24).
obtained with reference solution (b) and the area of the
corresponding peak in the chromatogram obtained with Comparison acetylcholine chloride CRS.
the test solution (2 ppm VIV), C. Examine the chromatograms obtained in the test for
— any other impurity, for each impurity, not m ore than the related substances.
difference between the area o f the peak due to impurity A Results T h e principal zone in the chromatogram obtained
in the chromatogram obtained with reference solution (a) with test solution (b) is similar in position, colour and size to
and the area o f the corresponding peak in die the principal zone in the chromatogram obtained with
chromatogram obtained with the test solution reference solution (b).
(0.05 p er cent VIV). D . T o 15 m g add 10 m L of dilute sodium hydroxide solution R,
M a tte r in so lu b le in w a te r 2 m l. o f 0.02 M potassium permanganate and heat.
D ilute 1.0 m L to 20 m L w ith water R. T he solution is clear T h e vapours formed change the colour o f red litmus paper R
(2.2.1). to blue.
2016 Acetylcysteine 1-59

E. 0.5 m L of solution S (see Tests) gives reaction (a) o f A SSAY

chlorides (2.3.1). Dissolve 0.200 g in 20 m L of carbon dioxide-free water R.
TESTS Neutralise with 0.01 M sodium hydroxide using 0.15 m l. of
S o lu tio n S
phenolphthalein solution R as indicator. Add 20.0 m L o f 0.1 M
Dissolve 5.0 g in carbon dioxide-free water R and dilute to
sodium hydroxide and allow to stand for 30 min. T itrate with
0.1 M hydrochloric acid.
50 m L with the same solvent.
1 m L of 0 . 1 M sodium ftydroxide is equivalent to 18.17 mg of
A p p e a ra n c e o f so lu tio n
C 7H 16C IN 0 2.
Solution S is clear (2.2.1) and not more intensely coloured
than reference solution Y 6 o r BY6 (2.2.2, Method II). STO R A G E
A cid ity In ampoules, protected from light.
D ilute 1 m L o f solution S to 10 m L with carbon dioxide-free IM P U R IT IE S
water R. A dd 0.05 m L of phenolphthalein solution R. N o t more
than 0.4 m L o f 0.01 M sodium hydroxide is required to H3C + CH3
change the colour of the indicator to pink. \ N^ Cf
ch 3
R e la te d su b s ta n c e s
Thin-layer chromatography (2.2.27). Prepare the solutions A. 2- chloride
immediately before use. (choline chloride),
Test solution (a) Dissolve 0.30 g of the substance to be
examined in methanol R and dilute to 3.0 m L with the same
solvent.
Test solution (b) D ilute 1 m L of test solution (a) to 10 m L
with methanol R.
B. 2-(acetyloxy)-N>N-dimethylethanaminium chloride,
Reference solution (a) Dilute 1 m L of test solution (a) to
100 m L with methanol R. ch 3
Reference solution (b) Dissolve 20.0 mg of acetylcholine
chloride CRS in methanol R and dilute to 2.0 m L with the H3C ch 3

same solvent.
Reference solution (c) Dissolve 20 mg of choline chloride R in C. NjN-dimethylmethanamine.
methanol R, add 0.4 m L of test solution (a) and dilute to PhEur
2.0 m L with methanol R.
Plate TLC silica gel plate R.
Mobile phase Mix 20 volumes of a 40 g/L solution o f
ammonium nitrate R, 20 volumes of methanol R and Acetylcysteine ★* * ★★
60 volumes of acetonârUe R. ★ ★
Application 5 nL as bands o f 10 mm by 2 mm. (Ph. Eur. monograph 0967) *****
Development Over 2/3 o f the plate.
ch 3
Detection Spray with potassium iodobismuthate solution R3.
System suitability T h e chromatogram obtained with reference
0={
H NH
solution (c) shows 2 clearly separated zones.
co 2h
Limits:
— any impurity: any zones in the chromatogram obtained
with test solution (a), apart from the principal zone, are C 5H 9N 03 S 163.2 616-91-1
n o t more intense than the principal zone in the
Action and use
chrom atogram obtained with reference solution (a)
Sulfydryl donor; antidote to paracetamol poisoning;
(1 per cent).
mucolytic.
T rim e th y la m in e
P r e p a r a tio n
Dissolve 0.1 g in 10 m L of sodium carbonate solution R and
Acetylcysteine eye drops
heat to boiling. N o vapours appear which turn red litmus
paper R blue. Acetylcysteine Injection

H eav y m e ta ls (2.4.8) PhEur.

M axim um 10 ppm .
D E F IN IT IO N
12 m L o f solution S complies with test A. Prepare the (2i?)-2-(Acetylamino)-3-sulfanylpropanoic add.
reference solution using lead standard solution (1 ppm Pb) R.
C o n te n t
L oss o n d ry in g (2.2.32)
98.0 per cent to 101.0 per cent (dried substance).
M axim um 1.0 p er cent, determined on 1.000 g by drying in
an oven at 105 °C for 3 h. CHARACTERS
A p p e a ra n c e
S u lfa te d a sh (2.4.14)
W hite or almost white, crystalline powder or colourless
M axim um 0.1 p er cent, determined on the residue obtained
crystals.
in the test for loss on drying.
S o lu b ility
Freely soluble in water and in ethanol (96 per cent),
practically insoluble in methylene chloride.
 1-60 Acetylcysteine 2016

IDENTIFICATION Injection 20 (iL, 3 times; inject 0.01 M hydrochloric acid as a

First identification A , C. blank.
Second identification A , B, D, E. Run time 5 times the retention time o f acetylcysteine (about
A. Specific optical rotation (see Tests). 30 min).
B. Melting point (2.2.14): 104 °C to 110 °C. Retention time Im purity A = about 2.2 m in; impurity B =
about 2.4 min; 2-methyl-2-thiazoline-4-carboxylic acid,
C. Infrared absorption spectrophotometry (2.2.24).
originating in test solution (c) = about 3.3 min;
Preparation Discs of potassium bromide R. acetylcysteine = about 6.4 min; im purity C = about 12 min;
Comparison acetylcysteine CRS. impurity D = about 14 min.
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 T he principal peak in the chromatogram obtained impurities A and B and minim um 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
reference solution (b). A \ x m 2 x 100
E. T o 0.5 m L of solution S (see Tests) add 0.05 m L of a A-----------
11 = ----~ x mi
50 g/L solution of sodium nitroprusside R and 0.05 m L of
_ A 3 x m 3 x 100
concentrated ammonia R. A dark violet colour develops. 12 = ----------------
A* x m i
TESTS
Solution S
A1 = peak area o f individual im purity (impurity A,
Dissolve 1.0 g in carbon dioxide-free water R and dilute to
impurity B, impurity C and im purity D) in the
20 m L with the same solvent.
chromatogram obtained with test solution (a);
Appearance of solution A 2 = peak area o f the corresponding individual im purity
Solution S is clear (2 . 2 . 1 ) and colourless (2.2.2, Method II). (impurity A, im purity B, im purity C and impurity
pH (2.2.3) D ) in the chrom atogram obtained with reference
2.0 to 2.8. solution (a);
T o 2 m L o f solution S add 8 m L of carbon dioxide-free A 3 = peak area of unknown impurity in the chromatogram
water R and mix. obtained with test solution (a);
A 4 = peak area o f acetylcysteine in the chromatogram
Specific optical rotation (2.2.7)
obtained with reference solution (b);
+ 21.0 to + 27.0 (dried substance).
mi = mass o f the substance to be examined in test
In a 25 m L volumetric flask, mix 1.25 g with 1 m L of a solution (a);
10 g/L solution of sodium edetate R. A dd 7.5 m L o f a 40 g/L m2 = mass of the individual im purity in reference solution
solution o f sodium hydroxide R, mix and dissolve. Dilute to (a);
25.0 m L with phosphate buffer solution pH 7.0 R2. m3 = mass of acetylcysteine in reference solution (b).
Related substances Limits:
Liquid chrom atography (2.2.29). Except where otherwise — impurities A , B, C, D: for each im purity, maximum
prescribed, prepare the solutions immediately before use. 0.5 per cent;
Test solution (a) Suspend 0.80 g of the substance to be — arty other impurity: for each impurity, maximum
examined in 1 m L o f 1 M hydrochloric acid and dilute to 0.5 per cent;
100.0 m L with water R. — total: maximum 0.5 p er cent;
Test solution (b) Dilute 5.0 m L of test solution (a) to — disregard Umar. 0.1 times the area o f the principal peak in
100.0 m L with water R. Dilute 5.0 m l. of this solution to the chromatogram obtained with reference solution (b)
50.0 m L w ith water R. (0.05 per cent); disregard any peak w ith a retention time
Test solution (c) Use test solution (a) after storage for at least of about 3.3 m in due to 2-methyl-2-thiazoline-4-
1 h. carboxylic ad d .
Reference solution (a) Suspend 4.0 m g o f acetylcysteine CRS, H eav y m e ta ls (2.4.8)
4.0 mg of L-cystme R (impurity A), 4.0 mg o f l -cysteine R M aximum 10 ppm .
(impurity B), 4.0 m g of acetylcysteine impurity C CRS and 2.0 g complies with test C. Prepare the reference solution
4.0 mg of acetylcysteine impurity D CRS in 1 m L o f 1 M using 2 m L o f lead standard solution (10 ppm Pb) R.
hydrochloric add and dilute to 100.0 m L with water R. Z in c
Reference solution (b) Suspend 4.0 mg o f acetylcysteine CRS in Maximum 10 ppm.
1 m L of 1 M hydrochloric add and dilute to 100.0 m l. with Atomic absorption spectrometry (2.2.23, Method II).
water R.
Test solution Dissolve 1.00 g in 0.001 M hydrochloric add and
Column: dilute to 50.0 m L with the same ad d .
— sizer. I = 0.25 m, 0 = 4 mm;
Reference solutions Prepare the reference solutions using zinc
— stationary phase: octadecylsifyl silica gel for chromatography R
(5 pm).
standard solution (5 mglmL Zn) R, diluting with 0.001 M
hydrochloric add.
Mobile phase Stir 3 volumes of acetonitrüe R and 97 volumes
Source Zinc hollow-cathode lamp.
o f water R in a beaker; adjust to pH 3.0 with phosphoric
add R. Wavelength 213.8 nm .
Flow rate 1.0 mL/min. Atomisation device Air-acetylene flame.
Detection Spectrophotom eter at 220 nm . U se a correction procedure for non-specific absorption.
2016 Acetyldigoxin 1-61

Loss o n d ry in g (2.2.52)
Acetyldigoxin ★■k+ic★
M aximum 1.0 per cent, determined on 1.000 g by drying in ★ ★
★ ★
an oven in vacuo at 70 °C for 3 h. fl}~Acetyldigoxin, Ph Eur monograph 2168)
S u lfated a s h (2.4.14)
Maximum 0.2 per cent, determined on 1.0 g.
ASSAY
Dissolve 0.140 g in 60 m L o f water R and add 10 m L of
dilute hydrochloric acid R. After cooling in iced water, add
10 m L of potassium iodide solution R and titrate with 0.05 M
iodine, using 1 m L of starch solution R as indicator.
1 m L of 0.05 M iodine is equivalent to 16.32 m g of
C5H 9 N 0 3S.
STORAGE
Protected from light.
IMPURITIES
Specified impurities A, B, C, D

H NH,
HOjC
COjH
H NH2 C43H660 15 823 5355-48-6

A. 3,3 '-disulfenediylbis [(2J?)-2-aminopropanoic acid] Action and use

(L-cystine), Cardiac Glycoside.

PhEur_______________
H NH,
DEFINITION
co 2h
3 P- [(4- O-Acetyl-2,6-dideoxy-P-D-n&o-hexopyranosyl- (1 -> 4)-
2,6-dideoxy-P-D-nZ>o-hexopyranosyl-(l->4)-2,6-dideoxy-P-D-
B. (2i^-2-am ino-3-sulfanylpropanoic a d d (L-cysteine), n&o-hexopyranosyl)oxy]-l 2P, 14-dihydroxy-5P-card-20(22)-
enolide.
ch 3
Content
0=\ 97.0 p er cent to 102.0 p er cent (dried substance).
H NH
ho 2c CHARACTERS
> r ^ S ' Sx— X 'COjH
Appearance
H NH
W hite or alm ost white powder.
°= <
ch 3 Solubility
Practically insoluble in water, sparingly soluble in methylene
C. (2i?,2/i?)-3>3 /-disulfanediylbis[2-(acetylamino)propanoic chloride, slightly soluble in ethanol (96 per cent).
add] (N ^'-diacetyl-L-cystine), IDENTIFICATION
Infrared absorption spectrophotometry (2.2.24).
ch 3 Comparison ^-acetyldigoxin CRS.
0=< TESTS
H NH
h3C^ S p ecific o p tic a l ro ta tio n (2.2.7)
Yo COjH
+ 26.2 to + 28.2 (dried substance).
Dissolve 0.50 g in a mixture of equal volumes o f methanol R
and methylene chloride R and dilute to 25.0 m L with the same
D. (2i?)-2-(acetylamino)-3-(acetylsulfenyl)propanoic a d d m ixture o f solvents.
(2V,.S-diacetyl-L-cysteine).
Related substances
PhEur
L iquid chrom atography (2.2.29). Prepare the solutions
immediately before use.
Solvent mixture M ix equal volumes of methanol R2 and
acetomtrUe for chromatography R.
Test solution Dissolve 50.0 mg of the substance to be
examined in the solvent mixture and dilute to 100.0 m L w ith
the solvent m ixture.
Reference solution (a) Dissolve 10.0 m g o f /?-acetyldigoxin CRS
in the solvent m ixture and dilute to 20.0 m L with the solvent
mixture.
Reference solution (b) Dilute 1.0 m L of the test solution to
20.0 m L w ith th e solvent mixture. D ilute 1.0 m L of this
solution to 10.0 m L with the solvent mixture.
 1-62 Acetyldigoxin 2016

Reference solution (c) Dissolve 5 m g o f gitoxin CRS Loss o n d ry in g (2.2.32)

(impurity D ) in the solvent mixture and dilute to 100.0 m L M aximum 1.5 per cent, determ ined on 1.000 g by drying in
with the solvent mixture. T o 5.0 m L o f this solution, add an oven at 105 °C.
0.5 m L of reference solution (a) and dilute to 100.0 m L with S u lfa te d a s h (2.4.14)
the solvent mixture. Maximum 0.1 per cent, determ ined on the residue obtained
Reference solution (d) Dissolve 5.0 m g o f (¡-acetyldigoxin for in the test for loss on drying.
peak identification CRS (containing impurities A and B) in ASSAY
10.0 m L o f the solvent mixture. Liquid chromatography (2.2.29) as described in the test for
Column: related substances with the following modification.
— sizer. I = 0.125 m , 0 = 4.0 mm; Injection T est solution and reference solution (a).
— stationary phase: octadecylsQyl silica gelfor chromatography R Calculate the percentage content o f C 43H 660 15 from the
(4 nm).
declared content of fi-acetyldigoxin CRS.
Mobile phase:
STO RA G E
— mobile phase A: water for chromatography R',
— mobile phase B: acetonitrUe for chromatography R\ Protected from light.
IM P U R IT IE S
Specified impurities A, B, D.
Time Mobile phase A Mobile phase B
(min) (per cent V/V) (percent V/V) Other detectable impurities (the following substances would, if
0 - 10 70 30 present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general
1 0 -2 0 70->35 30-» 65
acceptance criterion for other/unspecified impurities. It is
20 - 20.1 35-» 70 65-» 30 therefore not necessary to identify these impurities for
20.1 - 25 70 30 demonstration o f compliance. See also 5.10. Control of
impurities in substances for pharmaceutical use): C, E, F, G, H.

Flow rate 1.5 mlVmin.

Detection Spectrophotom eter at 2 2 5 nm .
Injection 10 pL o f 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 W ith reference to ^-acetyldigoxin (retention
time = about 9 min): im purity B = about 0.3;
impurity A = about 0 .7 ; impurity D = about 1.2.
System suitability: reference solution (c):
— resolution: minim um 1.5 between the peaks due to
P-acetyldigoxin and impurity D;
— symmetry factor, maximum 2 .5 for the peak due to
^-acetyldigoxin.
CH3
Limits:
— impurities A , B: for each impurity, not more than the area A. 3|3-[(3-O-acetyl-2,6-dideoxy-p-D-rt&0-hexopyranosyl-
o f the principal peak in the chromatogram obtained with (1 -> 4)-2, 6-dideoxy-P-D-ribo-hexopyranosyl- (1 -*•4)-2, 6-
reference solution (b) (0 .5 per cent); dideoxy-P-D-n&o-hexopyranosyl)oxy]-12(3,14-dihydroxy-5 P-
— impurity D: not m ore than 0 .6 times the area of the card-20 (22)-enolide (a-acetyldigoxin),
principal peak in the chromatogram obtained with
reference solution (b) (0 .3 per cent);
— any other impurity: for each impurity, not m ore than
0 .4 times the area o f the principal peak in the
chromatogram obtained with reference solution (b)
(0.2 per cent);
— sum of impurities other than A , B and D~. not m ore than
1 .2 times the area of the principal peak in the
chromatogram obtained with reference solution (b)
(0.6 per cent);
— total: not m ore than 3 times the area o f the principal peak
in the chromatogram obtained with reference solution (b)
(1 .5 per cent);
— disregard limit. 0.1 times the area o f the principal peak in
the chromatogram obtained with reference solution (b)
(0 .0 5 per cent).
T he thresholds indicated under Related substances B. 3 P- [(2, 6-dideoxy-P-D-n&o-hexopyranosyl-( 1-*-4)-2,6-
(Table 2 0 3 4 .-1 ) in the general m onograph Substances for dideoxy-P-D-n&o-hexopyranosyi-( 1->4)-2, 6-dideoxy-P-D-nJo-
pharmaceutical use (2034) do n o t apply. hexopyranosyl)oxy]-12j},14-dihydroxy-5(3-card-20(22)-enolide
(digoxin),
2016 Acetyldigoxin 1-63

C. 3ß, 12ßjl4-trihydroxy-5ß-card-20(22)-enolide
(digoxigenin),

CH3

F . 3 ß- [(3,4-0-diacetyl-2,6-dideoxy-ß-i>nk>-hexopyranosyl-
( 1-^4)-2,6-dideoxy-ß-D-rÄö-hexopyranosyl-(l ->4)-2j6-
dideoxy-ß-D-rÄo-hexopyranosyl) oxy] -12 ßj 14-dihydroxy-5 ß-
card-20 (22)-enolide (diacetyldigoxin),

D . 3ß-[(2j6-dideoxy-ß-D-rÄo-hexopyranosyl-(l^4)-2j6-
dideoxy-ß-D-rÄo-hexopyranosyl-(l-^4)-2j6-dideoxy-ß-D-rÄo-
hexopyranosyl)oxy]-14jl6ß-dihydroxy-5ß-card-20(22)-enolide
(gitoxin),

ch3

G . 3 ß- [(3-0-acetyl-2,6-dideoxy-ß-i>nZ>o-hexopyranosyl-
(1 -+4)-2j6-*dideoxy-ß-D-r&o-hexopyranosyl-(l -+4)-2,6-
dideoxy-ß-D-rÄo-hexopyranosyl) oxy] -14-hydroxy-5 ß-caid-
20 (22)-enolide (a-acetyldigitoxin)j

E. 3ß- [(2,6-dideoxy-ß-i>rjfcö-hexopyranosyl-(l -+4)-2,6-

dideoxy-ß-D-r&o-hexopyranosyl-(l-+4)-2j6-dideoxy-ß-D-ri&o-
hexopyranosyl)oxy]- 14-hydroxy-5 ß-card-20(22)-enolide
(digitoxin).

H . 3ß-[(4-0-acetyl-2j6-dideoxy-ß-i>nZ>o-hexopyranosyl-
(1 ^4)-2,6-dideoxy-ß-D-rÄo-hexopyranosyl-(l -+4)-2,6-
dideoxy-ß-D-r&o-hexopyranosyl) oxy] -14-hydroxy-5 ß-card-
20(22)-enolide (ß-acetyldigitoxm).
___________________ :______________________________________ PhEur
 1-64 Acetyltryptophan 2016

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

Acetyltryptophan ***** for substances hydrolysable only with difficulty.
(N-Acetyltryptophan, Ph Eur monograph 1383) * TESTS
A p p e a ra n c e o f so lu tio n
o T he solution is clear (2.2. T) and n o t m ore intensely coloured
CH and enanttomer. than reference solution or GY 7 (2.2.2, Method II).
COjH Dissolve 1.0 g in a 40 g/L solution o f sodium hydroxide R and
dilute to 100 m L with the same alkaline solution.
O p tic a l ro ta tio n (2.2.7)
C 13H 14N203 246.3 87-32-1
- 0 . 1° to + 0 . 1°.
PhEtr___________________________________________________________
Dissolve 2.50 g in a 40 g/L solution o f sodium hydroxide R
D E F IN IT IO N and dilute to 25.0 m L with the same alkaline solution.
(i?5)-2-Acetylamino-3-( 1#-indol-3-yl)propanoic add. R e la te d su b s ta n c e s
C o n te n t Liquid chromatography (2.2.29). Prepare the test and reference
99.0 p er cent to 101.0 per cent (dried substance). solutions immediately before use.
P R O D U C T IO N Buffer solution pH 2.3 Dissolve 3.90 g o f sodium dihydrogen
T ryptophan used for the production o f N-acetyltiyptophan
phosphate R in 1000 m L o f water R. A dd about 700 m L o f a
2.9 g/L solution o f phosphoric add R and adjust to p H 2.3
complies with the test for impurity A and other related
with the same a d d solution.
substances in the monograph on Tryptophan (1272).
Solvent mixture acetonitrile R, water R (10:90 VIV).
CHARACTERS
Test solution Dissolve 0.10 g o f the substance to be examined
A p p e a ra n c e
in a mixture o f 50 volumes o f acetonitrile R and 50 volumes
W hite or almost white, crystalline powder, or colourless
o f water R and dilute to 20.0 m L with the same mixture of
crystals.
solvents.
S o lu b ility
Reference solution (a) Dilute 1.0 m L o f the test solution to
Slightly soluble in water, very soluble in ethanol
100.0 m L w ith the solvent mixture.
(96 p er cent). It dissolves in dilute solutions of alkali
hydroxides. Reference solution (b) Dilute 4.0 m L o f reference solution (a)
to 100.0 m L with the solvent mixture.
mp
Reference solution (c) Dissolve the contents o f a vial o f 1,1'-
A bout 205 °C.
ethyUdenebisttyptophan CRS in 1 m L o f reference solution (b).
ID E N T IF IC A T IO N Column:
First ideritification A , B. — sizer. I = 0.25 m , 0 = 4.6 mm;
Second identification A , C, D, E. — stationary phase: octadecylsUyl silica gel for chromatography R
A Optical rotation (see Tests). (5 nm);
— temperature: 40 °C.
B. Infrared absorption spectrophotom etry (2.2.24).
Mobile phase:
Comparison N-acetyltryptophan CRS.
— mobile phase A: acetonitrile R, buffer solution p H 2.3
C. Thin-layer chromatography (2.2.27). (115:885 VIV);
Test solution Dissolve 50 mg o f the substance to be examined — mobile phase B: acetonitrile R, buffer solution p H 2.3
in 0.2 m L of concentrated ammonia R and dilute to 10 m L (350:650 VIV)’,
with footer R.
Reference solution (a) Dissolve 50 mg o f N- Mobile phase A Mobile phase B
Time
acetyltryptophan CRS in 0.2 m L o f concentrated ammonia R (min) (percent V/V) (per cent V/V)
and dilute to 10 m L with water R. 0 -1 0 100 0
Reference solution (b) Dissolve 10 m g o f tryptophan R in the 0+100
10-45 100 -> 0
test solution and dilute to 2 m l. with the test solution.
4 5 -6 5 0 100
Plate TLC silica gel F 254 plate R.
Mobile phase glacial acetic add R, water R, butanol R
(25:25:40 VIVJV). Flow rate 0.7 mL/min.
Application 2 |iL. Detection Spectrophotom eter at 220 nm .
Development Over a path of 10 cm. Injection 20 p L o f the test solution and reference solutions (a)
Drying In an oven at 100-105 °C for 15 min. and (c).
Detection Examine in ultraviolet light at 254 nm. Retention time N-acetyltryptophan = about 29 min;
System suitability, reference solution (b): 1,1 '-ethylidenebis(tryptophan) = about 34 min.
— the chrom atogram shows 2 clearly separated spots. System suitability, reference solution (c):
Results T he principal spot in the chrom atogram obtained with — resolution: minimum 8.0 between the peaks due to
the test solution is similar in position and size to the prindpal N -acetyltryptophan and 1,1 '-ethylidenebis(tryptophan);
spot in the chrom atogram obtained w ith reference if necessary, adjust the time program m e for die elution
solution (a). gradient (an increase in the duration o f elution with
mobile phase A produces longer retention times and a
D . Dissolve about 2 m g in 2 m L o f water R Add 2 m L o f
b etter resolution);
dimethylaminobenzcddekyde solution R 6 . H eat on a water-bath.
A blue or greenish-blue colour develops.
2016 Acetyltryptophan 1-65

— symmetry factor, maximum 3.5 for the peak due to

ljl'-ethylidenebistcyptophan in the chromatogram
obtained with reference solution (c).
Limits:
— impurities A , B, C, D, E, F, G, H, I, J, K, L: for each
impurity, n o t more than 0.25 times the area o f the C. R = H : (S)-2-amino-4-(2-aminophenyI)-4-oxobutanoic
principal peak in the chromatogram obtained with a d d (kynurenine),
reference solution (a) (0.25 per cent); E. R = C H O : (5)- 2-amino-4-[2- (formylamino)phenyl]-4-
— total: not m ore than 0.5 times the area of the principal oxobutanoic a d d (AT-fonnylkynurenine) 3
peak in the chromatogram obtained with reference
solution (a) (0.5 per cent);
— disregard limit: 0.01 times the area of the principal peak
the chrom atogram obtained with reference solution (a)
(0.01 per cent).
A m m o n iu m (2.4.1, Method B) HO
Maximum 200 ppm , determined on 0.10 g.
Prepare the standard using 0.2 m L o f ammonium standard D . (S)- 2 -amino-3-(5-hydroxy - 1H-indol-3-yl)propanoic a d d
solution ( 1 0 0 ppm N H J R. (5-hydroxytryptophan),
Ir o n (2.4.9)
M aximum 10 ppm . h Hv Nh2
Dissolve 1.0 g in 50 m L o f hydrochloric acid R1, with heating
at 50 °C. Allow to cool. In a separating funnel, shake with
3 quantities, each of 10 m L, of methyl isobutyl ketone R1,
shaking for 3 min each time. T o the combined organic layers
add 10 m L o f water R and shake for 3 min. Examine the F . (S)-2-amino-3-(phenylamino)propanoic a d d
(3-phenylaminoalanine),
aqueous layer.
H eavy m e ta ls (2.4.8)
M aximum 10 ppm .
2.0 g complies with test C. Prepare the reference solution
using 2 m L o f lead standard solution (10 ppm Pb) R.
L oss o n d ry in g (2.2.32)
M axim um 0.5 per cent, determined on 1.000 g by drying in G. 0S)-2-amino-3-(2-hydroxy-lH-indol-3-yl)propanoic a d d
an oven at 105 °C. (2-hydroxytryptophan),
S u lfa te d a sh (2.4.14)
M aximum 0.1 per cent, determined on 1.0 g. R
A SSAY
Dissolve 0.200 g in 5 m L o f methanol R. Add 50 m L of
anhydrous ethanol R Titrate with 0.1 M sodium hydroxide,
determining the end-point potentiometrically (2 .2 . 2 0 ).
1 m L of 0.1 M sodium hydroxide is equivalent to 24.63 m g of
H . R = H : (3i?5)-l,2,3,4-tetrahydro-9/f-P-carboline-3-
C 13H14N20 3 .
carboxylic ad d ,
STORA GE I. R = C H 3: 1-m ethyl-1,2,3,4-tetrahydro-9/f-P-carboline-3-
Protected from light. carboxylic ad d ,
IM P U R IT IE S
Specified impurities A, B, C , D , E, F, G, H , I, J, K , L

c o 2h

A. (S)-2-amino-3-(lH-indol-3-yl)propanoic acid
(tryptophan), J. R = C H O H -C H 2-O H: (5)-2-amino-3-[2- [2,3-dihydroxy-1-
(l/f-indol-3-yl) propyl] - lH-indol-3-yl]propanoic add,
K. R = H : (5)-2-amino-3-[2-(1 //-indol-3-ylmethyl)-1H-
and epimer at C*
indol-3-yl] propanoic ad d .
CO2H

B. (S)-2-amino-3-[(3i?S)-3-hydroxy-2-oxo-2,3-dihydro-li/-
indol-3-yl]propanoic a d d (dioxyindolylalanine),
 1-66 Acetyltyrosine 2016

spot in the chrom atogram obtained with th e reference

solution.
D. Solution S (see Tests) is strongly a d d (2.2.4).
TESTS
S o lu tio n S
Dissolve 2.50 g in water R and dilute to 100.0 m L with the
same solvent.
A p p e a ra n c e o f so lu tio n
L. 1-(lH-indol-3-yimet±iyl)-li2j3,4-tetxahydro-9f/-ß- Solution S is d e a r (2.2.1) and colourless (2.2.2, Method II).
carboline-3-carboxylic ad d . S p ecific o p tic a l r o ta tio n (2.2.7)
___________________________________________________________PhEur + 46 to + 49 (dried substance).
Dilute 10.0 m L o f solution S to 25.0 m L with water R.
R e la te d su b s ta n c e s
Liquid chromatography (2.2.29). Cany out the test protected
from light.
Acetyltyrosine ? **%
** * Test solution Dissolve 50.0 m g of die substance to be
(N-Acetyltyrosine, Ph Eier monograph 1384) ** examined in mobile phase A and dilute to 50.0 m L with
mobile phase A.
Reference solution (a) Dilute 1.0 m L o f the test solution to
100.0 m L with mobile phase A. D ilute 1.0 m l. o f this
solution to 10.0 m L with mobile phase A.
Reference solution (b) Dissolve 20.0 m g o f tyrosine CRS
(impurity A) in 2 m L of a 40 g/L solution o f sodium
Cn H 13N 0 4 223.2 537-55-3 hydroxide R and dilute to 20.0 m L w ith water R. Dilute
1.0 m l. o f this solution to 10.0 m L with water R.
PhEir ___________________________________________________________
Reference solution (c) Dilute 1.0 m L o f reference solution (b)
D E F IN IT IO N to 10.0 m L with mobile phase A.
(2S)-2-(Acetylamino)-3-(4-hydroxyphenyI)propanoic ad d .
Reference solution (d) Dilute 1.0 m L o f reference solution (b)
C o n te n t to 20.0 m L with the test solution.
98.5 p er cent to 101.0 p er cent (dried substance). Column:
CHARACTERS — size. I = 0.15 m , 0 = 3 mm;
A p p e a ra n c e — stationary phase, spherical octadecylsUyl silica gel for
W hite or almost white, crystalline powder or colourless chromatography R (3 pm);
crystals. — temperature. 40 °C.
S o lu b ility
Mobile phase:
Fredy soluble in water, practically insoluble in cyclohexane. — mobile phase A: mix 1.0 m L of phosphoric add R and
1000 m L of water for chromatography R;
ID E N T IF IC A T IO N — mobile phase B: acetonitrile Rl;
First ideruification A , B
Second identification A , C, D Time Mobile phase A Mobile phase B
A Specific optical rotation (see Tests). (min) (per cent V/V) (per cent V/V)
B. Infrared absorption spectrophotom etry (2.2.24). 0 -2 97 3
Comparison N-acetyltyrosine CRS. 2 - 15 97 -» 62 3 -» 38
C. Thin-layer chrom atography (2.2.27).
Test solution Dissolve 80 mg of the substance to be examined
Flow rate 0.7 mL/min.
in a mixture o f 3 volumes of glacial acetic acid R, 3 volumes
of water R and 94 volumes o f anhydrous ethanol R, and dilute Detection Spectrophotom eter at 219 nm .
to 10 m L with the same mixture of solvents. Iiyection 2 [iL o f the test solution and reference solutions (a),
Reference solution Dissolve 80 m g o f N-acetyltyrosine CRS in a (c) and (d).
m ixture of 3 volumes o f glacial acetic acid R, 3 volumes of Relative retention W ith reference to N-acetyltyrosine (retention
water R and 94 volumes o f anhydrous ethanol R , and dilute to time = about 6 min): impurity A = about 0.5.
10 m L with the same mixture o f solvents. System suitability Reference solution (d):
Plate TLC silica gel F 254 plate R. — resolution: m inim um 5.0 between the principal peak and
the peak due to im purity A.
Mobile phase water R, glacial acetic add R} ethyl acetate R
(10:15:75 V/VIV). Limits:
— impurity A: n o t m ore than 0.8 times the area o f the
Application 5 [iL.
corresponding peak in the chrom atogram obtained with
Development Over 2/3 o f the plate. reference solution (c) ( 0.8 per cent);
Drying In air. — unspecified impurities: for each impurity, n o t m ore than the
Detection Examine in ultraviolet light at 254 nm. area o f the principal peak in the chrom atogram obtained
Results T he p rin d p al spot in the chrom atogram obtained with with reference solution (a) (0.10 per cent);
the test solution is similar in position and size to. the p rindpal — total: m axim um 1.0 p er cent;
2016 Aciclovir 1-67

— disregard, limit. 0.5 times the area of the principal peak in

the chromatogram obtained with reference solution (a)
(0.05 per cent).
COjH
C h lo rid e s (2.4.4)
M aximum 200 ppm .
B. (25)-2-(acetylamino)-3-[4-(acetoxy)phenyl]propanoic a d d
Dilute 10 m L o f solution S to 15 m L with water R.
(diacetyltyrosine).
S u lfates C2.4.13)
PhEur
M axim um 200 ppm .
Dissolve 1.0 g in distilled water R and dilute to 20 m L with
the same solvent.
A m m o n iu m (2.4.1, Method B)
Aciclovir ★+** ★
Maximum 200 ppm , determined on 0.100 g. ★ ★
Prepare the standard using 0.2 m L of ammonium standard (Ph Eur monograph 0968) *****
solution (100 ppm N H J R.
Ir o n (2.4.9)

"¿0
M axim um 20 ppm .
OH
In a separating funnel, dissolve 0.5 g in 10 m L of dilute
h 2n
hydrochloric add R. Shake with 3 quantities, each o f 10 m L,
of methyl isobutyl ketone R l, shaking for 3 m in each time.
T o the combined organic layers add 10 m L o f water R and
C 8H 11N 5O 3 225.2 592 7 7 -8 9 -3
shake for 3 m in. T h e aqueous layer complies with the test.
H eav y m e ta ls (2.4.8) A c tio n a n d u se
Maximum 10 ppm . Purine nucleoside analogue; antiviral (herpesviruses).
Dissolve 2.0 g in water R and dilute to 20 m L with the same P re p a r a tio n s
solvent. 12 m L o f the solution complies with test A. Prepare Aciclovir C ream
the reference solution using lead standard solution Aciclovir Eye O intm ent
(1 ppm Pb) R.
Acidovir Infusion
L oss o n d ry in g (2.2.32)
A d d o v ir Oral Suspension
M aximum 0.5 p er cent, determined on 1.000 g by drying in
an oven at 105 °C. Adclovir Tablets
Dispersible A dd o v ir Tablets
S u lfa te d a sh (2.4.14)
M aximum 0.1 per cent, determined on 1.0 g. P h E u r __________________________________________________________________________________________
B a c te ria l e n d o to x in s (2.6.14)
D E F IN IT IO N
Less than 25 IU /g, if intended for use in the manufacture of
2-Amino-9- [(2-hydroxyethoxy) methyl] -1,9-dihydro-6//-purin-
parenteral preparations without a further appropriate
6-one.
procedure for the removal of bacterial endotoxins.
C o n te n t
A SSAY 98.5 per cent to 101.0 per cent (anhydrous substance).
Dissolve 0.180 g in 50 m L o f carbon dioxide-free water R.
T itrate with 0.1 M sodium hydroxide, determining the CHARACTERS
end-point potentiometrically (2 . 2 . 2 0 ). A p p e a ra n c e ^
W hite or almost white, crystalline powder.
1 m L of 0.1 M sodium hydroxide is equivalent to 22.32 m g of
C n H 13N 0 4. S o lu b ility
Slightly soluble in water, very slightly soluble in ethanol
STORA GE
(96 per cent), practically insoluble in heptane. It dissolves in
Protected from light. If the substance is sterile, store in a dilute solutions o f mineral adds and alkali hydroxides.
sterile, airtight, tam per-proof container.
ID E N T IF IC A T IO N
IM P U R IT IE S
Infrared absorption spectrophotometry (2.2.24).
Specified impurities A
Comparison aciclovir CRS.
Other detectable impurities (the following substances would, if
present at a sufficient level, be detected by one or other of TESTS
the tests in the monograph. They are limited by the general A p p e a ra n c e o f so lu tio n
acceptance criterion for other/unspecified impurities and/or T he solution is clear (2.2.1) and n o t more intensely coloured
by the general monograph Substances for pharmaceutical use than reference solution Y7 (2.2.2, Method IT).
(2034). It is therefore not necessary to identify these Dissolve 0.25 g in a 4 g/L solution of sodium hydroxide R and
impurities for demonstration o f compliance. See also 5.10. dilute to 25 m L with the same solvent.
Control of impurities in substancesfor pharmaceutical use): B. . R e la te d su b sta n c e s
Liquid chromatography (2.2.29). Prepare the solutions
immediately before use.
Solvent mixture dimethyl sulfoxide R, water R (20:80 VIV).
Phosphate buffer solution pH 2.5 Dissolve 3.48 g of dipotassium
A. (26)-2-amino-3-(4-hydroxyphenyl)propanoic acid hydrogen phosphate R in 1000 m L of water R and adjust to
(tyrosine), p H 2.5 with phosphoric add R.
 1-68 Aciclovir 2016

Phosphate btcffer solution pH 3.1 Dissolve 3.48 g of dipotassium Limits:

hydrogen phosphate R in 1000 m L o f water R and adjust to — correction factor, for the calculation o f content, multiply the
p H 3.1 with phosphoric acid R. peak area o f im purity I by 1.5;
Test solution Dissolve 25 m g o f the substance to be examined — impurity B: not more than 7 times the area o f the
in 5.0 mT- o f dimethyl sulfoxide R and dilute to 25.0 m L with principal peak in the chrom atogram obtained with
water R. reference solution (b) (0.7 per cent);
— sum of impurities O and Q: n o t m ore than 3 times the area
Reference solution (a) Dissolve 5 mg o f addovir for system
o f the principal peak in the chrom atogram obtained with
suitability CRS (containing impurities A, B, J, K , N , O and
reference solution (b) (0.3 per cent);
P) in 1 m L o f dimethyl sulfoxide R and dilute to 5.0 m L with
— sum cf impurities K and R: n o t m ore than twice the area of
water R.
the principal peak in the chrom atogram obtained with
Reference solution (b) Dilute 1.0 m L o f the test solution to reference solution (b) (0.2 per cent);
100.0 m L with the solvent mixture. Dilute 1.0 mT. o f this — impurities A , G, J, N, F. for each impurity, n o t m ore than
solution to 10.0 m L with the solvent mixture. twice the area o f the principal peak in the chromatogram
Reference solution (c) Dissolve the contents of a vial o f aciclovir obtained with reference solution (b) (0.2 per cent);
for peak identification 1 CRS (containing impurities C and I) — impurities C, F, I: for each impurity, not m ore than the
in 200 pL o f dimethyl sulfoxide R and dilute to 1.0 m L with area o f the principal peak in the chrom atogram obtained
water R. with reference solution (b) ( 0.1 per cent);
Reference solution (d) Dissolve the contents o f a vial of — unspecified impurities: for each impurity, n o t m ore than
aciclovir for peak identification 2 CRS (containing impurities F 0.5 times the area o f the principal peak in the
and G ) in 1.0 m L of reference solution (a). chrom atogram obtained with reference solution (b)
Column’. (0.05 per cent);
— size: I = 0.25 m , 0 = 4.6 mm; — total: n o t m ore than 15 times the area o f the principal
— stationary phase: end-capped octadecylsüyl silica gel for peak in the chromatogram obtained with reference
chromatography R (5 pm). solution (b) (1.5 per cent);
Mobile phase: — disregard limit. 0.3 times the area o f the p rin d p al peak in
— mobile phase A: acetonitrile R, phosphate buffer solution the chrom atogram obtained with reference solution (b)
p H 3.1 (1:99 VIV); (0.03 per cent).
— mobile phase B: acetonitrile R, phosphate buffer solution W a te r (2.5.12)
p H 2.5 (50:50 VIV)’, M aximum 6.0 per cent, determ ined on 0.500 g.
S u lfa te d a s h (2.4.14)
Time Mobile phase A Mobile phase B M aximum 0.1 per cent, determ ined on 1.0 g.
(min) (per cent V/V) (per cent V/V) B a c te ria l e n d o to x in s (2.6.14, Method D)
0-5 100 0 Less than 0.50 IU/m g, if intended for use in th e manufacture
5 - 27 100 -» 80 0 -» 20 o f parenteral preparations without a further appropriate
procedure for the removal o f bacterial endotoxins.
27 - 40 80 20
A SSA Y
Dissolve 0.150 g in 60 m L o f anhydrous acetic add R. T itrate
Flow rate 1.0 mL/min. with 0.1 M perchloric add, determining the end-point
Detection Spectrophotom eter at 254 nm . potentiometrically (2.2.20). C an y out a blank titration.
Injection 10 pL o f the test solution and reference 1 m L o f 0.1 Mperchloric add is equivalent to 22.52 m g
solutions (b), (c) and (d). 0f C 8H u N 5O 3.
Identification of impurities Use the chrom atogram supplied IM P U R IT IE S
with aciclovir for peak identification 1 CRS and the Specified impurities A, B, C, F, G , I, J, K, N , O , P, Q, R
chrom atogram obtained with reference solution (c) to identify Other detectable impurities (the following substances would, if
the peaks due to impurities C and I; use the chrom atogram
present at a suffirient levd, be detected by one or other o f
supplied with addovir for peak identification 2 CRS and the
the tests in the monograph. T hey are limited by the general
chrom atogram obtained with reference solution (d) to
acceptance criterion for other/unspecified impurities and/or
identify the peaks due to impurities A, B, F, G, J, K, N , O
by the general monograph Substances for pharm aceutical use
and P.
(2034). It is therefore not necessary to identify these
Relative retention W ith reference to aciclovir (retention impurities for demonstration o f compliance. See also 5.10.
time = about 13 min): impurity B = about 0.4; Control of impurities in substances for pharmaceutical use): L, M.
im purity P = about 0.7; im purity C = about 0.9;
im purity N = about 1.37; impurities O and Q = about 1.42; O
im purity I = about 1.57; im purity J = about 1.62;
N O
im purity F = about 1.7; im purity A = about 1.8;
impurities K and R = about 2.5; impurity G = about 2.6. > p
N / -' CH3
System suitability: ^—o
— resolution: minimum 1.5 between the peaks due to
im purity C and aciclovir in the chrom atogram obtained A. 2 -[( 2 -am ino- 6-oxo- l,6-dihydro-9/i-purin-9-
with reference solution (c); m inim um 1.5 between the yl)methoxy] ethyl acetate,
peaks due to impurities F and A and minimum 1.5
between the peaks due to impurities K and G in the
chrom atogram obtained with reference solution (d).
2016 Aciclovir 1-69

H2N
i V>
h N N
1 aV>
h3c ^ n^ n^ n

B. 2-amincHl ,7-dihydro-6i7-purin-6-one (guanine),

h y o
ch 3

L. N - (9-acetyl-6-oxo-6,9-dihydro-1.ff-purin-2-yl) acetamide
o -O OH (2^9-diacetylguanine),

1 1 / VJ
H2N N N 0
/^°x P ~ich3
C. 2-am ino-7- [(2-hydroxyethoxy)methyl]-l ,7-dihydro-6/i-
purin-6-onej
h3c^X ^JL^JL^
n n n
H

o
M . 2- [ [2-(acetylamino)-6-oxo-1,6-dihydro-7H-puiin-7-
yl]methoxy] ethyl acetate,

h3c ^ X a JCn> ;
" nA
H
^ n
\ — 0'
N . unknown structure,
O. unknown structure,

F. N-[9-[(2-hydroxyethoxy)methyl]-6-oxo-6,9-dihydro-l/i- 0

HNX .
purin-2-yl] acetamide,

o h2n ^ lX > n^ n^ ^ oh
O
i iY> ^
/

h3c ^ n ^ n ^ - n j—f ( 3
ch
P. 2-amino-9-(2-hydroxyethyl)-1,9-dihydro-6/f-purin-6-onej
H \— o
0

G. 2- [ [2-(acetylamino)-6-oxo-1,6-dihydro-9H-purin-9-
yl]methoxy] ethyl acetate.
x YN > N OH
V

HN JLn O
+

i xN >
H2N
x V>
h -O
/ \
OH
N NH2

I. 2-am ino-7-[ [2- [(2-amino-6-oxo-1,6-dihydro9H -purin-9- Q. mixture o f 2-amino-9-[[2-

yl)methoxy]ethoxy]methyl]-l,7-dihydro-6i/-purin-6-one, (hydroxymethoxy) ethoxy] methyl] -1 j9-dihydro-6//-purin-6-
one and 2-amino-9-[[2-(hydroxyethoxy)methoxy]methyl]-l,9-
dihydro-6//-purin-6-one,

H2N
a N
I> N / -----\
ccSl
N N NH2
'—O O—’

J. 9 ,9 '- [ethylenebis(oxymethylene)]bis(2-amino-l,9-dihydro-
6//-purin-6-one),

R. 9 ,9 '-
[methylenebis(oxyethyleneoxymethylene)]bis(2-amino-l,9-
dihydro-6//-purin-6-one).
PhEur

K. 2,2'-(methylenediimino)bis[9-[(2-hydroxyethoxy)methyl]-
1,9-dihydro-6/i-purin-6-one],
 1-70 Acitretin 2016

TESTS
Acitretin ******
Related substances
(Ph Eur monograph 1385) * Liquid chrom atography (2.2.29). Maintain the sampler at
4 °C.
Test solution (a) Dissolve 2 5 .0 m g o f the substance to be
examined in 5 m L of tetrahydrofuran R and dilute
immediately to 1 0 0 .0 m L with anhydrous ethanol R.
Test solution (b) Dilute 1 0 .0 m L o f test solution (a) to
2 5 .0 m l. with anhydrous ethanol R.
Reference solution (a) Dissolve 2 5 .0 m g o f acitretin CRS in
Cj i H ^ O s 326.4 55079-83-9 5 m L of tetrahydrofuran R and dilute immediately to
100.0 m L with anhydrous ethanol R. D ilute 10.0 m L o f this
A ctio n a n d u se solution to 2 5 .0 m l. with anhydrous ethanol R
Vitamin A analogue (retinoid); treatm ent o f psoriasis; Reference solution (b) Dissolve 1 .0 m g of tretinoin CRS in
ichthyosis; D arier’s disease. anhydrous ethanol R and dilute to 2 0 .0 m L with the same
P re p a r a tio n solvent. M ix 5 .0 m L o f this solution with 2 .5 m L o f
A dtretin Capsules reference solution (a) and dilute to 1 0 0 .0 m L with anhydrous
ethanol R
PhEtr___________________________________________________________
Reference solution (c) Dilute 2 .5 m L o f reference solution (a)
D E F IN IT IO N to 5 0 .0 m L with anhydrous ethanol R. Dilute 3 .0 m L o f this
(aU-£)-9-(4-Methoxy-2,3, 6-trimethylphenyl)-3,7 - solution to 2 0 .0 m l. with anhydrous ethanol R
dimethylnona-2j4j6j8-tetraenoic ad d . Column’.
C o n te n t — size I = 0 .2 5 m , 0 = 4 mm;
98.0 per cent to 102.0 per cent (dried substance). — stationary phase: microparticulate octadecylsQyl silica gel for
chromatography R (5 pm) with a specific surface area of
CHARACTERS 2 0 0 m 2/g, a pore size o f 15 n m and a carbon loading o f
A p p e a ra n c e 20 per cent;
Yellow or greenish-yellow, crystalline powder. — temperature: 25 °C.
S o lubility Mobile phase A 0 .3 per cent V/V solution o f glacial acetic
Practically insoluble in water, sparingly soluble in acid R in a mixture o f 8 volumes o f water R and 9 2 volumes
tetrahydrofuran, slightly soluble in acetone and in ethanol o f anhydrous ethanol R.
(96 per cent), very slightly soluble in cyclohexane. Flow rate 0 .6 mL/min.
It is sensitive to air, heat and light, espedally in solution. Detection Spectrophotom eter at 3 6 0 nm .
It shows polymorphism. Injection 10 pL o f test solution (a) and reference solutions (b)
Cany ota all operations as rapidly as possible and avoid exposure and (c).
to actinic light; use freshly prepared solutions. Run time 2 .5 times the retention time o f adtretin.
ID E N T IF IC A T IO N Retention time Impurity A = about 4 .8 min; tretinoin = about
First identification B 5 .2 m in; adtretin = about 6 .2 min; impurity B = about
Second identification A , C 10.2 min.
A. Ultraviolet and visible absorption spectrophotom etry System suitability: reference solution (b):
(2.2.25). — resolution: minim um 2.0 betw een the peaks due to
Test solution Dissolve 15.0 m g in 10 m L o f tetrahydrofuran R adtretin and tretinoin; if necessary, adjust the
and dilute immediately to 100.0 m L with the same solvent. concentration o f anhydrous ethanol R
D ilute 2.5 m L o f this solution to 100.0 m L with Limits:
tetrahydrofuran R — impurities A , B: for each im purity, n o t more than the area
o f the peak due to adtretin in the chrom atogram obtained
Spectral range 300-400 nm.
w ith reference solution (c) (0 .3 p er cent);
Absorption maximum A t 358 nm . — total: n o t more than the area o f the peak due to ad tretin
Specific absorbance at the absorption maximum 1350 to 1475. in the chrom atogram obtained with reference solution (b)
B. Infrared absorption spectrophotom etry (2.2.24). (1.0 per cent);
Preparation Discs. — disregard limit. 0.1 times the area o f the principal peak in
the chrom atogram obtained with reference solution (c).
Comparison acitretin CRS.
If the spectra obtained in the solid state show differences, Palladium
dissolve die substance to be examined and the reference M axim um 10 ppm.
substance separately in 2-propanol R heating under reflux, A tomic absorption spectrometry (2.2.23, Method I).
filter, evaporate to dryness and record new spectra using the Test solution Introduce 2 .0 g into a quartz beaker and add
residues. 3 m l. o f magnesium nitrate solution R. H eat in a muffle
C. Examine the chrom atograms obtained in the assay. furnace to 3 5 0 °C at a rate o f 4 0 °C/min to incinerate the
Results T he prindpal peak in the chrom atogram obtained content. Ignite at about 4 5 0 °C for 8 h and then at
5 5 0 ± 5 0 °C for a further hour. Dissolve the residue in a
with test solution (b) is similar in retention time to the
m ixture o f 0 .7 5 m L o f hydrochloric add R and 0 .2 5 m L o f
principal peak in the chrom atogram obtained with reference
solution (a).
nitric acid R, warming gently. Cool, then transfer the solution
2016 Adapalene 1-71

into a volumetric flask contain in g water R and dilute to

50.0 m L w ith the same solvent.
Adapalene *****

Reference solution Dissolve 0.163 g o f heavy magnesium oxide R (Ph. Eur. monograph 2445) *
in a mixture o f 0.5 m L of nitric acid R, 1.5 m L o f hydrochloric
acid R and 50 m L o f water R, add 2.0 m L o f palladium
standard solution (20 ppm Pd) R and dilute to 100.0 m L with
water R.
Source Palladium hollow-cathode lamp.
Wavelength 247.6 nm.
Atomisation device Air-acetylene flame.
H eavy m e ta ls {2.4.8)
M aximum 20 ppm. C ss H a A 412.5 J 06685-40-9
2.0 g complies with test C. Prepare the reference solution A ctio n a n d u se
vising 2 m l. o f lead standard solution (10 ppm Pb) R. Vitamin A analogue (retinoid); treatm ent of acne.
L oss o n d ry in g (2.2.32) P re p a r a tio n s
M aximum 0.5 per cent, determined on 1.000 g by drying Adapalene Cream
in vacuo at 100 °C for 4 h. Adapalene Gel
S u lfated a s h (2.4.14)
M aximum 0.1 per cent, determined on 1.0 g. Ph Eur____________ _____________________________________________

ASSAY DEFINITION
Cany out the assay protected from light, use amber volumetric 6-(4-Methoxy-3-tricyclo [3.3.1.13,7] dec-1-
flasks and prepare the solutions immediately before use. ylphenyI)naphthalene-2-carboxylic acid.
Liquid chromatography (2.2.29) as described in the test for C o n te n t
related substances with the following modifications. 98.0 per cent to 102.0 per cent (dried substance).
Injection T est solution (b) and reference solution (a). CHARACTERS
System suitability: A p p e a ra n c e
— repeatability: maximum relative standard deviation of White or almost white powder.
1.0 per cent after 6 injections o f reference solution (a); Solubility
if necessary, adjust the integration parameters.
Practically insoluble in water, sparingly soluble in
Calculate the percentage content of C 21H 26O 3 from the tetrahydrofuran, practically insoluble in ethanol
declared content of acitrean CRS. (96 per cent).
STORAGE ID E N T IF IC A T IO N
In an airtight container, protected from light, at a Infrared absorption spectrophotometry (2.2.24).
tem perature o f 2 °C to 8 °C. Comparison adapalene CRS.
I t is recom mended that the contents o f an opened container
TESTS
be used as soon as possible and any unused part be protected
by an atmosphere o f inert gas. A p p e a ra n c e o f solution
T h e solution is clear (2.2.1) and not more intensely coloured
IM P U R IT IE S than reference solution BY6 (2.2.2, Method II).
Specified impurities A, B. Dissolve 0.2 g in tetrahydrofuran R and dilute to 20 m l. with
the same solvent.
ch 3 ch3 ch 3
R e la te d su b sta n c e s
Liquid chromatography (2.2.29).

h3co
Solvent mixture tetrahydrofuran R, acetomtrUe R, water R
(20:37:43 V/V/V).
ch 3
Test solution (a) Dissolve 40.0 mg o f the substance to be
examined in 10 m L o f tetrahydrofuran R, add 7 m L of the
A. (2Zj4£,6£’,8£)-9-(4-methoxy-2,3,6-trimethylphenyl)-3,7-
solvent mixture and dilute to 20.0 m L with tetrahydrofuran R.
dim ethylnona-2,4,6,8-tetraenoic add.
Test solution (b) Dissolve 20.0 m g o f the substance to be
examined in 50 m L of tetrahydrofuran R, add 35 m l. of the
solvent mixture and dilute to 100.0 m L with
tetrahydrofuran R. Dilute 5.0 m L o f the solution to 50.0 m L
with the solvent mixture.
Reference solution (a) Dilute 1.0 m L o f test solution (a) to
10.0 m L with tetrahydrofuran R. D ilute 1.0 m l. o f this
solution to 100.0 m l, with the solvent mixture.
B. ethyl (all-£)-9-(4-methoxy-2,336-trimethylphenyl)-3,7-
Reference solution (b) Dissolve 2.4 m g of adapalene
dim ethylnona-2,4,6,8-tetraenoate.
impurity C CRS in 2 m L of tetrahydrofuran R and dilute to
—-________________________________________________________ PhEur 20.0 m L with the same solvent. Dilute 2.0 m L o f the
solution to 20.0 m L with the solvent mixture. T o 2.0 m L of
this solution add 2.0 m L of reference solution (a) and dilute
to 20.0 m l. with the solvent mixture.
 1-72 Adapalene 2016

Reference solution (c) Dissolve the contents of a vial of — disregard Omit: 0.5 times the area of the principal peak in
adapalene for peak identification CRS (containing impurities A, the chrom atogram obtained w ith reference solution (a)
C and D ) in 0.5 m L o f tetrahydrqfuran R and dilute to (0.05 per cent).
1.0 m L with the solvent mixture. H eav y m e ta ls (2.4.8)
Reference solution (d) Dissolve 20.0 m g o f adapalene CRS in M axim um 20 ppm .
50 m L of tetrahydrqfuran R> add 35 m L o f the solvent 0.250 g complies with test G . Prepare the reference solution
m ixture and dilute to 100.0 m L with tetrahydrofuran R. u sin g 0.5 m L of lead standard solution (10 ppm Pb) R.
D ilute 5.0 m L of the solution to 50.0 m L with die solvent
L oss o n d ry in g (2.2.32)
mixture.
M axim um 0.5 p er cent, determ ined on 1.000 g by drying in
Column: an oven at 105 °C for 4 h.
— size. I = 0.25 m , 0 = 4.6 mm;
— stationary phase: end-capped phenylsQyl silica gel for S u lfa te d a s h ( 2.4.14)
chromatography R (5 tun) with a carbon loading of M axim um 0.1 per cent, determ ined on 1.0 g.
7.5 per cent; A SSA Y
— temperature: 30 °C. Liquid chromatography (2.2.29) as described in the test for
Mobile phase: related substances w ith the following modification.
— mobile phase A: glacial acetic add R, water R (0.1:100 VIV)\ Irtjection T est solution (b) and reference solution (d).
— mobile phase B: tetrahydrofuran R, acetomtrUe R
Calculate the percentage content o f adapalene from the
(35:65 VIV)\
declared content o f adapalene CRS.
IM P U R IT IE S
Time Mobile phase A Mobile phase B
(min)
Specified impurities A, C , D
(percent V/V) (per cent V/V)
0 -2 .5 50 50 Other detectable impurities (the following substances would, if
present at a sufficient level, be detected by one or other o f
23 - 40 50-» 28 50*72
the tests in the m onograph. T hey are limited by die general
40 - 42 28 72 acceptance criterion for other/unspecified impurities and/or
by the general m onograph Substances for pharmaceutical use
(2034). It is therefore n o t necessary to identify these
Flow rate 1 .2 mL/min. impurities for dem onstration o f compliance. See also 5.10.
Detection Spectrophotom eter at 2 7 0 nm . Control of impurities in substances for pharmaceutical use): B.
Injection 2 5 |iL of test solution (a) and reference solutions (a),
(b) and (c).
Identification of impurities Use the chrom atogram supplied
with adapalene for peak identification CRS and the
chrom atogram obtained with reference solution (c) to identify
the peaks due to impurities A, C and D.
Relative retention W ith reference to adapalene (retention
tim e = about 2 0 min): impurity A = about 0 .3 ; A. 2 ,2 /-binaphthalene- 6,6 '-dicarboxylic acid,
impurity C = about 0 .9 ; im purity D = about 1.9.
System suitability: reference solution (b):
— resolution: m in im u m 4 .5 between the peaks due to
impurity C and adapalene;
— signal-to-noise ratio: minimum 1 0 for the peak due to
im purity C.
Limits: COjH
— correction factors: for the calculation of content, multiply
the peak areas o f the following impurities by the B. 6-[3-(3-hydroxytricydo[3.3.1.1 3,7]dec-l-yl)-4-
corresponding correction facto r im purity A = 0 .7 ; methoxyphenyl] naphthalene- 2-carboxylic ad d ,
impurity C = 7; impurity D = 1.4;
- — impurity A: not m ore than 3 times die area o f the
principal peak in the chrom atogram obtained with
reference solution (a) (0 .3 p er cent);
— impurity D: n o t m ore than twice die area o f the principal
peak in the chrom atogram obtained with reference
solution (a) (0.2 per cent);
C. l-(2-m ethoxyphenyi)tricydo[3.3.1. l 3,7]decane,
— impurity C: n o t m ore than 1.5 times the area o f the
principal peak in die chrom atogram obtained with
reference solution (a) (0 .1 5 p er cent);
— unspecified impurities: for each impurity, n o t m ore than the
area o f the principal peak in the chrom atogram obtained
w ith reference solution (a) (0.10 per cent);
— total: n o t m ore th an 5 times the area o f the principal peak
in the chrom atogram obtained with reference solution (a) D . 1, 1 '- [4,4 '-bis(methoxy)biphenyl-3,3
(0 .5 per cent); diyl]bis(tricyclo [3.3.1.13’7] decane).
______________________________________________■ PhEut
2016 Adenine 1-73

Adenine ★★* ★★ Test solution (b) Dilute 1 m L of test solution (a) to 10 m L

★ ★ with dilute acetic add R.
(Ph Eur monograph 0800) ***** Reference solution (a) Dissolve 10 m g of adenine CRS in dilute
acetic add R, with heating if necessary, and dilute to 10 m L
nh 2
with the same ad d .
nV ^ v Reference solution (b) Dilute 1 m L o f test solution (b) to
I I /) 20 m L with dilute acetic add R.
Reference solution (c) Dissolve 10 m g of adenine CRS and
C5H5N5 135.1 73-24-5
10 mg o f adenosine R in dilute acetic add R, with heating if
necessary, and dilute to 10 mL w ith the same add.
Action and use Apply to the plate 5 jiL o f each solution. Develop over a
C onstituent of anticoagulant and preservative solutions for path of 12 cm using a mixture of 20 volumes of concentrated
-blood. ammonia R, 40 volumes o f ethyl acetate R and 40 volumes of
propanol R. Dry the plate in a current of warm air and
PhEir. examine in ultraviolet light at 254 nm. Any spot in the
DEFINITION chromatogram obtained with test solution (a), apart from the
Adenine contains not less than 98.5 p er cent and not more prindpal spot, is not m ore intense than the spot in the
than the equivalent of 101.0 per cent o f 7H-purin-6-amine, chromatogram obtained with reference solution (b)
calculated w ith reference to the dried substance. (0.5 per cent). T h e test is not valid unless the chromatogram
obtained with reference solution (c) shows two clearly
CHARACTERS separated spots.
A white or almost white powder, very slightly soluble in
Chlorides (2.4.4)
water and in alcohol. It dissolves in dilute m in eral a d d s and
T o 10 m L o f solution S add 1 m L of concentrated ammonia R
in dilute solutions of alkali hydroxides.
and 3 m L o f silver nitrate solution R2. Filter. Wash the
IDENTIFICATION predpitate with a little water R and dilute the filtrate to
First identification A. 15 m L with water R. T h e solution complies with the limit
Second identification B, C. test for chlorides (100 ppm). W hen carrying out the test, add
2 m L of dilute nitric add R instead o f 1 m L of dilute nitric
A. Examine by infrared absorption spectrophotometry
(2.2.24), comparing with the spectrum obtained with add R.
adenine CRS. Examine the substances prepared as discs. Sulfates (2.4.13)
Dilute 10 m L o f solution S to 15 m L with distilled water R.
B. Examine the chromatograms obtained in the test for
T he solution complies with the limit test for sulfates
related substances. The principal spot in the chromatogram
(300 ppm).
obtained with test solution (b) is similar in position and size
A m m on iu m
to the prindp al spot in the chromatogram obtained with
reference solution (a). Prepare a cell consisting of two watch-glasses 60 mm in
diameter placed edge to edge. T o the inner wall of the upper
C. T o 1 g add 3.5 m L of propionic anhydride R and boil for
watch-glass stick a piece o f red litmus paper R 5 mm square
15 min with stirring. Cool. T o the resulting crystalline mass
and wetted with a few drops of water R. Finely powder the
add 15 m L o f light petroleum R and heat to boiling with
substance to be examined, place 0.5 g in the lower watch-
vigorous stirring. Cool and filter. W ash the predpitate with
glass and suspend in 0.5 mL of water J?. T o the suspension
two quantities, each o f 5 mT, of light petroleum R. Dissolve
add 0.30 g of heavy magnesium oxide R. Briefly triturate with
the predpitate in 10 m L of water R and boil for 1 min. Filter
a glass rod. Immediately close the cell by putting the two
the mixture at 30 °C to 40 °C. Allow to cool. Filter, and dry
watch-glasses together. H eat at 40 °C for 15 min. T he litmus
the predpitate at 100 °C to 105 °C for 1 h. T he melting
paper is n o t more intensdy blue coloured than a standard
point (2.2.14) o f the predpitate is 237 °C to 241 °C.
prepared at the same time and in the same manner using
TESTS 0.05 m L o f ammonium standard solution (100 ppm NH 4) R,
Solution S 0.5 m L o f water R and 0.30 g of heavy magnesium oxide R
Suspend 2.5 g in 50 m L o f distilled water R and boil for (10 ppm).
3 m in. Cool and dilute to 50 m L with distilled water R. Filter. Heavy metals (2.4.8)
Use the filtrate as solution S. 1.0 g complies with test C for heavy metals (20 ppm).
Appearance o f solution Prepare the reference solution using 2 m L of lead standard
Dissolve 0.5 g in dilute hydrochloric acid R and dilute to solution (10 ppm Pb) R.
50 m L with the same ad d . The solution is clear (2.2.1) and Loss on drying (2.2.32)
colourless (2.2.2, Method II). N o t more than 0.5 per cent, determined on 1.000 g by
Acidity or alkalinity drying in an oven at 105 °C.
T o 10 m L o f solution S add 0.1 mL o f bromothymol blue Sulfated ash (2.4.14)
solution R1 and 0.2 m L of 0.01 M sodium hydroxide. N ot more than 0.1 per cent, determined on 1.0 g.
The solution is blue. Add 0.4 m L of 0.01 M hydrochloric acid. ASSAY
T he solution is yellow.
Dissolve 0.100 g in a mixture o f 20 m L of acetic anhydride R
Related substances and 30 m L of anhydrous acetic acid R. T itrate with 0.1 M
Examine by thin-layer chromatography (2.2.27), using silica perchloric add, determining the end-point potentiometrically
gel GF2 5 4 R as the coating substance. (2.2.20).
Test solution (a) Dissolve 0.10 g of the substance to be 1 m L of 0 . 1 M perchloric add is equivalent to 13.51 m g of
examined in dilute acetic acid R, with heating if necessary, and CsHsNs.
dilute to 10 m L with the same add. __________________________________________________________ PhEur
 1-74 Adenosine 2016

★ ★ Reference solution (a) Dilute 1.0 m L o f the test solution to

Adenosine ★ ★ 100.0 m L with the mobile phase. Dilute 1.0 m L o f this
(Ph. Eur. monograph 1486) ***** solution to 10.0 m L with the mobile phase.
Reference solution (b) Dissolve 5 m g o f adenine R (impurity A)
NH2 and 5 m g o f inosine R (impurity G) in the mobile phase and
dilute to 50 m L with the mobile phase. D ilute 4 m L o f this
solution to 100 m L with the mobile phase.
Column:
— size: I = 0.25 m , 0 = 4.6 mm;
— stationary phase: end-capped octadecykHyl silica gel for
chromatography R (5 (im).
OH OH
Mobile phase water R, solvent mixture (40:60 VIV).
Flow rate 1.5 mlVmin.
C 10H 13N 5O4 267.2 58-61-7
Detection Spectrophotom eter at 254 nm.
Action and use Injection 20 pL.
Antiarrhythmic. Run time 1.5 times the retention time o f adenosine.
P h E tr ____________________
Relative retention W ith reference to adenosine (retention
time = about 13 min): impurity A = about 0.3;
DEFINITION im purity G = about 0.4.
9-P-D-Ribofuranosyl-9ii-purin-6-araine. System suitability, reference solution (b):
Content — resolution: minim um 1.5 between the peaks due to
99.0 per cent to 101.0 per cent (dried substance). impurities A and G.
CHARACTERS Limits:
Appearance — correction factors: for the calculation o f content, multiply
W hite or alm ost white, crystalline powder. the peak areas o f the following impurities by the
corresponding correction facto r impurity A = 0.6;
Solubility
im purity G = 1.4;
Slightly soluble in water, soluble in h o t water, practically
— impurity A: n o t m ore than twice the area o f the prindpal
insoluble in ethanol (96 per cent) and in methylene chloride.
peak in the chromatogram obtained with reference
It dissolves in dilute mineral adds.
solution (a) ( 0.2 per cent);
mp — impurity G: n o t more than the area o f the prin d p al peak
A bout 234 °C. in the chrom atogram obtained with reference solution (a)
IDENTIFICATION (0.1 p er cent);
Infrared absorption spectrophotom etry (2.2.24). — unspecified impurities: for each impurity, no t m ore than the
area o f the prin d p al peak in the chrom atogram obtained
Comparison adenosine CRS.
with reference solution (a) (0.10 per cent);
TESTS — total: n o t m ore than 5 times the area o f the prin d p al peak
Solution S in the chrom atogram obtained with reference solution (a)
Suspend 5.0 g in 100 m L o f distilled water R and heat to (0.5 p er cent);
boiling. Allow to cool, filter with the aid o f vacuum and — disregard ltmir. 0.5 times the area of the prin d p al peak in
dilute to 100 m L w ith distilled water R. the chrom atogram obtained with reference solution (a)
Appearance o f solution (0.05 p er cent).
Solution S is colourless (2.2.2, Method II). C h lo rid e s (2.4.4)
Acidity or alkalinity M axim um 100 ppm .
T o 10 m L o f solution S, add 0.1 mT. o f bromocresolpurple D ilute 10 m L o f solution S to 15 m L with water R.
solution R and 0.1 m L o f 0.01 M hydrochloric add. S u lfa te s (2.4.13)
T he solution is yellow. Add 0.4 m L o f 0.01 M sodium M axim um 200 ppm , determined on solution S.
hydroxide. T h e solution is violet-blue.
A m m o n iu m (2.4.1, Method B)
Specific optical rotation (2.2.7) M axim um 10 ppm , determined on 0.5 g.
- 4 5 to - 4 9 (dried substance).
Prepare the standard using 5 m L o f ammonium standard
Dissolve 1.25 g in / M hydrochloric acid and dilute to solution (1 ppm NH 4 ) R.
50.0 m L with the same ad d . Examine within 10 min o f
L o ss o n d ry in g (2.2.32)
preparing the solution.
Maximum 0.5 per cent, determined on 1.000 g by drying in
Related substances an oven a t 105 °C.
Liquid chrom atography (2.2.29).
S u lfa te d a s h (2.4.14)
Solvent mixture Dissolve 6.8 g o f potassium hydrogen sulfate R M axim um 0.1 p er cent, determined on 1.0 g.
and 3.4 g o f tetrabutylammonium hydrogen sulfate R in water R,
adjust to p H 6.5 with a 60 g/L solution o f potassium A SSA Y
hydroxide R and dilute to 1000 m L with the same solvent. Dissolve 0.200 g, warming slightly if necessary, in a mixture
U se a freshly prepared solvent mixture. o f 20 m L o f acetic anhydride R and 30 m L o f anhydrous acetic
Test solution Dissolve 20 m g o f the substance to be examined add R. 1111316 w ith 0.1 M perchloric add, determ ining the
end-point potentiometrically (2 . 2 . 2 0 ).
in the mobile phase and dilute to 20 m L with the mobile
phase.
2016 Adipic A dd 1-75

1 m L of 0 . 1 M perchloric acid is equivalent to 26.72 m g ★ ★

o f C 10H 13N 5O 4.
Adipic Acid ★ ★
* .★
(Fh Eur monograph 1586) *★*
IM P U R IT IE S
Specified impitrities A, G.
Other detectable impurities (the following substances would, if HO2C'
present at a sufficient levd, be detected by one or other of
the tests in the monograph. They are limited by the general
C6H10O4 146.1 124-04-9
acceptance criterion for other/unspecified impurities and/or
by the general monograph Substances for pharmaceutical use A c tio n a n d u se
(2034). It is therefore not necessary to identify these Exdpient.
impurities for demonstration of compliance. See also 5.10.
Control of impurities in substances for pharmaceutical use): F, H. PhEur.

D E F IN IT IO N
nh2
Hexanedioic ad d .
C o n te n t
CT> N
N N
H
99.0 per cent to 101.0 per cent (dried substance).
CHARACTERS
A p p e a ra n c e
A. 7H-purin-6-amine (adenine).
W hite or alm ost white, crystalline powder.
S o lu b ility
Sparingly soluble in water, soluble in boiling water, fredy
soluble in ethanol (96 per cent) and in methanol, soluble in
acetone.
ID E N T IF IC A T IO N
A. M d tin g point {2.2.14): 151 °C to 154 °C.
B. Infrared absorption spectrophotometry {2.2.24).
OH OH
Comparison adipic acid CRS.
F. l-^i>ribofuranosylpyrim idine-2j4(l//j3/i)-dione TESTS
(uridine), S o lu tio n S
Dissolve 5.0 g with heating in distilled water R and dilute to
50 m L with the same solvent. Allow to cool and to
crystallise. Filter through a sintered-glass filter (40) {2.1.2).
HN W ash the filter with distilled water R. Collect the filtrate and
the washings until a volume of 50 m L is obtained.
HO A p p e a ra n c e o f so lu tio n
T he solution is clear {2.2.1) and colourless (2.2.2,
Method II).
Dissolve 1.0 g in methanol R and dilute to 20 m l. with the
OH OH
same solvent.

G . 9-P-D-ribofuranosyl-l,9-dihydro-6H-purin-6-one (inosine), R e la te d su b sta n c e s

Liquid chromatography (2.2.29).
0 Test solution Dissolve 0.20 g of the substance to be examined
in the mobile phase and dilute to 10.0 m L with the mobile
phase.
h2n
Reference solution (a) Dissolve 20 m g of ghitaric acid R in
ho 1.0 m L o f the test solution and dilute to 10.0 m L with the
mobile phase.

V? OH oh
Reference solution (b) Dilute 1.0 m L o f the test solution to
100.0 m L with the mobile phase, dilute 1.0 m L o f the
solution to 10.0 m L with the mobile phase.
Column:
H . 2-amino-9-P-D-ribofuranosyl-1,9-dihydro-6H-purin-6-one — size. I = 0.125 m , 0 = 4.0 mm,
(guanosine). — stationary phase, spherical octadecylstiyl silica gel for
PhEur chromatography R (5 Jim) with a specific surface area o f
350 m 2/g and a pore size of 10 nm,
— temperature: 30 °C.
Mobile phase M ix 3 volumes of acetonitrüe R and 97 volumes
o f a 24.5 g/L solution o f dilute phosphoric acid R.
Flow rate 1 m L/m in.
Detection Spectrophotometer at 209 nm.
Injection 20 (iL.
 1-76 Adrenaline 2016

Run time 3 times the retention time o f adipic ad d . IM P U R IT IE S

System suitability: reference solution (a):
— resolution: minimum 9.0 between the peaks due to glutaric HO2C
acid and adipic a d d .
Limits'. A R = C H 2- C 0 2H: pentanedioic a d d (glutaric ad d ),
— any impurity: not m o re than the area o f the p rindpal peak B. R = C 0 2H : butanedioic a d d (succinic a d d ),
in the chrom atogram obtained with reference solution (b) C. R = [ C H J 3-C O 2H : heptanedioic a d d (pimelic ad d ).
(0.1 per cent),
________________________________:___________________________ PhEur
— total: not m ore th an 5 times the area o f the prindpal peak
in the chrom atogram obtained with reference solution (b)
(0.5 per cent),
— disregard limit: 0.5 times the area o f th e prindpal peak in
the chrom atogram obtained with reference solution (b) Adrenaline / Epinephrine ** * ***
(0.05 per cent). ** **
(Ph Eur monograph 2303) *
C h lo rid e s (2.4.4)
M axim um 200 ppm .
D ilute 2.5 m L o f solution S to 15 m L with water R
N itra te s
M aximum 30 ppm .
T o 1 m L of solution S add 2 m L of concentrated ammonia R,
C9H 13N 0 3 183.2 51-43-4
0.5 m L of a 10 g/L solution o f manganese sulfate R, 1 m L of
a 10 g/L solution o f sulfanilamide R and dilute to 20 m L with A c tio n a n d u se
water R. Add 0.10 g o f zinc powder R and cool in iced water Adrenoceptor agonist
for 30 min; shake from time to time. Filter and cool 10 m L
P re p a r a tio n s
of the filtrate in iced water. A dd 2.5 m L o f hydrochloric
Adrenaline Eye Drops/Epinephrine Eye D rops
add R1 and 1 m L o f a 10 g/L solution of
naphthylethylenediamine dihydrochloride R. Allow to stand at D ilute Adrenaline Injection (I in 10,000)/Dilute Epinephrine
room tem perature. A fter 15 m in the mixture is no t more Injection (1 in 10,000)
intensely coloured th a n a standard prepared at the sam e time
Ph Eur ---------------------------------------------------------------------------------
and in the sam e m anner, using 1.5 m L o f nitrate standard
solution ( 2 ppm NO j) R instead o f 1 m L o f solution S. D E F IN IT IO N
T he test is invalid if a blank solution prepared at the same 4- [(1R) - 1-Hydroxy-2-(methyiamino) ethyl] benzene-1,2-diol.
time and in the same m anner, using 1 m L o f water R instead Synthetic p ro d u c t
of 1 m L of solution S, is more intensely coloured than a
C o n te n t
2 m g/L solution o f potassium permanganate R.
99.0 per cent to 101.0 per cent (dried substance).
S u lfates (2.4.13)
CHARACTERS
Maximum 500 ppm.
A p p e a ra n c e
D ilute 3 m L o f solution S to 15 m L w ith distilled water R. White or alm ost white crystalline powder, becoming coloured
Ir o n (2.4.9) on exposure to air and lig h t
Maximum 10 ppm , determ ined on solution S.
S o lu b ility
H eav y m e ta ls (2.4.8) Practically insoluble in water, in ethanol (96 per cent) and in
M aximum 10 ppm . methylene chloride. It dissolves in hydrochloric ad d .
12 m L of solution S complies with test A. Prepare the ID E N T IF IC A T IO N
reference solution using lead standard solution (1 ppm Pb) R. A Infrared absorption spectrophotom etry (2.2.24).
L oss o n d ry in g (2.2.32) Comparison adrenaline CRS.
M aximum 0.2 per cent, determined on 1.000 g by drying in
B. Spedfic optical rotation (see Tests).
an oven at 105 °C.
S u lfa te d a s h (2.4.14) TESTS
M axim um 0.1 per c e n t S o lu tio n S
Dissolve 1.000 g in a 25.75 g/L solution o f hydrochloric add R
M d t 1.0 g completely over a gas burner, then ignite the
and dilute to 50.0 m L with the same solvent Examine the
m d te d substance with the burner. After ignition, lower or
solution immediately.
remove the flame in order to prevent th e substance from
boiling and keep it burning until completely carbonised. A p p e a ra n c e o f so lu tio n
Carry out the test for sulfated ash using the residue. Solution S is not more opalescent than reference
suspension II (2.2.1) and n o t m ore intensely coloured than
A SSA Y reference solution BY5 (2.2.2, Method II).
Dissolve 60.0 m g in 50 m L o f water R. A dd 0.2 m L o f
S pecific o p tic a l ro ta tio n (2.2.7)
phenolphthalein solution R and titrate w ith 0.1 M sodium
- 5 0 .0 to - 5 4 .0 (dried substance), determined on solution S.
hydroxide.
1 m L of 0.1 M sodium hydroxide is equivalent to 7.31 m g of R e la te d su b s ta n c e s
Liquid chromatography (2.2.29). Prepare the solutions protected
C6H 10O4.
from light.
Solvent mixture A Dissolve 5.0 g o f potassium dihydrogen
phosphate R and 2.6 g o f sodium octanesulfonate R in waterfor
2016 Adrenaline 1-77

chromatography R and dilute to 1000 m L with the same corresponding correction factor: impurity D = 0.7;
solvent (it is usually necessary to stir for at least 30 min to impurity E = 0.6;
achieve complete dissolution). Adjust to p H 2.8 with — impurities B, C, F: for each impurity, not more than twice
phosphoric acid R. the area of the principal peak in the chromatogram
Solvent mixture B acetomtrUe R l, solvent mixture A obtained with reference solution (a) (0.2 per cent);
(13:87 VIV). — impurities D, E: for each impurity, not more than the area
Test solution Dissolve 40 mg of the substance to be examined o f the principal peak in the chromatogram obtained with
reference solution (a) (0.1 p er cent);
in 5 m L o f 0.1 M hydrochloric add and dilute to 50.0 m L
with solvent mixture B.
— unspedfied impurities: for each impurity, not more than the
area o f the principal peak in the chromatogram obtained
Reference solution (a) Dilute 1.0 m L of the test solution to with reference solution (a) (0.10 per cent);
100.0 m L with solvent mixture B. Dilute 1.0 m L o f this
— total: not m ore than 5 times the area o f the principal peak
solution to 10.0 m L with solvent mixture B.
in the chromatogram obtained with reference solution (a)
Reference solution (b) Dissolve 1.5 mg of noradrenaline (0.5 per cent);
tartrate CRS (impurity B) and 1.5 m g of adrendone — disregard limit. 0.5 times the area of the principal peak in
hydrochloride R (impurity C) in solvent mixture B, add the chromatogram obtained with reference solution (a)
1.0 m L of the test solution and dilute to 100 m L with (0.05 per cent).
solvent mixture B.
Loss on drying (2.2.32)
Reference solution (c) Dissolve the contents o f a vial o f M axim um 0.5 per cent, determined on 1.000 g by drying
adrenaline impurity mixture CRS (containing impurities D over diphosphorus pentoxide R at a pressure not exceeding
and E) in 1.0 m L o f the blank solution. 0.7 kPa for 18 h.
Reference solution (d) Dissolve 4 m g o f adrenaline with Sulfated ash (2.4.14)
impurity F CRS in 0.5 m L of 0.1 M hydrochloric add and M axim um 0.1 per cent, determined on 1.0 g.
dilute to 5 m L with solvent mixture B.
Blank solution 0.1 M hydrochloric acid, solvent mixture B ASSAY
(1:9 VIV). Dissolve 0.150 g in 50 m L of anhydrous acetic acid R. T itrate
with 0 . 1 M perchloric add, determining the end-point
Column:
potentiometrically (2 . 2 .2 0 ).
— size. I = 0.10 m , 0 = 4.6 mm;
— stationary phase, end-capped octadecylsOyl silica gel for 1 m L of 0.1 M perchloric add is equivalent to 18.32 mg
chromatography R (3 |xm); o f C 9H 13N 0 3.
— temperature: 50 °C. STORAGE
Mobile phase: U nder nitrogen, protected from light.
— mobile phase A: acetomtrUe R l, solvent mixture A
IM P U R IT IE S
(5:95 VIV)-,
Specified impurities B, C , D , E, F
— mobile phase B: acetonitrUe R l, solvent mixture A
(45:55 VIV)-,
HO
Time Mobile phase A Mobile phase B
(min) (per cent V7V) (per cent V/V) HO
0 - 15 92*50 8*50

15-20 50*92 50*8 B. (1 i?)-2-am ino-1-(3,4-dihydroxyphenyl)ethanol

2 0 -2 5 92 8 (noradrenaline),

O
Flow rate 2.0 mL/min.
Detection Spectrophotom eter at 210 nm.
Injection 20 (lL.
Identification of impurities Use the chromatogram supplied
with adrenaline impurity mixture CRS and the chromatogram C . 1-(3,4-dihydroxyphenyl)-2-(methylamino) ethanone
obtained with reference solution (c) to identify the peaks due (adrenalone),
to impurities D and E; use the chromatogram supplied with
adrenaline with impurity F CRS and the chromatogram
obtained with reference solution (d) to identify the peak due
to im purity F.
Relative retendon W ith reference to adrenaline (retention
time = about 4 min): impurity F = about 0.2;
impurity B = about 0.8; impurity C = about 1.3;
impurity D = about 3.3; impurity E = about 3.7.
System suitability: reference solution (b): D . 4-[(li?)-2-(benzyImethylamino)-l-hydroxyethyl]benzene-
— resolution: m inim um 3.0 between the peaks due to 1,2-diol,
im purity B and adrenaline.
Limits:
— correction factors: for the calculation of content, multiply
the peak areas o f the following impurities by the
 1-78 Adrenaline Acid Tartrate 2016

(2.2.7) of the residue (adrenaline base) is —53.5 to —50,

determ ined using a 20.0 g/L solution in 0.5 M hydrochloric
add.
B. Infrared absorption spectrophotom etry (2.2.24).
Preparation Discs o f adrenaline base prepared as described
under identification test A.
E. 2-(beri2ylmethylamino)-l-(3,4-dihydroxyphenyl)ethanonej Comparison U se adrenaline base prepared as described under
identification test A from 50 m g o f adrenaline tartrate CRS
H O ,s h H dissolved in 5 m L o f a 5 g/L solution o f sodium
metabisulfite R. Keep the mixture at room tem perature for at
least 30 m in . Filter through a sintered-giass filter (2.1.2).
C. 0.2 m L o f the filtrate obtained in identification test A
gives reaction (b) of tartrates (2.3.1).
F. (li?)-l-(3,4-dihydroxyphenyl)-2-
(methylamino)ethanesulfonic ad d . TESTS
Appearance of solution
PhEur
T he solution is not m ore opalescent than reference
suspension II (2 . 2 . 1 ) and n o t m ore intensely coloured than
reference solution BY5 (2.2.2, Method II).
Dissolve 0.5 g in water R and dilute to 10 m L w ith the same
Adrenaline Acid Tartrate / * * * * *
★ ★ solvent. Examine the solution im m ediatdy.

Epinephrine Acid Tartrate ***** Related substances

Liquid chromatography (2.2.29). Prepare the solutions protected
(Adrenaline Tartrate, Ph Eur monograph 0254)
from light.
H OH
H OH
Solvent mixture A Dissolve 5.0 g o f potassium dihydrogen
COjH
phosphate R and then 2.6 g o f sodium octanesidfonate R in
HOjC water for chromatography R, and dilute to 1000 m L with the
H OH same solvent (it is usually necessary to stir for at least 30 min
to achieve complete dissolution). Adjust to p H 2.8 with
C 13H 19NO 9 333.3 51-42-3 phosphoric acid R.
Solvent mixture B acetonitrUe R l, solvent m ixture A
Action and use (130:870 V/V).
Adrenoceptor agonist.
Test sdution Dissolve 75 m g of the substance to be examined
Preparations in 5 m L o f 0.1 M hydrochloric add and dilute to 50 m L with
Adrenaline Injection/Epinephrine Injection solvent mixture B.
D ilute Adrenaline Injection (1 in 10,000)/Dilute Epinephrine Reference solution (a) Dilute 1.0 m L o f the test solution to
Injection (1 in 10,000) 100.0 m L with solvent mixture B. Dilute 1.0 m L o f this
Adrenaline Solution/Epinephrine Solution solution to 10.0 m L with solvent mixture B.
Adrenaline and Cocaine Intranasal Solution Reference solution (b) Dissolve 1.5 mg of noradrenaline
Bupivacaine and Adrenaline Injection/Bupivacaine and tartrate CRS (impurity B) and 1.5 m g of adrenalone
Epinephrine Injection hydrochloride R (impurity C) in solvent mixture B, add
1.0 m L o f the test solution and dilute to 100.0 m L with
Lidocaine and Adrenaline Injection/Lidocaine and
solvent m ixture B.
Epinephrine Injection
Reference solution (c) Dissolve the contents o f a vial of
PhEur___________________________________________________________ adrenaline impurity mixture CRS (impurities D and E) in
D E F IN IT IO N 0.1 m L o f 0.1 M hydrochloric add and 0.9 m L o f solvent
mixture B.
(li?)-l-(3j4-Dihydroxyphenyl)-2-(m ethylam ino)ethanol
hydrogen (2i?,3i?)-2,3-dihydroxybutanedioate. Reference solution (d) Dissolve 7.5 mg o f adrenaline tartrate
with impurity A CRS in 0.5 m L o f 0.1 M hydrochloric add and
Content
dilute to 5.0 m L with solvent mixture B.
98.5 per cent to 101.0 per cent (dried substance).
Blank solution 0.1 M hydrochloric add, solvent mixture B
CHARACTERS (1:9 VIV).
Appearance Column:
W hite or greyish-white, crystalline powder. — size: I = 0.10 m , 0 = 4.6 mm;
Solubility — stationary phase: end-capped octadecylsilyl silica gel for
F red y soluble in water, slightly soluble in ethanol chromatography R (3 pm);
(96 per cent). — temperature: 50 °C.
ID E N T IF IC A T IO N Mobile phase:
A. Dissolve 5 g in 50 m L of a 5 g/L solution of sodium — mobile phase A: acetomtrUe R l, solvent mixture A
metabisidfite R and m ake alkaline by addition of ammonia R. (5:95 V/V);
Keep, the mixture at room tem perature for a t least 15 min — mobile phase B: acetomtrUe R l, solvent mixture A
and filter. Reserve the filtrate for identification test C . W ash (45:55 V/V);
the predpitate with 3 quantities, each o f 10 mL, of
methanol R. D ry at 80 °C. T h e specific optical rotation
 I

2016 Agar 1-79

Tune Mobile phase A Mobile phase B IM P U R IT IE S

(min) (per cent V/V) (per cent V/V) Specified impurities A, B, C , D , E
0 - 15 92->50 8 -> 50
A. unknown structure,
15 -2 0 50->92 50 -> 8
H OH
20 - 25 92 8

Flow rate 2.0 mL/min.

Detection Spectrophotom eter at 210 nm. B. ( 1i?)-2-amino-1-(3,4-dihydroxyphenyl)ethanol
Injection 20 |xL. (noradrenaline),
Identification of impurities Use the chromatogram supplied
with adrenaline impurity mixture CRS and the chromatogram
obtained w ith reference solution (c) to identify the peaks due
to impurities D and E; use the chromatogram supplied with
adrenaline tartrate with impurity A CRS and the chromatogram
obtained w ith reference solution (d) to identify the peak due
to impurity A. , C. 1-(3,4-dihydroxyphenyl)-2-(methyiamino)ethanone
(adrenalone),
Relative retention W ith reference to adrenaline (retention
time = about 4 min): impurity B = about 0.8;
impurity C = about 1.3; impurity A = about 3.2;
impurity D = about 3.3; impurity E = about 3.7.
System suitability: reference solution (b):
— resolution: minimum 3.0 between the peaks due to
impurity B and adrenaline.
Limits:
— correction factors: for the calculation o f content, multiply D . 4-[(li?)-2-(benzylmethylamino)-l-hydroxyethyl]benzene-
the peak areas o f the following impurities by the 1, 2-diol,
corresponding correction factor impurity D = 0.7;
im purity E = 0.6;
— impurity A: not more than 3 times the area of the
principal peak in the chromatogram obtained with
reference solution (a) (0.3 per cent);
— impurities B, C: for each impurity, no t more than twice
the area o f the principal peak in the chromatogram
obtained w ith reference solution (a) ( 0.2 per cent);
— impurities D, E: for each impurity, n o t more than the area E. 2-(benzyimethylamino)-l-(3,4-dihydroxyphenyl)ethanone.
of the principal peak in the chromatogram obtained with — ___ ____________________________________________________ PhEtr
reference solution (a) (0.1 per cent);
— unspecified impurities: for each impurity, not more than the
area o f the principal peak in the chromatogram obtained
with reference solution (a) (0.10 per cent); ★ ★
— total: n o t more than 6 times the area of the principal peak Agar ★ ★
in the chromatogram obtained with reference solution (a) (Ph. Eur. monograph 0310) *****
(0.6 per cent);
— disregard limit. 0.5 times the area o f the principal peak in Action and use
the chrom atogram obtained with reference solution (a) Excipient.
(0.05 p er cent).
Ph Eur_______________________
Loss on drying ('2.2.32)
M aximum 0.5 per cent, determined on 1.000 g by drying DEFINITION
in 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
whilst hot, concentrated and dried.
ASSAY
Dissolve 0.300 g in 50 m L 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 m L of crystal violet Powder or crumpled strips 2-5 m m wide or sometimes flakes,
solution R as indicator. colourless or pale yellow, translucent, somewhat tough and
difficult to break, becoming more britde on drying.
1 m L of 0.1 M perchloric acid is equivalent to 33.33 mg
of C 13H 19N O 9. Mucilaginous taste.

STORAGE IDENTIFICATION
In an airtight container, or preferably in a sealed tube under A. Examine under a microscope. W hen m ounted in 0.005 M
vacuum or u n d er an inert gas, protected from light. iodine, the strips or flakes are partly stained brownish-violet.
Magnified 100 times, they show the following diagnostic
characters: numerous m inute, colourless, ovoid or rounded
1-80 Air 2016

grains on an am orphous background; occasional brown, solution add 5 m L o f picric acid solution R. N o turbidity
round or ovoid spores with a reticulated surface, measuring appears within 10 min.
up to 60 nm, may be present. Reduce to a powder, if L o ss o n d ry in g (2.2.32)
necessary. T h e pow der is yellowish-white. Examine un d er a M axim um 20.0 per cent, determ ined on 1.000 g o f the
microscope using 0.005 M iodine. T h e powder presents pow dered herbal drug (355) (2.9.12) by drying in an oven at
angular fragments w ith num erous grains similar to those seen 105 °C.
in the strips and flakes; some o f the fragments are stained
T o ta l a s h (2.4.16)
brownish-violet.
M axim um 5.0 per c e n t
B. Dissolve 0.1 g with heating in 50 m L of vocaer R. Cool.
M ic ro b ia l c o n ta m in a tio n
T o 1 m L of the mucilage carefully add 3 m L of water R so as
T A M C : acceptance criterion 103 C FU /g (2.6.12).
to form 2 separate layers. A dd 0.1 m L o f 0.05 M iodine.
A dark brownish-violet colour appears at the interface. Mix. T Y M C : acceptance criterion 102 C FU /g (2.6.12).
T he liquid becomes pale yellow. Absence o f Escherichia coli (2.6.13).
C. H eat 5 m L o f the mucilage prepared for identification Absence o f Salmonella (2.6.13).
test B on a w ater-bath with 0.5 m L o f hydrochloric acid R for
L A B E L L IN G
30 min. A dd 1 m L o f barium chloride solution R l. A white
T h e label states th e swelling index.
turbidity develops within 30 min.
___________________________________________________________ PhEur
D. H eat 0.5 g with 50 m L o f water R on a water-bath until
dissolved. Only a few fragments rem ain insoluble. D uring
cooling, the solution gels between 35 °C and 30 °C. H eat the
gel thus obtained on a water-bath; it does not liquefy below
80 °C. Medical Air *****
TESTS
(Medicinal Air\ Ph Eur monograph 1238) *
Sw elling in d e x (2.8.4)
M inim um 10 and within 10 p er cent o f the value stated on
When Medical Air is intended for use in a room in which
magnetic resonance imaging (MRJ) is being performed, the
the label, determ ined on the pow dered herbal drug (355)
cylinder and fittings should be made from suitable non-
(2.9.12).
ferromagnetic materials and labelled accordingly.
In so lu b le m a t te r
PhEur___________________________________________________________
M aximum 1.0 per cent.
T o 5.00 g o f the powdered herbal drug (355) (2.9.12) add D E F IN IT IO N
100 m i. of water R and 14 m L o f dilute hydrochloric add R. Compressed am bient air.
Boil gendy for 15 m in with frequent stirring. Filter the hot C o n te n t
liquid through a tared, sintered-glass filter (160) (2 . 1 . 2 ), rinse 20.4 per cent VIV to 21.4 per cent VIV o f oxygen (O 2).
the filter with hot water R and dry at 100-105 °C.
CHARACTERS
T h e residue weighs a maximum o f 50 mg.
A p p e a ra n c e
G elatin Colourless gas.
T o 1.00 g add 100 m L of water R and heat on a water-bath
until dissolved. Allow to cool to 50 °C. T o 5 m L o f this S o lu b ility
A t 20 °C at a pressure of 101 kPa, 1 volume dissolves in
about 50 volumes of water.

t
Photomultiplier
t
Amplifier

Figure 1238.-1. - UV fluorescence analyser

2016 Air 1-81

PRODUCTION than 0.05 ppm V/V of nitrogen monoxide and nitrogen

Carbon dioxide dioxide.
M axim um 500 p pm V/V, determined using an infrared Reference gas (b) U se a mixture o f 2 ppm V/V o f nitrogen
analyser (2.5.24). monoxide R in nitrogen R l.
Gas to be examined Filter the substance to be examined to Calibrate the apparatus and set the sensitivity using reference
avoid stray light phenomena. gases (a) and (b). Measure the content o f nitrogen monoxide
Reference gas (a) Use a mixture of 21 per cent V/V o f and nitrogen dioxide in the gas to be examined.
oxygen R and 79 per cent VIV of nitrogen R l, containing less Water
than 1 ppm V/V o f carbon dioxide R l. Maximum 67 ppm V/V, determined using an electrolytic
Reference gas (b) Use a mixture of 21 per cent VIV of hygrometer (2.5.28), except where the com petent authority
oxygen R and 79 per cent ViV of nitrogen R l, containing decides that the following limit applies to medicinal air
500 ppm VIV o f carbon dioxide R l. generated on-site and distributed in pipe-line systems
Calibrate the apparatus and set the sensitivity using reference operating at a pressure not greater than 10 bars and a
gases (a) and (b). M easure the content of carbon dioxide in tem perature not less than 5 °C: m axim um 870 ppm V/V,
the gas to be examined. determined using an electrolytic hygrometer (2.5.28).
Carbon monoxide Assay
M axim um 5 p p m VIV, determined using an infrared analyser Determ ine the concentration of oxygen in air using a
(2.5.25). paramagnetic analyser (2.5.27).
Gas to be examined Filter the substance to be examined to
avoid stray light phenomena.
Reference gas (a) U se a mixture of 21 per cent V/V o f
oxygen R and 79 per cent VIV of nitrogen R l, containing less
than 1 ppm VIV o f carbon monoxide R.
Reference gas (b) U se a mixture of 21 per cent VIV of
oxygen R and 79 per cent VIV of nitrogen R l, containing
5 ppm V/V o f carbon monoxide R.
Calibrate the apparatus and set the sensitivity using reference
gases (a) and (b). M easure the content of carbon monoxide
in the gas to be examined.
Sulfur dioxide
M axim um 1 ppm V/V, determined using an ultraviolet
fluorescence analyser (Figure 1238.-1).
T h e apparatus consists o f the following:
— a system generating ultraviolet radiation with a
wavelength o f 210 nm , made up o f an ultraviolet lamp, a
collimator, and a selective filter; the beam is blocked
periodically by a chopper rotating at high speeds;
— a reaction chamber, through which flows the gas to be
examined;
— a system that detects radiation emitted at a wavelength of
350 nm , m ade up o f a selective filter, a photomultiplier
tube and an amplifier.
Gas to be examined Filter the substance to be examined.
Reference gas (a) U se a mixture of 21 per cent V/V of
oxygen R and 79 per cent V/V of nitrogen R l.
Reference gas (b) U se a mixture of 21 per cent V/V of
oxygen R and 79 per cent VIV of nitrogen R l, containing
0.5 ppm VIV to 2 ppm V/V of sulfur dioxide R l.
Calibrate the apparatus and set the sensitivity using reference
gases (a) and (b). M easure the content of sulfur dioxide in
the gas to be examined
Oil
M axim um 0.1 m g/m 3, determined using an oil detector tube
(2. 1 . 6 ), when an oil-lubricated compressor is used for the
production.
Nitrogen m onoxide and nitrogen dioxide
M axim um 2 p pm V/V in total, determined using a
chemiluminescence analyser (2.5.26).
Gas to be examined T h e substance to be examined.
Reference gas (a) U se a mixture of 21 per cent V/V of
Figure 1238.-2. - Gas burette
oxygen R and 79 per cent V/V o f nitrogen R l, containing less
 1-82 Air 2016

ID E N T IF IC A T IO N IM P U R IT IE S
First identification C. A. C O 2: carbon dioxide,
Second identification A , B. B. S 0 2: sulfur dioxide,
A. In a conical flask containing the substance to be C. N O : nitrogen monoxide,
examined, place a glowing wood splinter. T he splinter D . N 0 2: nitrogen dioxide,
rem ains glowing. E. oil,
B. U se a gas burette (Figure 1238.-2) o f 25 m L capacity in F. CO: carbon monoxide,
the form of a cham ber in the middle o f which is a tube
G . H 20 : water.
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 Ph Eur

conical barrel. T he lower tap is joined to a tube with an

olive-shaped nozzle and is used to introduce the gas into the
apparatus. A cylindrical funnel above the upper tap is used to ★ ★
introduce die absorbent solution. W ash the burette with Synthetic Air ★ ★
water R and dry. O pen the 2 taps. C onnect the nozzle to the *★ ★*
(Synthetic Medicinal Air, Ph Eur monograph 1684) *
source of the gas to be examined and set the flow rate to
When Synthetic Air is intended for use in a room in which
1 L/m in. Flush the burette by passing the gas to be examined
magnetic resonance imaging (MBI) is being performed, the
through it for 1 min. Close the lower tap of the burette and
cylinder and fittings should be made from suitable non­
immediately afterwards the upper tap. Rapidly disconnect the
ferromagnetic materials and labelled accordingly.
burette from the source o f the gas to be examined. Rapidly
give a half turn to the upper tap to eliminate any excess P h E u r ___________________________________________________________________________________________

pressure in the burette. Keeping the burette vertical, fill the D E F IN IT IO N

funnel with a freshly prepared mixture of 21 m L of a 560 g/L M ixture of Nitrogen (1247) an d Oxygen (0417).
solution of potassium hydroxide R and 130 m L o f a 200 g/L
C o n te n t
Irolution o f sodium dithionite R. O pen the upper tap slowly.
95.0 per cent to 105.0 per cent o f the nominal value which is
T he solution absorbs the oxygen and enters the burette.
between 21.0 per cent V/V to 22.5 per cent V/V o f oxygen
Allow to stand for 10 min w ithout shaking. Read the level of
the liquid meniscus on the graduated part of the burette. (Oa).
This figure represents the percentage V/V of oxygen. CHARACTERS
T he value read is 20.4 to 21.4. Colourless and odourless gas.
C. It complies with the limits o f the assay. S o lu b ility
TESTS A t a tem perature of 20 °C and a pressure o f 101 kPa,
C a rb o n dioxide 1 volume dissolves in about 50 volumes o f water.
M axim um 500 ppm V/V, determ ined using a carbon dioxide P R O D U C T IO N
detector tube (2. 1 . 6 ). W a te r (2.5.28)
S u lfu r dioxide M axim um 67 ppm V/V.
M aximum 1 ppm V/V, determ ined using a sulfur dioxide A ssay (2.5.27)
detector tube (2. 1 . 6). Carry out the determination o f oxygen in gases.
O il ID E N T IF IC A T IO N
M axim um 0.1 mg/m3, determ ined using an oil detector tube First identification C
(2 . 1 . 6 ), when an oil-lubricated compressor is used for the
Second identification A, B
production.
A. In a conical flask containing the substance to be
N itro g e n m o n o x id e a n d n itro g e n diox id e
examined, place a glowing splinter o f wood. T h e splinter
M axim um 2 ppm V/V, determ ined using a nitrogen
remains glowing.
monoxide and nitrogen dioxide detector tube (2 . 1 . 6 ).
B. U se a gas burette (Figure 1684.-1) o f 25 m L capacity in
C a rb o n m o n o x id e the form o f a chamber, in the middle o f which is a tube
M axim um 5 ppm V/V, determ ined using a carbon monoxide graduated in 0.2 per cent between 19.0 per cent and
detector tube (2 . 1 . 6 ). 23.0 per cent, and isolated at each end by a tap with a
W a te r'v a p o u r conical barrel. T h e lower tap is joined to a tube with an
M axim um 67 ppm V/V, determ ined using a water vapour olive-shaped nozzle and is used to introduce the gas into the
detector tube (2 . 1 . 6 ), except where the competent authority apparatus. A cylindrical funnel above the upper tap is used to
decides that the following limit applies to medicinal air introduce the absorbent solution. W ash the burette with
generated on-site and distributed in pipe-line systems water R and dry. O pen both taps. C onnect the nozzle to the
operating at a pressure not greater than 10 bars and a source of the substance to be examined and set the flow rate
tem perature n o t less than 5 °C: maximum 870 ppm V/V, to 1 L/min. Flush the burette by passing the substance to be
determined using a w ater vapour detector tube (2 . 1 . 6 ). examined through it for 1 m in. Close the lower tap o f the
STORAGE burette and immediately afterwards the upper tap. Rapidly
disconnect the burette from the source of the substance to be
As a gas, in suitable containers complying with the legal
examined. Rapidly give a h alf tu rn o f the upper tap to
regulations or as a gas supplied by a pipe network.
eliminate any excess pressure in the burette. Keeping the
L A B E L L IN G b urette vertical, fill the funnel with a freshly prepared mixture
W here applicable, the label states the production m ethod, as of 2 1 m L o f a 560 g/L solution o f potassium hydroxide R and
regards to the use o f an oil - lubricated compression. 130 m L o f a 200 g/L solution o f sodium dithionite R. O pen
the upper tap slowly. T he solution absorbs the oxygen and
2016 Alanine 1-83

STORAGE
As a compressed gas in suitable containers complying w ith
the legal regulations or as a compressed gas supplied by a
pipe network, after mixing of the components.
L A B E L L IN G
T he label states the nominal content of 0 2 in per cent VIV.
IMPURITIES
A. H 20 : water.
__________________________________________________________ PhEur

★★*★★
Alanine ★ ★
(Ph Eitr monograph 0752) *****

H NH,
V
HjC COjH

C3H7N 0 2 89.1 5 6 -4 1 -7

A ctio n a n d u se
Amino ad d .

PhEur

D E F IN IT IO N
(2S)-2-Aminopropanoic add.
C o n te n t
98.5 per cent to 101.0 per cent (dried substance).
CHARACTERS
A p p e a ra n c e
W hite or almost white, crystalline powder or colourless
crystals.
S o lu b ility
F red y soluble in water, very slightly soluble in ethanol
(96 per cent).
ID E N T IF IC A T IO N
First identification A , B
Second identification A , C, D
A. Specific optical rotation (see Tests).
B. Infrared absorption spectrophotometry (2.2.24).
Comparison alanine CRS.
C. Thin-layer chromatography (2.2.27).
Test solution Dissolve 10 mg of the substance to be examined
in water R and dilute to 50 m L w ith the same solvent.
Reference solution. Dissolve 10 mg of alanine CRS in water R
and dilute to 50 m L with the same solvent.
Plate TLC silica gel plate R.
Figure 1684.-1,- Gas burette
Mobile phase glacial acetic add R, water R, butanol R
(20:20:60 V/V/V).
enters the burette. Allow to stand for 10 min without Application 5 |xL.
shaking. Read the level of the liquid meniscus on the Development Over 2/3 of the plate.
graduated part o f the burette. T his figure represents the
Drying In air.
percentage VIV o f oxygen. T h e value read is 95.0 per cent to
105.0 p er cent o f the nominal value. Detection Spray with ninhydrin solution R and heat at 105 °C
for 15 min.
C. It complies w ith the limits of the assay.
Results T h e p rindpal spot in the chromatogram obtained with
TESTS the T est solution is similar in position, colour and size to the
W a te r v a p o u r prindpal spot in the chromatogram obtained with the
M axim um 67 p p m VIV, determined using a water vapour reference solution.
detector tube (2 . 1 . 6 ).
 1-84 Alanine 2016

D . Dissolve 0.5 g in a mixture o f 0.25 m L o f hydrochloric — total: maximum 0.5 per cent;
add R l, 0.5 m L of a 100 g/L solution of sodium nitrite R and — reporting threshold: 0.05 per cent.
1 m L of water R. Shake; gas is given off, Add 2 m L o f dilute C h lo rid e s (2.4.4)
sodium hydroxide solution R, followed by 0.25 m L o f iodinaxed M aximum 200 ppm.
potassium iodide solution R. After about 30 min, a yellow
Dilute 5 m L of solution S to 15 m L with water R.
precipitate is formed.
S u lfates (2.4.13)
TESTS M aximum 300 ppm.
S o lu tio n S
Dilute 10 m L of solution S to 15 m L with dxstHled water R.
Dissolve 2.5 g in distilled water R and dilute to 50 m L with
the same solvent. A m m onium
Amino a d d analysis (2.2.56) as described in the test for
A p p e a ra n c e o f so lu tio n
ninhydrin-positive substances with the following
T he solution is clear ('2.2.1) and not more intensely coloured
modifications.
than reference solution BY 6 (2.2.2, Method II).
Injection T est solution, reference solution (c) and blank
Dilute 10 m L of solution S to 20 m L with water R.
solution.
S pecific o p tic a l r o ta tio n (2.2.7) Limit:
+ 13.5 to + 15.5 (dried substance). — ammonium at 570 ran: not more than the area of the
Dissolve 2.50 g in hydrochloric add R l and dilute to 25.0 m L corresponding peak in the chrom atogram obtained with
with the same acid. reference solution (c) (0.02 per cent), taking into account
N in h y d rin -p o sitiv e su b sta n c e s the peak due to ammonium in the chrom atogram
Amino a d d analysis (2.2.56). F or analysis, use M ethod 1. obtained with the blank solution.
T he concentrations o f the test solution and the reference Ir o n (2.4.9)
solutions may be adapted according to the sensitivity of the M aximum 10 ppm.
equipm ent used. T h e concentrations o f all solutions are In a separating funnd, dissolve 1.0 g in 10 m L of dilute
adjusted so that the system suitability requirements described hydrochloric add R. Shake with 3 quantities, each of 10 mL,
in general chapter 2.2.46 are fulfilled, keeping the ratios of of methyl isobutyl ketone R l, shaking for 3 m in each time.
concentrations between all solutions as described. T o the combined organic layers add 10 m L of water R and
Solution A dilute hydrochloric acid R l or a sample preparation shake for 3 min. Use the aqueous layer.
buffer suitable for the apparatus used. H eav y m e ta ls (2.4.8)
Test solution Dissolve 30.0 m g o f the substance to be M aximum 10 ppm.
examined in solution A and dilute to 50.0 m L with Dissolve 2.0 g in water R and dilute to 20 m L with the same
solution A. solvent. 12 m L of the solution complies with test A. Prepare
Reference solution (a) D ilute 1.0 m L of the test solution to the reference solution using lead standard solution
100.0 m L with solution A Dilute 2.0 m L of this solution to (1 ppm Pb) R.
10.0 m L with solution A. L oss o n d ry in g (2.2.32)
Reference solution (b) Dissolve 30.0 mg of proUne R in Maximum 0.5 per cent, determined on 1.000 g by drying in
solution A and dilute to 100.0 m L with solution A. Dilute an oven at 105 °C.
1.0 m L of the solution to 250.0 m L with solution A. S u lfa te d a s h (2.4.14)
Reference solution (c) Dilute 6.0 m L o f ammonium standard Maximum 0.1 per cent, determined on 1.0 g.
solution (100 ppm NH 4 ) R to 50.0 m L with solution A. D ilute
A SSAY
1.0 m L of this solution to 100.0 m L with solution A.
Dissolve 80.0 mg in 3 mL o f anhydrous formic add R.
Reference solution (d) Dissolve 30 m g o f isoleucine R and Add 30 m l. of anhydrous acetic add R. T itrate with 0.1 M
30 m g of leudne R in solution A and dilute to 50.0 m L with
perchloric add, determining the end-point potentiometrically
solution A. Dilute 1.0 m L o f the solution to 200.0 m L with
(2.2.20).
solution A.
1 m L of 0.1 M perchloric acid is equivalent to 8.91 m g of
Blank solution Solution A.
c 3h 7n o 2.
Inject suitable, equal am ounts of the test, blank and reference
solutions into the amino a d d analyser. Run a program STO RA G E
suitable for the determination of physiological amino adds. Protected from light.
System suitability Reference solution (d): IM P U R IT IE S
— resolution: minimum 1.5 between the peaks due to Other detectable impurities (the following substances would, if
isoleucine and leucine. present at a suffident levd, be detected by one or other of
Calculation of percentage contents: the tests in the monograph. They are limited by the general
— for any ninhydrin-positive substance detected at 570 nm , acceptance criterion for other/unspecified impurities and/or
use the concentration of alanine in reference solution (a); by the general monograph Substances for pharmaceutical use
— for any ninhydrin-positive substance detected at 440 nm , (2034). It is therefore no t necessary to identify these
use the concentration o f proline in reference solution (b); impurities for demonstration of compliance. See also 5.10.
if a peak is above the reporting threshold at both Control of impurities in substances for pharmaceutical use): A , B.
wavelengths, use the result obtained at 570 nm for
quantification. H nh 2

Limits: HO!° ' ~ X c0 !h

— any ninhydrin-positive substance: for each impurity,
maximum 0.10 p er cent;
A. (25)-2-aminobutanedioic acid (aspartic ad d ),
2016 Albendazole 1-85

Column:
h o 2c ' — size. I = 0.25 m , 0 = 4.6 mm;
— stationary phase’, spherical end-capped octadecylsUyl silica gel
B. (25)-2-am inopentanedioic ad d (glutamic ad d ). for chromatography R (5 pm) with a pore size of 10 nm
PhEur and a carbon loading o f 19 p er cent.
Mobile phase Mix 300 volumes of a 1.67 g/L solution of
ammonium dihydrogen phosphate R and 700 volumes of
methanol R.
★ ★ Flow rate 0.7 mL/min.
Albendazole ★ ★
Detection Spectrophotom eter at 254 nm.
(Ph Eur monograph 1386) *****
Injection 20 |iL.
ch 3 Run time 1.5 times the retention time of albendazole.
Relative retention W ith reference to albendazole:
-NH impurity D = about 0.40; impurities B and C = about 0.43;
impurity E = about 0.47; impurity F = about 0.57;
impurity A = about 0.80.
C 12H 15N 3O 2S 265.3 54965-21-8 System suitability", reference solution (b):
— resolution', m inim um 3.0 between the peaks due to
A ctio n a n d u se
albendazole and oxibendazole.
Benzimidazole antihelminthic.
Limits:
P re p a r a tio n s — impurities A, B, C, D, E, F: for each impurity, not more
Albendazole Oral Suspension than 1.5 times the area of the principal peak in the
Albendazole Oral Suspension with Minerals chromatogram obtained with reference solution (a)
(0.75 p er cent);
PhEir.
— total: n o t more than 3 times the area o f the prindpal peak
D E F IN IT IO N in the chrom atogram obtained with reference solution (a)
M ethyl [5-(propylsulfanyl)-1 /f-benzimidazol-2 -yl] carbamate. (1.5 p er cent);
C o n te n t — disregard limit: 0.1 times the area of the prindpal peak in
98.0 per cent to 102.0 per cent (dried substance). the chromatogram obtained with reference solution (a)
(0.05 p er cent).
CHARACTERS
Loss on drying (2.2.32)
A p p e a ra n c e
M aximum 0.5 p er cent, determined on 1.000 g by drying in
W hite or slightly yellowish powder.
an oven at 105 °C for 4 h.
S o lu b ility
Sulfated ash (2.4.14)
Practically insoluble in water, freely soluble in anhydrous
M aximum 0.2 p er cent, determ ined on 1.0 g.
formic a d d , very slightly soluble in methylene chloride,
practically insoluble in ethanol (96 p er cent). ASSAY
In order to avoid overheating during the titration, mix thoroughly
ID E N T IF IC A T IO N
throughout and stop the titration immediately after the end-point
Infrared absorption spectrophotometry (2.2.24).
has been reached.
Preparation Discs.
Dissolve 0.250 g in 3 m L of anhydrous formic acid R and add
Comparison albendazole CRS. 40 m L o f anhydrous acetic add R. Titrate with 0.1 M
TESTS perchloric add, determining the end-point potentiometrically
A p p e a ra n c e o f so lu tio n (2.2.20).
T he solution is clear (2.2.1) and not m ore intensely coloured 1 m L of 0.1 M perchloric add is equivalent to 26.53 mg
than reference solution BY 6 (2.2.2, Method II). OfCi2Hj5N302S.
Dissolve 0.10 g in a mixture of 1 volume o f anhydrous formic STORAGE
add R and 9 volumes o f methylene chloride R and dilute to Protected from light.
10 m L w ith the same mixture of solvents.
IMPURITIES
R e la te d s u b s ta n c e s
Specified impurities A, B, C , D , E, F.
liq u id chrom atography (2.2.29).
Test solution Dissolve 25.0 m g o f die substance to be
examined in 5 m L o f methanol R containing 1 per cent VIV
-NH,
o f sulfuric add R and dilute to 50.0 m L with the mobile
phase.
Reference solution (a) Dissolve 10.0 m g o f the substance to be
examined in 10 m L of methanol R containing 1 p er cent VIV A. R = S-CH2-CH2-CH3: 5-(propylsulfanyl)-IH -
o f sulfuric acid R and dilute to 100.0 m L with the mobile benzimidazol- 2-am ine,
phase. D ilute 0.5 m L of this solution to 20.0 m L with the D . R = SO 2-C H 2-C H 2-C H 3: 5-(propyisulfonyI)-lii-
mobile phase. benzimidazol- 2-amine,
Reference solution (b) Dissolve 50.0 m g o f the substance to be
examined and 50 m g o f oxibendazole CRS in 5 m L o f
methanol R c o n taining l per cent VIV o f sulfuric add R and
dilute to 100.0 m L with the mobile phase.
 1-86 Alcuronium Chloride 2016

O CH,
Reference solution Dissolve 10 m g o f alcuronium chloride CRS
1 y -o '
in methanol R and dilute to 10 m l. with the same solvent.
> -N H
Plate TLC silica gel plate R.
Mobile phase Mix 15 volumes o f a 58.4 g/L solution of sodium
B. R = SO -C H 2-C H 2-C H 3: methyi [5-(propylsuLfinyl)~ÎH- chloride R, 35 volumes of dilute ammonia R2 and 50 volumes
benzimidazol-2-yl]carfaamate, of methanol R.
C. R = SO 2-C H 2-C H 2-C H 3: methyl [5-(propylsulfonyl)-1H- Application 10 |iL.
benzimida 7ol- 2-yl] carbamate, Development Over a path of 15 cm.
E. R = H: methyl ( l.ff-benzimidazol-2-yl) carbamate, Drying In air for 10 min.
F. R = S-C H 3: methyl [5-(methylsulfanyl)-1/i-benziinidazol- Detection Spray with 0.1 M ammonium and cerium nitrate.
2-yl] carbamate. Results T he principal spot in the chrom atogram obtained with
__________________________________________________________ PhEur the test solution is similar in position, colour and size to the
principal spot in the chromatogram obtained with the
reference solution.
C. It gives reaction (a) of chlorides (2.3.1).
★*★
Alcuronium Chloride ★ ★ TESTS
★ ★
S o lu tio n S
(Ph Eur monograph 1285) *****
Dissolve 0.250 g in carbon dioxide-free water R and dilute to
25.0 m L with the same solvent.
A p p e a ra n c e o f so lu tio n
Solution S is clear (2.2.1) and n o t more intensely coloured
than reference solution Y6, BY 6 or B 6 (2.2.2, Method I).
A cid ity o r a lk alin ity
2 Cl T o 10 m L of solution S add 0.1 m L of methyl red solution R
and 0.2 m L of 0.01 M hydrochloric add T he solution is red.
Add 0.4 m L of 0.01 M sodium hydroxide. T he solution is
yellow.
Specific o p tic a l ro ta tio n (2.2.7)
—430 to —451 (anhydrous substance), determined on
solution S.
C44H 50C I2N 4O2 738 15180-03-7
P ro p a n -2 -o l (2.4.24, System A)
A ctio n a n d u se Maximum 1.0 per cent.
Non-depolarizing neuromuscular blocker. R e la te d su b s ta n c e s
Liquid chromatography (2.2.29).
PhEur______________________________________
Solvent mixture M ix 100 m L of methanol R, 200 m L o f
D E F IN IT IO N acetonitrile R and 200 m L of a 6.82 g/L solution of potassium
(lfl,3aS,10S,llaS,12J?,14aS,19aS,20bS,21S,22aS,23.E,26£)- dihydrogen phosphate R. Dissolve 1.09 g o f sodium
23,26-bis(2-Hydroxyethylidene)-l, 12-bis(prop-2-enyl)- laurylsulfonate for chromatography R in the mixture and adjust
2,3,11,1 la , 13,14,22,22a-octahydro-10tf,21/f-l, 21:10,12- the apparent p H to 8.0 with a 100 g/L solution of sodium
diethano-19aH ,20b/f- [1,5]diazocino [ 1,2,3-Zm: 5,6,7- hydroxide R
l'm '] dipyrrolo[2',3 '-d:2'',3":d ']dicaibazolediium dichloride
Test solution Dissolve 0.20 g o f the substance to be examined
(4 ,4 /-didesmethyl-4,4/-bis(prop-2-enyl)toxiferm I dichloride).
in the solvent mixture and dilute to 100.0 m L with the
C o n te n t solvent mixture.
98.0 per cent to 102.0 per cent (anhydrous substance).
Reference solution (a) Dilute 0.5 m L o f the test solution to
CHARACTERS 100.0 m L with the solvent mixture.
A p p e a ra n c e Reference solution (b) Dilute 4.0 m l, of reference solution (a)
W hite or slightly greyish-white, crystalline powder. to 10.0 m L with the solvent mixture.
S olubility Reference solution (c) Dilute 1.0 m L o f reference solution (a)
Freely soluble in water and in methanol, soluble in ethanol to 10.0 m L with the solvent mixture.
(96 per cent), practically insoluble in cyclohexane. Reference solution (d) T o 5.0 m L o f the test solution add
Carry out the identification, tests and assay as rapidly as possible 5.0 mg of aUylstrychnine bromide CRS, dissolve in the solvent
avoiding exposure to actinic light mixture and dilute to 100.0 m L with the solvent mixture.
ID E N T IF IC A T IO N Column:
First identification A , C — size. I = 0.25 m, 0 = 4 mm;
— stationary phase: octylsQyl silica gel for chromatography R
Second identification B, C
(5 jim).
A. Infrared absorption spectrophotometry (2.2.24).
Mobile phase Mix 200 m L o f methanol R, 400 m L of
Comparison alcuronium chloride CRS. acetonitrile R and 400 m L o f a 6.82 g/L solution o f potassium
B. Thin-layer chromatography (2.2.27). dihydrogen phosphate R Dissolve 2.18 g o f sodium
Test solution Dissolve 10 m g o f the substance to be exam ined laurylsulfonate for chromatography R in the mixture and adjust
in methanol R and dilute to 10 m L with the same solvent.
2016 Alfacalcidol 1-87

the apparent p H to 5.4 with a 100 g/L solution oiphosphoric

ch2
add R.
Flow rate 1.2 mL/min. ci'
Detection Spectrophotom eter at 254 nm.
Irgecdon 10 jxL~
Run time Twice the retention time o f alcuronium.
System suitability Reference solution (d):
B. (4bS, 1R,1 aS, 8 ai?, 13i?, 13ai?, 13 bS )-l 3-hydroxy-7-(prop-2-
— resolution: m inim um 4.0 between the peaks due to
enyI)-5,6,7 a, 8, 8a, 11,13,13a, 13b, 14-decahydro-7,9-methano-
N ’-allylstrychnine and alcuronium.
7H-oxepino [3,4-a] pyrrolo [2,3-d] carbazolium chloride
Limits: ((4 R, 17i?)-4-allyl-l 7,18-epoxy-17 -hydroxy-19,2 0-
— impurities A , B: for each impurity, not m ore than the area didehydrocuranium chloride).
of the principal peak in the chromatogram obtained with
reference solution (a) (0.5 per cent) and not m ore than
one of the peaks has an area greater than the area of the
principal peak in the chromatogram obtained with
reference solution (b) ( 0.2 per cent);
★
— total: not m ore than twice the area of the principal peak in ★ ★
Alfacalcidol ★ ★
the chromatogram obtained with reference solution (a)
*****
(1 per cent); (Ph Eur monograph 1286)
— disregard limit: th e area o f the principal peak in the
chromatogram obtained w ith reference solution (c)
(0.05 per cent).
W a te r (2.5.12)
Maximum 5.0 p er cent, determined on 0.500 g.
S u lfa te d ash (2.4.14)
M aximum 0.1 p er cent, determ ined on 1.0 g.
A SSA Y
Dissolve 0.300 g by stirring in 70 m L of acetic anhydride R
for 1 min. T itrate with 0.1 M perchloric add until the colour
changes from violet-blue to greenish-blue, using 0.1 m L of
crystal violet solution R as indicator.
C 27H 44O 2 400.6 41294-56-8
1 m L of 0.1 M perchloric add is equivalent to 36.9 m g
Of C44H5()Cl2N402. A ctio n a n d u se
STORA GE Vitamin D analogue.
In an airtight container under nitrogen, protected from light,
Ph Eur__________________________________________________________
at a tem perature o f 2 °C to 8 °C.
D E F IN IT IO N
IM P U R IT IE S
(5Z ,7£)-9,10-Secocholesta-5,7,10(19)-triene-l 0,3 P-diol.
Specified impurities A, B
C o n te n t
97.0 per cent to 102.0 per cent.
A reversible isomérisation to pre-alfacalcidol takes place in
solution, depending on temperature and time. T he activity is
due to b oth compounds (see Assay).
CHARACTERS
A p p e a ra n c e
W hite or almost white crystals.
S o lu b ility
Practically insoluble in water, freely soluble in ethanol
(96 per cent), soluble in fatty oils.
A. (l£,3aS,9Æ,9a£,10Æ,llaS,12Æ,14aS,19aS,20Æ,20aÆ,
20bS,21i?,22aS)-l, 12-bis(prop-2-enyl)- It is sensitive to air, heat and light.
2,3,9a,11,1 la,13,14,19a,20a,21,22,22a-dodecahydro- ID E N T IF IC A T IO N
10tf,2 0 b H -l,2 3 :12,27-dim ethano-9,10:20,21- A. Infrared absorption spectrophotometry (2.2.24).
bis (epoxyprop [2] eno)-9H,2 OH- [1,5] diazocino [ 1,2,3-lm: 5,6,7-
Comparison Ph. Eur. reference spectrum of aÿacalddol.
l'm '] dipyirolo[2 ',3 '-dr.2 " ,3 " :d']dicarbazolediium dichloride
B. Examine the chromatograms obtained in the test for
(4,4 '-diallylcaracurin V dichloride),
related substances.
Results T he principal peak in the chromatogram obtained
with the test solution is similar in retention time and size to
the principal peak in the chromatogram obtained with
reference solution (a).
 1-88 Alfacalcidol 2016

TESTS T h e contents of an opened container are to be used

Related substances immediately.
Liquid chromatography (2.2.29): use the normalisation IMPURITIES
procedure. Cany out the test as rapidfy as possible, avoiding Specified impurities A, B.
exposure to light and air.
Other detectable impurities (die following substances would, if
Test solution Dissolve 1.0 m g o f the substance to be examined present at a suffident levd, be detected by one or other o f
without heating in 10.0 m L o f the mobile phase.
the tests in the monograph. T hey are limited by the general
Reference solution (a) Dissolve 1.0 mg o f alfacalcidol CRS acceptance criterion for other/unspecified impurities and/or
without heating in 10.0 m L o f the mobile phase. by the general monograph Substances for pharmaceutical use
Reference solution (b) Dilute 1.0 m L of reference solution (a) (2034). It is therefore not necessary to identify these
to 100.0 m L w ith the mobile phase. Dilute 1.0 m L o f this impurities for demonstration o f compliance. See also 5.10.
solution to 20.0 m L with the mobile phase. Control of impurities m substances for pharmaceutical use): C.
Reference solution (c) In order to prepare pre-alfacalddol in
situ, dissolve the contents o f a vial o f alfacalcidolfor system
suitability CRS (containing impurities A and B) in 25 m L of
the mobile phase, heat in a water-bath at 80 °C under a
reflux condenser for 2 h and cool.
Column:
— sizer. I = 0.25 m , 0 = 4.6 mm;
— stationary phase: end-capped octadecylsUyl silica gel for
chromatography R (5 |im).
Mobile phase ammonia R, water R, acetomtrUe R
(1:200:800 V/V/V).^
Flow rate 2.6 mL/min.
A. (51%7E)-9,10-secocholesta-5,7,10(19)-triene-1a,3{$-diol
Detection Spectrophotom eter at 265 nm .
(zrans-alfacalddol),
Injection 100 (iL o f the test solution and reference
solutions (b) and (c).
Run time Twice the retention time o f alfacaladol.
Identification of impurities Use the chromatogram supplied
with alfacalcidol for system suitability CRS and the
chromatogram obtained with reference solution (c) to identify
the peaks due to impurities A and B.
Relative retention W ith reference to alfacaladol (retention
time = about 21 min): pre-alfacalddol = about 0 .88;
impurity A = about 0.93; impurity B = about 1.1.
System suitability: reference solution (c):
— resolution: m inim um 1.5 between the peaks due to pre-
alfacalddol and impurity A and minimum l .5 between B. (5Z ,7£)-9,10-secocholesta-5,7,10(19)-triene-l P,3fi-diol
the peaks due to im purity A and alfacaladol. ( 10-calddol),
Limitsr.
— impurities A , B: for each impurity, maximum 0.5 p er cent;
— unspecified impurities: for each impurity, maximum
0.10 per cent;
— total: maximum 1.0 per cent;
— disregard limit: the area o f the prindpal peak in the
chromatogram obtained with reference solution (b)
(0.05 per cent); disregard the peak due to pre-alfacalddol.
ASSAY
Liquid chromatography (2.2.29) as described in the test for
related substances w ith the following modifications.
Injection T est solution and reference solutions (a) and (c).
System suitability: reference solution (c): C. 6^-[(3S,5i?)-3,5-dihydroxy-2-methylcydohex-l-en-l-yl]-2-
— repeatability: maximum relative standard deviation o f phenyl-2,5,10-triaza-4,19-dinor-9^-cholest-7-ene-1,3-dione.
1 per cent for the peak due to alfacaladol after P b E tr

6 injections.
Calculate the percentage content of C 27H 44O 2 taking into
account the assigned content o f alfacaladol CRS and, if
necessary, the peak due to pre-alfacalddol.
STORAGE
U nder nitrogen, in an airtight container, protected from light,
at a tem perature of 2 °C to 8 °C.
2016 Alfadex 1-89

temperature. Add 10 m L of ammonium molybdate reagent R1

Alfadex f * \ and allow to stand for 15 min.
*★ ★*
A lphacydodextrin * Reference solution Prepare a reference solution at the same
(Ph. Eur. monograph 1487) time and in the same manner as the test solution, using
1 m L of a 0.02 g/L solution o f glucose R.
Measure the absorbance (2.2.25) o f the test solution and the
reference solution at the absorption maximum at 740 nm
using water R as the compensation liquid. T he 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 n o t greater than 0.05.
Related substances
Liquid chromatography (2.2.29).
Test solution (a) Dissolve 0.25 g o f the substance to be
[C6H 10O5]6 973 10016-20-3
examined in water R with heating, cool and dilute to
25.0 m L with the same solvent.
A ctio n a n d u se
Cyclodextran; carrier molecule for drug delivery systems. Test solution (b) Dilute 5.0 m L o f test solution (a) to
50.0 m L with water R.
PhEur__________________________________________________________
Reference solution (a) Dissolve 25.0 m g of betadex CRS
D E F IN IT IO N (impurity A), 25.0 mg of gammacydodextrin CRS
Cyclohexakis-(l -> 4)- (a-D-glucopyranosyl) (cyclom altohexaose (impurity B) and 50.0 mg of alfadex CRS in water R, then
o r a-cyclodextrin). dilute to 50.0 m L with the same solvent.
C o n te n t Reference solution (b) Dilute 5.0 m L o f reference solution (a)
97.0 per cent to 102.0 per cent (dried substance). to 50.0 m L w ith water R.

CHA RACTERS
Reference solution (c) Dissolve 25.0 m g of alfadex CRS in
water R and dilute to 25.0 m L with the same solvent.
A p p e a ra n c e
Column:
White or almost white, amorphous or crystalline powder.
— sizer. I = 0.25 m, 0 = 4.6 mm;
S o lu b ility — stationary phase:, octadecylsUyl silica gel for chromatography R
Freely soluble in water, slightly soluble in propylene glycol, (10 |im).
practically insoluble in anhydrous ethanol and in methylene
Mobile phase methanol R, water R (10:90 VIV).
chloride.
Flow rate 1.5 mL/min.
ID E N T IF IC A T IO N
Detection Differential refractometer.
A. Specific optical rotation (see Tests).
Equilibration W ith the mobile phase for about 3 h.
B. Examine the chromatograms obtained in the assay.
Injection 50 |JL of test solution (a) and reference solutions (a)
Results T he principal peak in the chromatogram obtained
and (b).
with test solution (b) is similar in retention time and size to
the principal peak in the chromatogram obtained with Run time 3.5 times the retention time of alfadex.
reference solution (c). Relative retention W ith reference to alfadex (retention
C. Dissolve 0.2 g in 2 m L of iodine solution R4 by warming time = about 10 min): impurity B = about 0.7;
on a water-bath, and allow to stand at room temperature; impurity A = about 2.2.
a yellowish-brown precipitate is formed. System suitability: reference solution (a):
— resolution: minimum 1.5 between the peaks due to
TESTS
impurity B and alfadex; if necessary, adjust the
S o lu tio n S concentration of methanol in the mobile phase.
Dissolve 1.000 g in carbon dioxide-free water R and dilute to
Limits:
100.0 m L with the same solvent
— impurities A , B: for each impurity, not more than
A p p e a ra n c e o f so lu tio n 0.5 times the area of the corresponding peak in the
Solution S is clear (2.2.1). chromatogram obtained with reference solution (b)
p H (2.2.3) (0.25 per cent);
5.0 to 8.0. — sum of impurities other than A and B: not more than
Mix 1 m L o f a 223.6 g/L solution o f potassium chloride R and 0.5 times the area of the peak due to alfadex in the
30 m L o f solution S. chromatogram obtained with reference solution (b)
(0.5 per cent).
S p ecific o p tic a l ro ta tio n (2.2.7)
+ 147 to + 152 (dried substance), determined on solution S. Heavy metals (2.4.8)
Maximum 10 ppm.
R e d u c in g s u g a rs
2.0 g complies with test C. Prepare the reference solution
M aximum 0.2 p e r cent.
using 2 m L o f lead standard solution (10 ppm Pb) R.
Test solution T o 1 m L o f solution S add 1 m L o f cupri-tartaric
solution R4. H eat on a water-bath for 10 min, cool to room
 1-90 Alfentanil Hydrochloride 2016

L oss o n d ry in g (2.2.32) ★ ★
M axim um 11 per cent, determ ined on 1.000 g by drying in
Alfentanil Hydrochloride .★ ★
an oven at 120 °C for 2 h. (Ph. Eur. monograph 1062) *****
S u lfa te d a s h (2.4.14)
M axim um 0.1 per cent, determ ined on 1.0 g.
n « nv ch3
A SSA Y J. N -V
Liquid chromatography (2.2.29) as described in the test for HCI
related substances with the following modifications.
Injection T est solution (b) and reference solutions (a) and (c). ch3

System suitability:
— repeatability: maximum relative standard deviation o f
2.0 per cent for the peak due to alfadex after 5 injections C21H33CIN6O3 453.0 69049-06-5
o f reference solution (a).
A c tio n a n d u se
Calculate the percentage content o f [ O H i o C ^ from the Opioid receptor agonist; analgesic.
assigned content of alfadex CRS.
PhEur_______________________________
STO R A G E
In an airtight container. D E F IN IT IO N
N -[l- [2-(4-Ethyl-4,5-dihydro-5-oxo-1H -tetrazol-1-yl) ethyl] -4-
IM P U R IT IE S
(methoxymethyl)piperidin-4-yl]-N-phenylpropanamide
Specified impurities A, B hydrochloride.

OH HO
C o n te n t
98.5 per cent to 101.5 per cent (anhydrous substance).
CHARACTERS
A p p e a ra n c e
W hite or alm ost white powder.
S o lu b ility
F red y soluble in water, in ethanol (96 p er cent) and in
methanol.
mp: about 140 °C, with decomposition.
ID E N T IF IC A T IO N
A. Infrared absorption spectrophotom etry (2.2.24).
Comparison Ph. Eur. reference spectrum of alfentanil
hydrochloride.
A. cycloheptakis-(l-»4)-(a-D-glucopyranosyl) (betadex or B. Dissolve 50 mg in a mixture of 0.4 m L o f ammonia R and
cyclomaltoheptaose or ß-cyclodextrin), 2 m L of water R. Mix, allow to stand for 5 min and filter.
A ddify the filtrate with dilute nitric add R. It gives
reaction (a) of chlorides (2.3.1).
TESTS
A p p e a ra n c e o f so lu tio n
T he solution is clear (2.2.1) and colourless (2.2.2,
Method IT).
Dissolve 0.2 g in water R and dilute to 20 m L with the same
solvent.
R e la te d su b s ta n c e s
Liquid chrom atography (2.2.29).
Test solution Dissolve 0.100 g of the substance to be
examined in methanol R and dilute to 10.0 m L with die same
solvent.
Reference solution (a) In order to produce impurity E in situ,
dissolve 10 mg of the substance to be examined in 10.0 m L
o f dilute hydrochloric add R. H eat on a water-bath under a
B. cyclooctakis-(l -*4)-(a-D-glucopyranosyl) reflux condenser for 4 h. Neutralise with 10.0 m L o f dilute
(cydomaltooctaose or y-cydodextrin). sodium hydroxide solution R. Evaporate to dryness on a water-
bath. Cool and take up the residue in 10 m L o f methanol R.
PhEur
Filter.
Reference solution (b) Dilute 1.0 m L o f the test solution to
100.0 m L with methanol R. Dilute 5.0 m L o f this solution to
20.0 m L w ith methanol R.
Column:
— size. I = 0.1 m , 0 = 4.6 mm;
2016 Alfentanil Hydrochloride 1-91

— stationary phase: octadecybüyl silica gel for chromatography R h2

(3 \im). Ar- = R-
Mobile phase.
— mobile phase A: 5 g/L solution o f ammonium carbonate R in
a mixture o f 10 volumes of tetrahydrqfuran R and
90 volumes o f water R; o
Ar -R
— mobile phase B: acetonitrüe R] I f N

Time Mobile phase A Mobile phase B o

V xe”3
v
(min) (per cent V/V) (percent V/V)
0 -15 90->40 10 -»60 A. cis-N- [ 1- [2-(4-ethyl-4,5-dihydro-5-oxo-1//-tetrazol-1-
1 5 -2 0 40 60 yI)ethyl]-4-(methoxymethyl)piperidin-4-yl]-iV-
phenylpropanamide N-oxide,
2 0 -2 5 40 ->90 60-» 10

r i^ x î r
Flow rate 1.5 mL/min. o
Detection Spectrophotom eter at 220 nm. ^C H 3
Equilibration W ith acetomtrSe R for at least 30 min and then
0 V e
with the mobile phase at the initial composition for at least
B . trans-N- [ 1- [2-(4-ethyl-4,5-dihydro-5-oxo- lH -tetrazol-1-
5 min.
yl)ethyl]-4-(methoxymethyl)piperidin-4-yl]-iV-
Irqection 10 jiL; inject methanol R as a blank. phenylpropanamide N-oxide,
Retention time Im purity E = about 6 min; alfentanil = about
7 min. Ar / x
I f NH
Identification of impurities Use the chromatogram obtained
with reference solution (a) to identify the peak due to
o *^0 - CH3
impurity E; disregard any other peak.
System suitability: reference solution (a):
— resolution: m in im u m 4.0 between the peaks due to C. N - [4-(methoxymethyl)piperidm-4-yl]-N-
alfentanil and impurity E; if necessary, adjust the phenylpropanamide,
concentration o f acetonitrile in the mobile phase or adjust
the time programme for the linear-gradient elution.
Limits:
— impurities A , B, C, D, E, F, G, H: for each impurity, not
m ore than the area of the principal peak in the
chrom atogram obtained with reference solution (b)
(0.25 p er cent); D . N -[l- [2-(4-ethyl-4,5-dihydro-5-oxo-1//-tetrazol-1-
— total: not more than twice the area of the principal peak in yl)ethyI]-4-(methoxymethyl)piperidm-4-yl]-N-
the chromatogram obtained with reference solution (b) phenylacetamide,
(0.5 per cent);
— disregard limit. 0.2 times the area of the principal peak in
the chrom atogram obtained with reference solution (b)
(0.05 p er cent); disregard any peak due to the blank.
W a te r (2.5.12)
3.0 per cent to 4.0 p er cent, determined on 0.500 g.
E. 1-ethyl-1,4-dihydro-4- [2-[ [4-(methoxymethyl)-4-
A SSA Y phenylamino] piperidin-1-yl] ethyl]-5/f-tetrazol-5-one,
Dissolve 0.350 g in 50 m L of a m ixture o f 1 volume of
ethanol (96 per cent) R and 4 volumes o f water R and add Ar / s ,
5.0 m L o f 0.01 M hydrochloric add. T itrate with 0.1 M 1 I N

sodium hydroxide, d eterm ining the end-point hO t n^

potentiometrically (2.2.20). Read the volume added between o k 0 .CH3
the 2 points of inflexion.
1 m L o f 0.1 M sodium hydroxide is equivalent to 45.30 mg o f F. N- [ 1-(2-hydroxyethyl)-4-(methoxymethyl)piperidin-4-yl] -
C21H33C 1N 60 3 . iV-phenylpropanamide,
STO RA G E
Protected from light.
IMPURITIES
Specified impurities A, B, C, D , E, F, G, H

G. N -[l- [2-(4-ethyl-4,5-dihydro-5-oxo-1 //-tetrazo l-1-

yl) ethyl] -4-(propanoyloxymethyl)piperidin-4-yI] -N-
phenylpropanamide,
 1-92 Alfuzosin Hydrochloride 2016

Reference solution (b) Dissolve 4 m g o f alfuzosin for system

suitability CRS (containing impurities A and D ) in the mobile
phase and dilute to 10 m L with the mobile phase.
Column:
— sizer. I = 0.15 m , 0 = 4.6 mm ;
H . N -[l-[2-(4-ethyl-4j5-dihydro-5-oxo-li/-tetrazol-1- — stationary phase: end-capped octadecylsUyl silica gel for
yl) ethyl] -4-(methoxymethyl)piperidin-4-yi] -N- chromatography R (5 Jim).
phenylbutanamide.
Mobile phase Mix 1 volume of tetrahydrcfuran R, 20 volumes
PhEur o f acetonitrile R and 80 volumes o f a solution prepared as
follows: dilute 5.0 m L o f perchloric add R in 900 m L o f
water R, adjust to p H 3.5 with dilute sodium hydroxide
solution R and dilute to 1000 m L with water R.
★ ★ Flow rate 1.5 mL/min.
Alfuzosin Hydrochloride ★ ★
Detection Spectrophotom eter at 254 nm.
(Ph Eur monograph 1287) *****
Injection 10 jiL.
Run time Twice the retention tim e o f alfuzosin.
Identification of impurities Use the chrom atogram supplied
HzC0' y ^ Y '
with alfuzosin for system suitability CRS and the chromatogram
h3c o - ^ Y n obtained with reference solution (b) to identify the peaks due
to impurities A and D.
nh2 and enantiomer
Relative retention W ith reference to alfuzosin (retention
tim e = about 8 min): impurity D = about 0.4;
C 19H 28Q N 5O 4 425.9 81403-68-1 impurity A = about 1.2.
A ctio n a n d u se System suitability: reference solution (b):
Alpha ^adrenoceptor antagonist. — peak-to-valUy ratio: minimum 5.0, where Hp = height
above the baseline o f the peak due to im purity A and
P re p a r a tio n s
Hv = height above the baseline o f the lowest point of the
Alfuzosin Tablets curve separating this peak from the peak due to alfuzosin.
Prolonged-release Alfuzosin Tablets Limits:
PhEir ________________________________
— impurity D: not more than twice the area o f the principal
peak in the chromatogram obtained with reference
D E F IN IT IO N solution (a) (0.2 per cent);
(2RS)-N- [3- [(4-Amino-6j7 -dimethoxyquinazolm- 2-yl) — unspecified impurities: for each impurity, n o t more than the
methylaminojpropyl] tetrahydrofuran- 2-carboxamide area o f die principal peak in the chromatogram obtained
hydrochloride. with reference solution (a) ( 0.10 per cent);
C o n te n t — total: n o t more than 3 times the area of the principal peak
99.0 per cent to 101.0 per cent (anhydrous substance). in the chromatogram obtained with reference solution (a)
(0.3 p e r cent);
CHARACTERS
— disregard limit: 0.5 times the area o f the principal peak in
A p p e a ra n c e
the chromatogram obtained with reference solution (a)
W hite or alm ost white, crystalline powder, slightly
(0.05 p er cent).
hygroscopic.
W a te r (2.5.12)
S o lu b ility
M axim um 0.5 per cent, determ ined on 1.000 g.
Freely soluble in water, sparingly soluble in ethanol
(96 per cent), practically insoluble in methylene chloride. S u lfa te d a sh (2.4.14)
M axim um 0.1 per cent, determ ined on 1.0 g.
ID E N T IF IC A T IO N
A Infrared absorption spectrophotometry (2.2.24). A SSA Y
Dissolve 0.300 g in a mixture o f 40 m L o f anhydrous acetic
Comparison alfuzosin hydrochloride CRS.
add R and 40 m L o f acetic anhydride R T itrate with 0.1 M
B. It gives reaction (a) of chlorides (2.3.1). perchloric add, determining the end-point potentiometrically
TESTS (2.2.20).
p H (2.2.3) 1 m l, o f 0.1 Mperchloric add is equivalent to 42.59 m g
4.0 to 5.5. o f C 19H 28CIN 5O 4.
Dissolve 0.500 g in carbon dioxide-free water R and dilute to STO R A G E
25.0 m L with the same solvent. Use a freshly prepared In an airtight container, protected from light.
solution.
IM P U R IT IE S
R e la te d su b s ta n c e s
Specified impurities D .
Liquid chromatography (2.2.29).
Other detectable impurities (the following substances would, if
Test solution Dissolve 40 mg o f the substance to be examined
present at a sufficient level, be detected by one or other of
in the mobile phase and dilute to 100.0 m L with the mobile
the tests in the monograph. T hey are limited by die general
phase.
acceptance criterion for other/unspecified impurities and/or
Reference solution (a) Dilute 1.0 m L o f the test solution to by the general monograph Substances for pharmaceutical use
100.0 m L with the mobile phase. Dilute 1.0 m L o f this (2034). It is therefore not necessary to identify these
solution to 10.0 m L with the mobile phase.
2016 Alginic Acid 1-93

impurities for dem onstration o f compliance. See also 5.10. IDENTIFICATION

Control of impurities in substances for pharmaceutical use): A , B, A. T o 0.2 g add 20 m L o f water R and 0.5 m L o f sodium
C, E. carbonate solution R. Shake and filter. T o 5 m L of the filtrate
add 1 m L of calcium chloride solution R. A voluminous
gelatinous mass is formed.
B. T o 5 m L o f the filtrate obtained in identification test A
add 0.5 m L o f a 123 g/L solution of magnesium sulfate R.
N o voluminous gelatinous mass is formed.
nh2 C . T o 5 m g add 5 m L of water R, 1 m L of a freshly
prepared 10 g/L solution of 1,3-dihydroxynaphthalene R in
A. N-[3-[(4-amino-6,7-dimethoxyquinazolin-2-yl) ethanol (96 per cent) R and 5 m L of hydrochloric add R. Boil
methylamino] propyl] furan-2-carboxamide3 gently for 3 min, cool, add 5 m L o f water R, and shake with
15 m L of di-isopropyl ether R. Carry out a blank test.
T h e upper layer obtained with the substance to be examined
exhibits a deeper bluish-red colour than that obtained with
the blank.

nh2 TESTS
Chlorides
B. R = Cl: 2<hloro-637-dimethoxyquina2olin-4-amine3 M axim um 1.0 p er cent.

D . R = N (C H 3)-[C H 2]3-N H 2: N-(4-amino-637- T o 2.50 g add 50 m L of dilute nitric acid R, shake for 1 h
dimethoxyquinazolin-2-yl) -N-methylpropane-1j3-diamine3 and dilute to 100.0 m L with dilute nitric add R. Filter.
T o 50.0 m L of the filtrate add 10.0 m L of 0.1 M silver nitrate
E. R = N (C H 3)-[C H 2]3-N H -CO -H : AT-[3-[(4-amino-6,7-
and 5 m L of toluene R. Titrate with 0.1 M ammonium
dimethoxyquinazolin-2-yl)methylamino]propyi]formamide,
thiocyanate, using 2 m L o f ferric ammonium sulfate solution R2
as indicator and shaking vigorously towards the end-point.
H H. / - , 1 m L of 0.1 M silver nitrate is equivalent to 3.545 mg o f Cl.
H’ °Y ^Y V N'v ^ NY b J Heavy m etals (2.4.8)
h 3c o
M axim um 20 ppm .
NH2 and enantiomer 1.0 g complies w ith test F . Prepare the reference solution
using 2 m L o f lead standard solution (10 ppm Pb) R.
C . (2RS)-N-[3-[(4-amino-6,7-dimethoxyquinazolin-2-yl) Loss on drying (2.2.32)
amino] propyl] -N-meiiiyltetrahydrofuran-2-carfaoxam ide. M axim um 15.0 p er cent, determined on 0.1000 g by drying
in an oven at 105 °C for 4 h.
PhEur
Sulfated ash (2.4.14)
Maximum 8.0 p er cent (dried substance), determined on
0.100 g.
M icrobial contamination
Alginic Acid ?**% T A M C : acceptance criterion 102 C FU /g (2.6.12).
(Ph Eur monograph 0591) * Absence o f Escherichia coli (2.6.13).
Absence o f Salmonella (2.6.13).
Action and use
T reatm ent o f gastro-oesophageal reflux disease; excipient; ASSAY
thickening agent. T o 0.2500 g add 25 m L of footer R, 25.0 m L o f 0.1 M
sodium hydroxide and 0.2 m L of phenolphthalein solution R.
PhEur__________________________________________________________ T itrate with 0.1 M hydrochloric acid.
DEFINITION 1 m L of 0.1 M sodium hydroxide is equivalent to 4.502 m g of
M ixture of polyuronic acids [(C6H80 6) J composed of carboxyl groups (-C 0 2H ).
residues of p-m annuronic and L-guluronic acids, obtained FUNCTIONALITY-RELATED CHARACTERISTICS
mainly from algae belonging to the Phaeophyceae. A small This section provides information on characteristics that are
proportion o f the carboxyl groups may be neutralised. recognised as being relevant control parameters for one or more
Content functions of the substance when used as an excipient (see chapter
19.0 per cent to 25.0 p er cent of carboxyl groups (-C 0 2H) 5.15). This section is a non-mandatory part of the monograph
(dried substance). and it is not necessary to verify the characteristics to demonstrate
CHARACTERS compliance. Control of these characteristics can however contribute
to the quality of a medicinal product by improving the consistency
Appearance
of the manufacturing process and the performance of the medicinal
W hite or pale yellowish-brown, crystalline or amorphous
product during use. Where control methods are cited, they are
powder.
recognised as bang suitable for the purpose, but other methods can
Solubility also be used. Wherever results for a particular characteristic are
Very slightly soluble o r practically insoluble in ethanol reported, the control method must be indicated
(96 per cent), practically insoluble in organic solvents.
The following characteristics may be relevant for alginic add used
It swells in w ater b u t does not dissolve; it dissolves in
as disintegrant and/or binder.
solutions of alkali hydroxides.
 1-94 Alimemazine 2016

P a rtic le -siz e d is trib u tio n (2.9.31 or 2.9.38). Dissolve 1.0 g in water R and dilute to 10 m L with the same
S e ttlin g v o lu m e solvent.
Place 75 m L o f water R in a 100 m L graduated cylinder and p H (2.2.3)
add 1.5 g of die substance to be examined in 0.5 g portions, 5.0 to 6.5. Carry out the test protected from light and use a
shaking vigorously after each addition. Dilute to 100.0 m L freshly prepared solution.
with water R and shake again until the substance is Dissolve 1.0 g in carbon dioxide-free water R and dilute to
homogeneously distributed. Allow to stand for 4 h and 50 m L with the same solvent.
determine the volume o f the settled mass.
Related substances
The following characteristic may be relevant for alginic acid used Liquid chromatography (2.2.29). Carry out the test protected
as gelling agent or viscosity-increasing agent. from light and use freshly prepared solutions.
A p p a re n t viscosity Solvent mixture acetomtrüe R, water R (20:80 VIV).
D eterm ine the dynamic viscosity using a rotating viscometer
Test solution Dissolve 35 m g o f the substance to be examined
(2.2.1Q).
in the solvent mixture and dilute to 100.0 m L with the
Prepare a 20 g/L suspension o f alginic a d d (dried substance) solvent mixture.
and add 0.1 M sodium hydroxide until a solution is obtained.
Reference solution (a) D ilute 1.0 m L o f the test solution to
__________________________________________________________ PhEur 100.0 m L with the solvent mixture. D ilute 1.0 m L of this
solution to 10.0 m L with the solvent mixture.
Reference solution (b) Dissolve 3.5 m g o f alimemazine for
system suitability CRS (containing impurities A, B and C) in
***** the solvent mixture and dilute to 10.0 m L with the solvent
Alimemazine Tartrate ★ ★ mixture.
(Alimemazine Hemitartrate Ph. Bur. monograph ***** Column:
2650) — size. I = 0.15 m , 0 = 4.6 mm;
— stationary phase, base-deactivated end-capped octadecylsüyl
silica gelfor chromatography R (3 |im );
H OH — temperature. 40 °C.
n' ^ v ^ ' n ' CH3 COjH Mobile phase acetomtrüe R, methanol R , 3.854 g/L solution o f
I I H i i and dnantiomer , HOî C
CH3 c h 3 ammonium acetate R (10:40:50 VÍVIV).
H OH
Flow rate 1.3 mL/min.
Detection Spectrophotom eter at 253 nm .
Injection 20 jiL.
C20H 25N 2O3S 373.5 4330-99-8
Run time Twice the retention time o f alimemazine.
A ctio n a n d u se Identification of impurities Use the chrom atogram supplied
Histamine H i, receptor antagonist; sedative with alimemazine for system suitability CRS and the
P re p a ra tio n s chromatogram obtained with reference solution (b) to
Paediatric Alimemazine Oral Solution identify the peaks due to impurities A, B and C.
Strong Paediatric Alimemazine Oral Solution Relative retention W ith reference to alimemazine (retention
Alimemazine Tablets time = about 27 min): impurity A = about 0.1;
impurity B = about 0.5; impurity C = about 1.4.
PhEur__________________________________________ System suitability: reference solution (b):
D E F IN IT IO N — resolution: minimum 5.0 between the peaks due to
(2RS)-N,N, 2-Trimethyi-3-( 1OH-phenothiazin-10-yl)propan- alimemazine and im purity C.
1-am ine hemi [(2Æ,3iQ-2,3-dihydroxybutanedioate]. Calculation of percentage contents:
C o n te n t — correction factors: multiply the peak areas o f the following
99.0 per cent to 101.0 per cent (dried substance). impurities by the corresponding correction facto r
impurity A = 4.4; impurity C = 0.4;
CHARACTERS — for each impurity, use the concentration o f alimemazine
A p p e a ra n c e in reference solution (a).
W hite or very slightly yellowish powder. Limits:
S o lu b ility — impurity B: máximum 0.3 per cent;
F red y soluble in water, sparingly soluble in ethanol — impurities A, C: for each impurity, maximum
(96 per cent), practically insoluble in toluene. 0.15 p er cent;
It deteriorates when exposed to air and light. — unspecified impurities: for each impurity, maximum
0.10 p er cent;
ID E N T IF IC A T IO N
— total: maximum 0.5 p er cent;
Infrared absorption spectrophotom etry (2.2.24). — reporting threshold: 0.05 per cent.
Comparison alimemazine hemitartrate CRS. Loss on drying (2.2.32)
TESTS M axim um 0.5 per cent, determined on 1.000 g by drying in
A p p e a ra n c e o f so lu tio n an oven at 105 °C for 3 h.
T he solution is not m ore opalescent than reference Sulfated ash (2.4.14)
suspension II (2.2.1) and n o t more intensely coloured than M axim um 0.1 per cent, determ ined on 1.0 g.
reference solution BY5 (2.2.2, Method IT).
2016 Allantoin 1-95

ASSAY It melts at about 225 °C, with decomposition.

D isso lv e 0.300 g in 50 m l , o f anhydrous acetic acid R. Titrate ID E N T IF IC A T IO N
with 0.1 M perchloric acid, determining the end-point First identification A.
potentiometrically (2.2.20).
Second identification B, C, D.
1 m L of 0.1 M perchloric acid is equivalent to 37.35 mg of
A. Examine by infrared absorption spectrophotometry
C 20H 25N 2O 3S .
(2.2.24), comparing with the spectrum obtained with
STORAGE allantoin CRS.
In an airtight container, protected from light. B. Examine the chromatograms obtained in the test for
IM P U R IT IE S related substances. T h e principal spot in the chrom atogram
Specified impurities A, B, C obtained with test solution (b) is similar in position, colour
and size to the principal spot in the chromatogram obtained
with reference solution (a).
C. Boil 20 m g w ith a mixture of 1 m L of dilute sodium
enantiomer hydroxide solution R and 1 m L of water R. Allow to cool.
s A CH3 CH3 Add 1 m L o f dilute hydrochloric add R. T o 0.1 m L of the
solution add 0.1 m L o f a 100 g/L solution of potassium
u bromide R, 0.1 m L o f a 20 g/L solution o f resorcmd R and
3 m L o f sulfuric add R. H eat for 5 min to 10 min on a water-
A. (2 i^ -iS />N,2-trimethyl-3-(5-oxido-l OH-phenothiazin-10- bath. A dark blue colour develops, which becomes red after
yl) propan-1-am ine, cooling and pouring into about 10 m L of water R.
D . H eat about 0.5 g. Ammonia vapour is evolved, which
turns red litmus paper R blue.

enantiomer TESTS
3 and
S o lu tio n S
Dissolve 0.5 g in carbon dioxide-free water R, with heating if
necessary, and dilute to 100 m L with the same solvent.
A cid ity o r a lk a lin ity
B . (2i?S}-ATj2-dimethyl-3-( 1OH-phenothiazin-10-yl)propan-1-
T o 5 m L o f solution S add 5 m L of carbon dioxide-free
amine,
water R, 0.1 m L o f methyl red solution R and 0.2 m L of
0.01 M sodium hydroxide. T he solution is yellow. Add 0.4 m L
o f 0.01 M hydrochloric acid The solution is red.
O p tic a l ro ta tio n (2.2.7)
T h e angle of optical rotation, determined on solution S, is
-0 .1 0 ° to + 0.10°.
R ed u c in g su b sta n c e s
Shake 1.0 g with 10 m L o f water R for 2 min. Filter.
C. 1OH-phenothiazine. Add 1.5 m L o f 0.02 Mpotassium permanganate. T he solution
Ph Bur m ust rem ain violet for at least 10 min.
R e la te d su b sta n c e s
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 o f the substance to be
(Ph. Eur. monograph 1288) * * * * * examined in 5.0 m L of water R with heating. Allow to cool.
Dilute to 10 m l, with methanol R. Use the solution immediately
o after preparation.
f H-
H. . y ' NH and enantiomer Test solution (b) Dilute 1 m L of test solution (a) to 10 m L
H,N with a mixture o f 1 volume of methanol R and 1 volume o f
water R.
Reference solution (a) Dissolve 10 m g of allantoin CRS in a
€4 ^ 403 158.1 97-59-6 mixture o f 1 volume o f methanol R and 1 volume of water R
and dilute to 10 m L with the same mixture of solvents.
A ctio n a n d u se
Astringent; keratolytic. Reference solution (b) Dissolve 10 m g of urea R in 10 m L o f
water R. Dilute 1 m L o f this solution to 10 m L with
PhEir ___________________ methanol R
D E F IN IT IO N Reference solution (c) M ix 1 m L o f reference solution (a) and
Allantoin contains n o t less than 98.5 per cent and not more 1 m L of reference solution (b).
than the equivalent o f 101.0 per cent of Apply to the plate 10 jiL o f test solution (a) and 5 |iL each
(RS) -(2 j 5-dioxoimidazolidin-4-yl)urea. o f test solution (b), reference solution (a), reference
solution (b) and reference solution (c). Develop over a path
CHARACTERS
o f 10 cm using a mixture of 15 volumes of glacial acetic
A white or alm ost white, crystalline powder, slightly soluble
add R, 25 volumes o f water R and 60 volumes of butanol R.
in water, very slightly soluble in alcohol.
 1-96 Allergen products 2016

Allow the plate to dry in air. Spray the plate with a 5 g/L prepared by dilution of aqueous or glycerinated extracts, or
solution of dimethylaminobenzaldehyde R in a mixture of by reconstitution o f unmodified freeze-dried extracts.
1 volume of hydrochloric add R and 3 volumes of methanol R. F or specific immunotherapy, allergen products may be either
Dry the plate in a current o f h o t air. Examine in daylight unmodified extracts or extracts modified chemically and/or
after 30 min. Any spot in the chromatogram obtained with by adsorption onto different carriers (for example, aluminium
test solution (a), apart from th e principal spot, is not more hydroxide, calcium phosphate or tyrosine).
intense than the spot in the chrom atogram obtained with
reference solution (b) (0.5 p er cent). T h e test is not valid P R O D U C T IO N
unless the chrom atogram obtained with reference solution (c) S O U R C E M A T E R IA L S
shows two clearly separated principal spots. Source materials for the preparation of allergen products are
products o f animal o r vegetable origin, mostly pollens,
L oss o n d ry in g ( 2.2.32)
moulds, mites, animal epithelia and outgrowths (such as hair
N ot more than 0.1 p e r cent, determined on 1.000 g by
and feathers) and/or dander, hymenoptera venoms, insects
drying in an oven at 105 °C.
and certain foods.
S u lfa te d a s h (2.4.14)
W here allergen products are m anufactured using materials of
N ot more than 0.1 p e r cent, determined on 1.0 g.
hum an or animal origin, the requirements o f chapter 5.1.7.
ASSAY Viral safety apply.
Dissolve 120.0 mg in 40 m L o f water R. T itrate with 0.1 M T h e source materials are defined by their origin, nature,
sodium hydroxide, determining the end-point m ethod o f collection or production and pretreatm en t
potentiometrically (2.2.20). Control m ethods and acceptance criteria relating to identity
1 m L of 0.1 M sodium hydroxide is equivalent to 15.81 m g of and purity are established. T h e acceptance criteria m ust
C 4H 6N 4O 3. ensure the consistency o f the allergenic source material from
a qualitative and quantitative point o f view. T h e source
IM P U R IT IE S
materials are stored under controlled conditions justified by
H CO2H stability data.
To T h e collection or production, as well as the handling o f the
source materials are such that uniform composition is
A. glyoxylic acid, ensured as far as possible from batch to batch.
T h e content of the relevant residual solvents, heavy metals
o and pesticides is determ ined on a num ber o f batches
according to a justified sampling plan. Residual solvents and
pesticides are limited according to the principles defined in
general chapter 2.4.24. Identification and control of residual
B. carbamide (urea).
solvents and 2.8.13. Pesticide residues respectively.
--------------------------------- — .— _____________________________PhEur
P o lle n s
Potential chemical contam inants, such as pesticides, heavy
metals and solvents, m ust be minimised. T h e content of
foreign pollen m ust be limited to 1 per cent o f total mixed
Allergen Products ***** pollens and 0.5 per cent o f any individual pollen as
determined by a microscopic particle count. D etectable
(Ph Eur. monograph 1063) * m ould spores m ust not exceed 1 per c e n t
Ph Fur _________________________________________ T h e contam ination with particles o f plant origin other than
This monograph does not apply to: chemicals that are used solely pollen m ust be kept to a minim um. T he m axim u m allowed
for diagnosis of contact dermatitis; chemically synthesised products; contam ination m ust be justified.
allergens derived by recombinant DNA technology. It does not M o u ld s
necessarily apply to allergen products for veterinary use. Biologically active contam inants such as mycotoxins in
D E F IN IT IO N moulds m ust be m in im ised and any presence justified.
Appropriate measures have to be im plem ented to avoid
Allergen products are pharm aceutical preparations derived
c o n tam inatio n by foreign m ould strains. C are m ust be taken
from extracts of naturally occurring source materials
to minimise any allergenic constituents o f the media vised for
containing allergens, which are substances that lead to and/or
provoke allergic reactions. T h e allergenic components are the cultivation of moulds as source materials. Culture media
th at contain substances of hum an or anim al origin m ust be
most often o f a proteinaceous nature. Allergen products are
intended for in vivo diagnosis o r treatm ent o f allergic diseases justified and, when required, m ust be suitably treated to
attributed to these allergens. ensure the inactivation or elimination of possible
transmissible agents of disease.
Allergen products are available as finished products, and as
finished products used on a nam ed-patient basis. Allergen T h e production m ethod is validated to demonstrate that
products are generally presented as parenteral preparations, allergen products obtained from moulds and intended for
parenteral administration, if tested* would comply with the
eye preparations, preparations for inhalation, preparations for
test for abnormal toxicity for immunosera and vaccines for
oral use, sublingual preparations or preparations for skin
tests. hum an use (2.6.9).

For in vivo diagnostic use, allergen products are usually M ite s

prepared as unmodified extracts in a 50 per cent VIV Appropriate measures have to be implem ented to avoid
solution of glycerol for skin testing. F o r intradermal diagnosis co n tam inatio n by foreign m ite strains. Care m ust be taken to
or for provocation tests by nasal, ocular or bronchial m in im ise any allergenic constituents o f the media used for
administration, suitable dilutions o f allergen products may be the cultivation of mites as source materials. Culture media
2016 Allergen products 1-97

th at contain substances of hum an or animal origin m ust be of suitable reagents, as well as the intended use. The characterised
justified and, when required, must be suitably treated to IHRP is used as the reference in the batch control of active
ensure the inactivation or elimination of possible substances and intermediates and, if possible, in the batch control
transmissible agents of disease. offinished products.
Anim al epithelia and outgrowths and/or dander T he IH R P is characterised by the protein content
T hey are obtained from healthy animals selected to avoid determination and a protein profile using appropriate
possible transmissible agents of disease. methods (such as isoelectric focusing, sodium dodecyl sulfate
Hymenoptera venoms polyacrylamide gel electrophoresis, Immunoelectrophoresis,
T h e species of hymenoptera from which the venom is capillary electrophoresis, chromatographic techniques and
mass spectrometry).
extracted is identified and specified. T h e m ethods of insect
collection and venom extraction are described and m ust Allergenic components may be detected by appropriate
ensure that the source material is of proper quality. methods (for example, immunoblotting or crossed radio-
im munoelecupphoresis). Characterisation of the allergenic
Food
components may include identification o f relevant allergens
T h e scientific nam e (species, variety, strain etc.) o f the
based on serological or other techniques using pooled or
an im al or vegetable species is indicated and the p art used is
individual sera from allergic patients, or allergen-specific
stated, if applicable. Foods must be o f a quality suitable for
polyclonal or monoclonal antibodies.
hum an consum ption. T he origin o f the food stuff as well as
its processing stage is stated. Determ ination o f the content of relevant allergens is
performed wherever possible. T his determination may be
MANUFACTURING PROCESS m ade using individual allergen-specific reference standards,
Allergen products are generally obtained by extraction, and w hen available. T h e choice of the relevant allergen
m ay be purified, from the source materials using appropriate components subjected to the determination m ust be justified.
m ethods shown to preserve the allergenic properties o f the Individual allergens are identified and named according to
com ponents. Allergens for which there are not enough internationally established nomenclature wherever possible.
patients to determine the total allergenic activity in vivo or in
T h e biological potency o f the first IH R P is determined in
vitro, the extraction ratio indicating the relative proportions
patients by in vivo techniques such as skin testing, and
(mlV) o f allergenic source materials and solvents is a
expressed in units of biological activity except when not
minimum requirem ent. Allergen products presented as
enough patients are available. In this case, the potency of the
parenteral preparations, eye preparations, preparations for
first IH R P is determ ined by an m vitro method.
inhalation and preparations for skin testing are m anufactured
Subsequently, the biological activity of future EHRPs is
u nder aseptic conditions.
demonstrated by m vitro methods by comparison with the
In the manufacture, packaging, storage and distribution o f results obtained with the first IH R P. T he in vitro potency
allergen products intended for administration by other routes, may be measured by a suitable immunoassay (for example,
suitable measures are taken to ensure their microbial quality; an assay based on the inhibition o f the binding capacity of
recom m endations on this aspect are provided in chapter specific immunoglobulin E antibodies).
5.1.4. Microbial quality of non-sterUe pharmaceutical preparations
and substances for pharmaceutical use. IDENTIFICATION
T h e tests for identification are performed as late as possible
All allergen preparations are m anufactured under conditions
in the manufacturing process. In the case of products used
designed to minimise exogenous and endogenous enzymatic
on a nam ed-patient basis, the control is performed on the
degradation.
active substance and/or at the intermediate stage between the
Any purification procedure is designed to minimise the active substance and the finished product.
content o f any potential irritant low molecular mass
Identity is confirmed by comparison with the IH R P using
com ponents and non-allergenic components.
protein profiling by appropriate methods (for example,
Allergen products m ay contain suitable antimicrobial isoelectric focusing, sodium dodecyl sulfate polyacrylamide
preservatives, the nature and concentration of which have to gel electrophoresis, Immunoelectrophoresis, immunoblotting,
be justified. liquid chromatography o r mass spectrometry).
T h e manufacturing process comprises various stages: In exceptional cases, if no IH R P is available, a representative
— source material; batch may be used to confirm identity.
— active substance: it is generally a modified or an
Identity may also be confirmed by comparison with
unm odified allergen extract; where applicable it is stored
individual allergen-specific reference standards, when
under conditions ensuring its stability, for example freeze-
available.
dried;
— finished product. TESTS
All other stages of the manufacturing process are considered T h e tests are perform ed as late as possible in the
as intermediates. manufacturing process. In the case o f products used on a
named-patient basis, th e control is performed on the active
IN-HOUSE REFERENCE PREPARATION substance and/or at the intermediate stage between the active
An appropriate representative preparation is selected as the substance and the finished product.
in-house reference preparation (IHRP), characterised and
Various biochemical and immunological tests have been
used to verify batch-to-batch consistency. T he IH R P is
developed in order to characterise allergens qualitatively and
stored in suitably sized aliquots under conditions ensuring its
quantitatively. In those cases where such m ethods cannot be
stability, for example freeze-dried.
applied, particularly for the determination o f allergenic
Characterisation o f the in-house reference preparation activity and allergen and/or protein profile, justification m ust
The extent of characterisation of the IHRP depends on the source be provided.
material, knowledge of the aUergejric components and availability
 1-98 Allopurinol 2016

W a te r (2.5.12) or (2 J J 2 ) the biological potency and/or the protein content and/or

M aximum 5 per cent for freeze-dried products. the extraction concentration;
In the case o f oral lyophilisates, the w ater content may be the route o f administration and die intended use;
higher than 5 per cent, where justified and authorised. the storage conditions;
where applicable, the name and am ount of added
S te rility (2.6.1)
antimicrobial preservative;
Allergen products presented as parenteral preparations, eye
where applicable, for freeze-dried preparations:
preparations, preparations for inhalation or preparations for
— the name, composition and volume o f the
skin testing comply with the test for sterility.
reconstituting liquid to be added;
M icro b ial c o n ta m in a tio n — the period o f tim e within which the preparation is to
For non-sterile allergen products, recommendations are be used after reconstitution;
provided in 5.1.4. Microbial quality of non-sterile pharmaceutical where applicable, that the preparation is sterile;
preparations and substances for pharmaceutical use. where applicable, the nam e and am ount of adsorbent.
P ro te in c o n te n t (2.5.33) PhEur
80 per cent to 120 p er cent o f die stated content, unless
otherwise justified and authorised. If the biological potency
can be determined then the test for protein content is
performed as a batch-to-batch consistency test and the ★*★
★ ★
protein content is within 50 per cent to 150 per cent o f the Allopurinol ★ ★
stated content. W hen the finished product contains
(Ph. Eur. monograph 0576) * * * * *
proteinaceous excipients, the test for protein content is
performed as late as possible during production before
addition o f the proteinaceous excipient.
P ro te in p ro file NH
T he protein profile determined by suitable methods J
corresponds to that o f the IH R P. T h e presence o f relevant
allergen components is verified, where possible. T he choice
of relevant allergen components to be tested for m ust be C5H4N4O 136.1 315-30-0
justified.
A c tio n a n d u se
Various additional tests, some with increasing selectivity, Xanthine oxidase inhibitor; treatm ent o f gout and
depending on the allergen product concerned can be applied, but in hyperuricaemia.
any casefor allergen products intended for therapeutic use, a
P re p a r a tio n s
validated test measuring the potency (total allergenic activity,
Allopurinol Oral Suspension
determination of individual allergens or any otherjustified tests)
must be applied. Allopurinol Tablets
A lu m in iu m (2.5.13) PhEur__________________________________________________________
80 per cent to 120 per cent o f the stated amount bu t in any
case not m ore than 1.25 m g per hum an dose unless D E F IN IT IO N
otherwise justified and authorised, w hen aluminium 1,5-Dihydro-4ii-pyrazolo [3,4 -d]pyrimidin-4-one.
hydroxide or aluminium phosphate is used as adsorbent. C o n te n t
C a lc iu m (2.5.14) 97.0 per cent to 102.0 per cent (dried substance).
80 per cent to 120 p er cent o f the stated amount when CHARACTERS
calcium phosphate is used as adsorbent. A p p e a ra n c e
A llergen p ro file W hite o r alm ost white powder.
Relevant allergenic components are identified by means of S o lu b ility
suitable techniques using allergen-specific hum an or animal Very slighdy soluble in water and in ethanol (96 p er cent).
antibodies. It dissolves in dilute solutions o f alkali hydroxides.
T o ta l allerg en ic a c tiv ity ID E N T IF IC A T IO N
50 per cent to 150 per cent o f the stated am ount as assayed First identification B
by inhibition o f the binding capacity o f specific
Second identification A , C, D
im munoglobulin E antibodies or a suitable equivalent in vitro
method. A. Ultraviolet and visible absorption spectrophotometry
(2.2.25).
In d iv id u a l allerg en s
50 per cent to 200 per cent o f the stated amount o f each Test solution Dissolve 10 mg in 1 m L o f a 4 g/L solution of
relevant allergen com ponent, determined by a suitable sodium hydroxide R and dilute to 100.0 m L with a 10.3 g/L
method. solution o f hydrochloric acid R. Dilute 10.0 m L o f this
solution to 100.0 m L with a 10.3 g/L solution o f hydrochloric
STO RA G E acid R.
Adsorbed allergen products are not to be frozen, unless
Spectral range 220-350 nm .
otherwise justified and authorised.
Absorption maximum A t 250 nm .
L A B E L L IN G
Absorption minimum At 231 nm.
The label states:
Absorbance ratio ^ 231^250 = 0-52 to 0.62.
— the nam e o f the allergen product;
B. Infrared absorption spectrophotom etry (2.2.24).
Comparison aOopurinol CRS.
2016 Allopurinol 1-99

C. Dissolve 0.3 g in 2.5 m L of düute sodium hydroxide Limits:

solution R and add 50 m L o f water R. Add slowly and with — impurity A: n o t more than twice the area of th e principal
shaking 5 m L o f silver nitrate solution R l. A white precipitate peak in the chromatogram obtained with reference
is formed which does not dissolve on the addition of 5 m L of solution (a) (0.2 per cent);
ammonia R. — impurity B: n o t more than the area of the principal peak
D . Thin-layer chromatography (2.2.27). in the chromatogram obtained with reference solution (a)
(0.1 per cent);
Test solution Dissolve 20 m g of the substance to be examined
— impurity C: n o t more than the area of the corresponding
in concentrated ammonia R and dilute to 10 m L with the same
peak in the chromatogram obtained with reference
solvent.
solution (b) (0.1 per cent);
Reference solution Dissolve 20 mg of aUopurinol CRS in — unspecified impurities: for each impurity, not m ore than the
concentrated ammonia R and dilute to 10 m L with the same area o f the principal peak in the chromatogram obtained
solvent. with reference solution (a) (0.10 per cent);
Plate TLC silica gel F2 5 4 plate R. — sum of impurities other than A , B and C: not m ore than
Mobile phase anhydrous ethanol R, methylene chloride R 3 times the area of the principal peak in the
(40:60 VIV). chrom atogram obtained with reference solution (a)
Application 10 |iL. (0.3 p er cent);
— disregard limit. 0.5 times the area of the principal peak in
Development Over 2/3 o f the plate.
the chromatogram obtained with reference solution (a)
Drying In air. (0.05 p er cent).
Detection Examine in ultraviolet light at 254 nm . Impurities D and E
Results T h e principal spot in the chromatogram obtained with Liquid chromatography (2.2.29). Use freshfy prepared solutions.
the test solution is similar in position and size to the principal Store and inject them at 8 °C} using a cooled autosampler.
spot in the chromatogram obtained with the reference
Solution A 1.25 g/L solution of potassium dihydrogen
solution.
phosphate R.
TESTS Test solution Dissolve 50.0 mg of the substance to be
Related substances examined in 5.0 m L of a 4 g/L solution o f sodium hydroxide R
Liquid chromatography (2.2.29). Use freshly prepared solutions. and dilute immediately to 100.0 m L with solution A.
Store and inject them at 8 °C, using a cooled autosampler. Reference solution Dissolve 5.0 m g o f aUopurinol
Test solution (a) Dissolve 25.0 mg of the substance to be impurity D CRS and 5.0 m g of aUopurinol impurity E CRS in
examined in 2.5 m L o f a 4 g/L solution of sodium hydroxide R 5.0 m L o f a 4 g/L solution of sodium hydroxide R and dilute
and dilute immediately to 50.0 m L with the mobile phase. immediately to 100.0 m L with solution A. Dilute 1.0 m L of
Test solution (b) Dissolve 20.0 mg o f the substance to be this solution to 100.0 m L with solution A.
examined in 5.0 m L o f a 4 g/L solution of sodium hydroxide R Column:
and dilute immediately to 250.0 m L with the mobile phase. — size: I = 0.05 m , 0 = 4.6 mm;
Reference solution (a) Dilute 2.0 m L o f test solution (a) to — stationary phase: base-deactivated octadecylsUyl silica gel for
100.0 m L with the mobile phase. Dilute 5.0 m L of this chromatography R (3 (am).
solution to 100.0 m L with the mobile phase. Mobile phase methanol R> 1.25 g/L solution of potassium
Reference solution (b) Dissolve 5 mg o f aUopurinol dihydrogen phosphate R (10:90 VIV).
impurity A CRS, 5 m g of aUopurinol impurity B CRS and Flow rate 2 mL/min.
5.0 mg o f aUopurinol impurity C CRS in 5.0 m L o f a 4 g/L Detection Spectrophotom eter at 230 nm.
solution o f sodium hydroxide R and dilute immediately to
Irtjection 20 |iL.
100.0 m L with the mobile phase. Dilute 1.0 m L o f this
solution to 100.0 m L with the mobile phase. Run time 1.5 times the retention time o f impurity E.
Reference solution (c) Dissolve 20.0 m g o f aUopurinol CRS in Retention times Im purity D = about 3.6 min;
5.0 m L o f a 4 g/L solution o f sodium hydroxide R and dilute im purity E = about 4.5 min.
immediately to 250.0 m L with the mobile phase. System suitability: reference solution:
Column: — resolution: minim um 2.0 between the peaks due to
— size: I = 0.25 m , 0 = 4.6 mm; impurities D and E.
— stationary phase: octadecylsSyl silica gel for chromatography R Limits:
(5 nm). — impurity D: n o t more than the area of the corresponding
Mobile phase 1.25 g/L solution of potassium dihydrogen peak in the chromatogram obtained with the reference
phosphate R. solution (0.1 p er cent);
— impurity E: n o t more than the area o f the corresponding
Flow rate 1.4 mL/min.
peak in the chromatogram obtained with the reference
Detection Spectrophotom eter at 230 nm . solution (0.1 p er cent).
Injection 20 |iL of test solution (a) and reference solutions (a) Impurity F
and (b). Liquid chromatography (2.2.29).
Run time Twice the retention time of aUopurinol. U nder the following conditions, any hydrazine in the sample
Elution order Im purity A, impurity B, impurity C , aUopurinol. reacts with benzaldehyde to give benzaldehyde azine.
Retention time AUopurinol = about 10 min. Solvent mixture M ix equal volumes o f dilute sodium hydroxide
System suitability: reference solution (b): solution R and methanol R.
— resolution: minim um 1.1 between the peaks due to
impurities B and C.
 I- 100 Almagate 2016

Solution A Dissolve 2.0 g of benzaldehyde R in the solvent

m ixture and dilute to 50.0 m L with the solvent mixture.
Prepare immediately before use.
N‘ m-
Test solution Dissolve 250.0 m g of the substance to be H N
examined in 5 m L o f the solvent mixture. Add 4 m L of
solution A, mix and allow to stand for 2.5 h at room A. R1 = N H 23 R2 = H : 5-am ino-li/-pyrazole-4-
tem perature. Add 5.0 m L o f hexane R and shake for 1 min. carboxamide,
Allow the layers to separate and use the upper layer. B. R1 = N H 2, R2 = C H O : 5-(fonnylam ino)-lH-pyrazole-4-
Reference solution Dissolve 10.0 mg o f hydrazine sulfate R in carboxamide,
the solvent mixture by sonicating for about 2 min and dilute D . R1 = 0 - C 2H 5, R2 = H: ethyl 5-am ino-lH-pyrazole-4-
to 50.0 m L with the solvent mixture. Dilute 1.0 m L to carboxylate,
20.0 m L with the solvent mixture. D ilute 1.0 m L o f this
E. R1 = O -C 2H 5, R2 = C HO : ethyl 5-(form ylam ino)-l//-
solution to 20.0 m L with the solvent mixture. T o 5.0 m L of
pyrazole-4-caiboxylate,
the solution obtained, add 4 m L o f solution A, mix and
allow to stand for 2.5 h at room temperature. Add 5.0 m L o f
hexane R and shake for 1 m in. Allow the layers to separate
and use the upper layer. nh2

Blank solution T o 5 m L of the solvent mixture add 4 m L of

solution A, mix and allow to stand for 2.5 h at room L /N
tem perature. Add 5.0 m L o f hexane R and shake for 1 min.
Allow the layers to separate and use the upper layer.
C. 5-(4H-l,2,4-triazol-4-yl)-lii-pyrazole-4-carboxam ide,
Column:
— sizer. I = 0.25 m ,0 = 4.0 mm; F. H 2N -N H 2: diazane (hydrazine).
— stationary phase', cyanosifyl silica gelfor chromatography R PhEur
(5 Jim) with a pore size o f 10 nm;
— temperature'. 30 °C.
Mobile phase 2-propanol R, hexane R (5:95 VIV).
★*★
Flow rate 1.5 mL/min. Almagate * •*
★ ★
Detection Spectrophotom eter at 310 nm . * * * * *
(Ph Eur monograph 2010)
Injection 20 pL.
Relative retention W ith reference to benzaldehyde (retention AhMftCsDaoHi^fflaO 630 66827-12-1
tim e = about 2.8 min): benzaldehyde azine = about 0.8. A c tio n a n d u se
System suitability: reference solution: Antacid.
— resolution: minimum 2 between the peaks due to
benzaldehyde azine and benzaldehyde; P h & i _____________________
— signal-to-noise ratio: minimum 20 for the peak due to DEFINmON
benzaldehyde azine.
H ydrated aluminium magnesium hydroxycarbonate.
Limit.
C o n te n t
— impurity F: the area of the peak due to benzaldehyde
— aluminium: 15.0 per cent to 17.0 per cent (calculated
azine in the chromatogram obtained with the test solution
as A120 3),
is not m ore than the area o f the corresponding peak in
— magnesium: 36.0 per cent to 40.0 per cent (calculated
the chromatogram obtained with the reference solution
as M gO ),
(10 ppm of hydrazine sulfate equivalent to 2.5 ppm of
— carbonic acid: 12.5 p er cent to 14.5 per cent (calculated
hydrazine).
as C O 2).
H eav y m e ta ls (2.4.8)
Maximum 20 ppm. CHARACTERS
A p p e a ra n c e
1.0 g complies with test C. Prepare the reference solution
W hite or almost white, fine, crystalline powder.
using 2 m L of lead standard solution (10 ppm Pb) R.
S o lu b ility
L o ss o n d ry in g (2.2.32)
Practically insoluble in water, in ethanol (96 per cent) and in
M axim um 0.5 per cent, determined on 1.000 g by drying in
methylene chloride. It dissolves with effervescence and
an oven at 105 °C.
heating in dilute mineral adds.
S u lfa te d a s h (2.4.14)
M axim um 0.1 per cent, determined on 1.0 g. IDENTIFICATION
A. Infrared absorption spectrophotom etry (2.2.24).
A SSA Y
Comparison Ph. Eur. reference spectrum of almagate.
Liquid chromatography ( 2.2.29) as described in the test for
related substances with the following modification. B. Dissolve 0.15 g in dilute hydrochloric add R and dilute to
20 m L with the same ad d . 2 m L o f the solution gives the
Injection T est solution (b) and reference solution (c).
reaction o f aluminium (2.3.1).
Calculate the percentage content o f C5H4N4O from the
C. 2 m L o f the solution prepared under identification test B
declared content of aUopurinol CRS.
gives the reaction o f magnesium (2.3.1).
IM P U R IT IE S
TESTS
Specified impurities: A, B, C, D , E, F.
p H (2.2.3)
9.1 to 9.7.
2016 Almagate 1-101

Disperse 4.0 g in 100 m L of carbon dioxide-free water R, stir Magnesium

for 2 m in and filter. Introduce 10.0 m L of solution A prepared in the assay of
N e u tra lisin g cap a c ity alum inium into a 500 m L conical flask, add 200 m L of
Carry out the testât 37 °C. Disperse 0.5 g in 100 m L of water R, 20 m L o f triethanolamine R with shaking, 10 m l. of
water R, heat, add 100.0 m L o f 0.1 M hydrochloric add, ammonium chloride buffer solution pH 10.0 R and 50 mg of
previously heated and stir continuously; the p H (2.2.3) o f the mordant black 11 triturate R Titrate with 0.05 M sodium
solution between 5 m in and 20 min is not less than 3.0 and edetate until the colour changes from violet to pure blue.
not greater than 4.5. Add 10.0 m L o f 0.5 M hydrochloric add, 1 m L of 0.05 M sodium edetate is equivalent to 2.015 mg
previously heated, stir continuously for 1 h and titrate with o f MgO.
0.1 M sodium hydroxide to p H 3.5; n o t m ore than 20.0 m L of C a rb o n ic a c id
0 . 1 M sodium hydroxide is required. 12.5 per cent to 14.5 per cen t
C h lo rid e s (2.4.4) Test sample Place 7.00 m g of the substance to be examined in
Maximum 0.1 per c e n t a tin capsule. Seal the capsule.
Dissolve 0.33 g in 5 m L of dilute nitric acid R and dilute to Reference sample Place 7.00 mg o f ahnagate CRS in a tin
100 m L with water R. Prepare simultaneously the standard capsule. Seal the capsule.
by diluting 0.7 m L o f dilute nitric add R to 5 m L with Introduce separately the test sample and the reference sample
water R and adding 10 m L o f chloride standard solution (5 ppm into a combustion chamber of a C H N analyser purged with
Cl) R. helium for chromatography R and maintained at a tem perature
S u lfates (2.4.13) o f 1020 °C. Simultaneously, introduce oxygen R at a pressure
M aximum 0.4 per cent. o f 40 kPa and a flow rate of 20 m I7m in and allow complete
Dissolve 0.25 g in 5 m L o f dilute hydrochloric add R and combustion o f the sample. Sweep the combustion gases
dilute to 100 m L with distilled water R. Prepare through a reduction reactor and separate the gases form ed by
simultaneously the standard by adding 0.8 m L o f dilute gas chromatography (2.2.28).
hydrochloric add R to 15 m L of sulfate standard solution Column:
(10 ppm SO 4) R — sizer. I = 2 m , 0 = 4 mm;
Sodium — stationary phase: ethylvinylbenzene-dxvinylbenzene
M aximum 150 ppm. copolymer R l.
Atomic absorption spectrometry (2.2.23, Method I). Carrier gas heHum for chromatography R.
Test solution Dissolve 0.25 g in 50 m L of a 103 g/L solution Flow rate 100 mL/min.
o f hydrochloric acid R. Temperature:
— column: 65 °C;
Reference solutions Prepare the reference solutions using sodium
— detector. 190 °C.
standard solution (200 ppm Na) R, diluted as necessary with a
103 g/L solution of hydrochloric add R. Detection Therm al conductivity.
H eavy m e ta ls (2.4.8) Run time 16 m in .
Maximum 20 ppm. System suitability:
Dissolve 1.0 g in dilute hydrochloric acid R and dilute to — average percentage o f carbon in 5 reference samples m ust
20.0 m L w ith the same acid. 12 m L o f the solution complies be within ± 0.2 per cent of the value assigned to
with test A. Prepare the reference solution using lead standard the CRS; the difference between the upper and the lower
solution (1 ppm Pb) R. values o f the percentage of carbon in these samples m ust
be below 0.2 per cent.
L oss o n ig n itio n
Calculate the percentage content o f carbonic acid in the test
43.0 per cent to 49.0 per cent, determined on 1.000 g by
sample according to the following formula:
ignition at 900 ± 50 °C.
M ic ro b ia l c o n ta m in a tio n
TA M C: acceptance criterion 103 C FU /g (2.6.12). CxKx —
m
TY M C: acceptance criterion 102 C FU /g (2.6.12).
Absence o f Escherichia colt (2.6.13). C percentage content o f carbonic a d d in the reference
Absence o f Pseudomonas aeruginosa (2.6.13). sample;
A SSAY K m ean value for the 5 reference samples of the ratio
A lu m in iu m o f the mass in milligrams to the area o f the peak
Dissolve 1.000 g in 5 m L o f hydrochloric acid R, heating if due to carbonic add;
necessary. Allow to cool to room tem perature and dilute to A area o f the peak due to carbonic a d d in the
100.0 m L with water R (solution A). Introduce 10.0 m L of chrom atogram obtained with the test sample;
solution A into a 250 m L conical flask, add 25.0 m L of sample mass, in milligrams.
0.05 M sodium edetate, 20 m L of buffer solution pH 3.5 R, STORAGE
40 m L o f ethanol R and 2 m L of a freshly prepared 0.25 g/L In an airtight container.
solution o f dithizone R in ethanol R. T itrate the excess of
PhEur
sodium edetate with 0.05 M zinc sulfate until the colour
changes from greenish-violet to pink.
1 m L of 0.05 M sodium edetate is equivalent to 2.549 mg
of AI2O 3.
 1-102 Almond Oil 2016

★* * — erucic add: maximum 0.1 per cent.

Virgin Almond Oil ★ ★
★ ★
S tero ls (2.4.23)
Almond Oil *****
Composition of sterolfraction of the oil:
(Ph. Eur. monograph 0261) — cholesterol: maximum 0.7 per cent,
P re p a r a tio n — campesterol: maximum 4.0 per cent,
Almond Oil E ar Drops — sugmasteroh maximum 3.0 per cent,
— f}-sitosterol: 73.0 per cent to 87.0 per cent,
PhEur_______________________
— A5-avenasterol: minimum 10.0 per cent,
D E F IN IT IO N — A7-stigmastenol: maximum 3.0 per cent,
Fatty oil obtained by cold expression from the ripe seeds of — A7-avenasterol: maximum 3.0 per cent,
Prunus dulcis (Mill.) D A .W ebb var. dulcis or Prunus dulds — brassicasterol: maximum 0.3 per cent.
(Mill.) D A .W ebb var. amara (DC.) Buchheim or a mixture W a te r (2.5.32)
of both varieties. Maximum 0.1 per cent, determ ined on 1.00 g.
CHARACTERS STORA GE
A p p e a ra n c e In a well-filled container, protected from light.
Yellow, d ear liquid.
PhEur
S o lu b ility
Slightly soluble in ethanol (96 per cent), m isdble with light
petroleum.
R elative d e n sity
About 0.916. Refined Almond Oil *****
*+ +*
It solidifies at about - 1 8 °C. (Ph. Eur. monograph 1064) *
ID E N T IF IC A T IO N PhEur__________________________________________________________
First identification A, C. D E F IN IT IO N
Second identification A, B. Fatty oil obtained from the ripe seeds of Prunus dulds
A Absorbance (see Tests). (Mill.) D A . Webb var. dulcis or Prunus dulcis (Mill.) D A .
B. Identification of fatty oils by thin-layer chromatography W ebb var. amara (DC.) Buchheim or a mixture o f both
(2.3.2). varieties by cold expression. It is then refined. A suitable
antioxidant may be added.
Results T he chromatogram obtained is similar to the
corresponding chromatogram shown in Figure 2.3.2.-1. CHA RACTERS
C. Composition of fatty adds (see Tests). A p p e a ra n c e
Pale yellow, d ear liquid.
TESTS
Specific a b so rb a n c e (2.2.25) S o lubility
M aximum 0.2, determined at the absorption maximum at Slightly soluble in ethanol (96 per cent), m isdble with light
270 nm . T he ratio o f the absorbance measured at 232 nm to petroleum.
that measured at 270 nm is greater than 7. R elativ e d en sity
T o 0.100 g add cyclohexane R and dilute to 10.0 m L with the A bout 0.916.
same solvent. Adapt the concentration of the solution so that It solidifies at about - 1 8 °C.
the absorbance lies between 0.5 and 1.5, measured in a 1 cm ID E N T IF IC A T IO N
cell.
A Identification o f fatty oils by thin-layer chromatography
A d d valu e (2.5.1) (2.3.2).
M aximum 2.0, determined on 5.0 g. Results T he chromatogram obtained is similar to the
P e ro x id e v alu e (2.5.5, Method A) corresponding chromatogram shown in Figure 2.3.2.-1.
Maximum 15.0. B. Composition of fatty a d d s (see Tests).
U n sap o n ifiab le m a tte r (2.5.7) TESTS
Maximum 0.9 per cent, determined on 5.0 g.
Specific a b so rb a n c e (2.2.25)
C o m p o sitio n o f fa tty acid s (2.4.22, Method A) Use the 0.2 to 6.0, determined at the absorption maximum at
mixture of calibrating substances in Table 2.4.22.-3. 270 nm.
Composition of the fatty-acid fraction of the oil: T o 0.100 g add cyclohexane R and dilute to 10.0 m L with the
— saturated fatty acids of chain length less than Cj^. maximum same solvent. Adapt the concentration o f the solution so that
0.1 per cent, the absorbance lies between 0.5 and 1.5, measured in a 1 cm
— palmitic acid: 4.0 per cent to 9.0 per cent, cell.
— palrmtoleic add: maximum 0.8 per cent,
A cid valu e (2.5.1)
— margaric add', maximum 0.2 per cent,
M aximum 0.5, determined on 5.0 g.
— stearic add: maximum 3.0 p er cent,
— oleic add: 62.0 per cent to 86.0 per cent, P e ro x id e v alu e (2.5.5, Method A)
— linoleic add: 20.0 per cent to 30.0 per cent, Maximum 5.0.
— linolenic acid: maximum 0.4 per cent, U n sap o n ifiab le m a tte r (2.5.7)
— arackidic add: maximum 0.2 per cent, M aximum 0.9 per cent, determined on 5.0 g.
— eicosenoic acid: maximum 0.3 per cent,
C o m p o sitio n o f fa tty a c id s (2.4.22, Method A)
— behenic add: maximum 0.2 per cent,
Use the mixture of calibrating substances in Table 2.4.22.-3.
2016 Aloxiprin 1-103

Composition of the fatty-acid fraction of the oü: TESTS

— saturated fatty adds of chain length less than Ci£ maximum H eav y m e ta ls
0.1 per cent; Carefully ignite 2 .0 g at a low tem perature until completely
— palmitic add: 4.0 per cent to 9.0 per cent; charred, cool, add 2 m L of nitric acid and 0 .25 m L of sulfuric
— paltmtoleic add: maximum 0.8 per cent; add, heat cautiously until white fumes are evolved and ignite
— margaric acid: maximum 0.2. per cent; at 5 00° to 60 0 °. Cool, add 2 m L of hydrochloric acid,
— stearic add: maximum 3.0 per cent; evaporate to dryness on a water bath and carry out the
— oleic acid: 62.0 per cent to 86.0 per cent; procedure for limit test C for heavy metals, Appendix VH,
— linoleic acid: 20.0 per cent to 30.0 per cent; beginning at the words Dissolve the residue...’. Use 2 m L of
— Imolenic add: maximum 0.4 per cent; lead standard solution (10 ppm Pb) to prepare die standard
— arachidic acid: maximum 0.2 per cent; (10 ppm).
— eicosenoic acid: maximum 0.3 per cent; F re e acetylsalicylic a cid
— behenic acid, maximum 0.2 per cent; T o a quantity containing the equivalent o f 1.0 g of total
— erucic acid: maximum 0.1 per cent.
salicylates add 5 0 m L of dry ether and shake for 3 0 minutes.
S tero ls (2.4.25) Filter quickly through fluted filter paper, wash the paper with
Composition of the sterolfraction of the oü: several portions o f dry ether and dilute the combined filtrate
— cholesterol: maximum 0.7 per cent; and washings to 1 0 0 m L with dry ether. T he absorbance o f the
— campesterol: maximum 5.0 per cent; solution at the maximum at 2 7 8 nm is n o t more than 0 .3 6 ,
— stigmasterol: maximum 4.0 per cent; Appendix I I B (0.5% , calculated with reference to the
— ^-sitosterol: 73.0 per cent to 87.0 per cent; content o f total salicylates).
— A5-avenasterol: minim um 5.0 per cent; S alicylic a c id
— A 7-sngmastenol: maximum 3.0 per cent; T he absorbance o f the solution used in the test for Free
— A 7-avenasterol: maximum 3.0 per cent; acetylsalicylic a d d at the maximum at 3 0 8 nm is not more
— brassicasterol: maximum 0.3 per cent. than 0 .5 0 , Appendix II B (0.15% , calculated with reference
W a te r (2.5.32) to the content o f total salicylates).
M aximum 0.1 per cent, determined on 1.00 g. C o m b in e d salicy late
STORA GE N o t more than 9.5% , calculated as salicylic ad d , C7H 60 3,
In a well-filled container, protected from light. with reference to the content o f total salicylates calculated as
__________________________________________________________________________________________ P h E tr
O-acetylsalicylic a d d when determined in the following
manner. T o 0.1 g add 4 0 m L o f a 0.5% w/v solution of
sodium fluoride in 0.1m hydrochloric add and shake for
5 minutes. Allow the solution to stand for 10 minutes,
shaking at frequent intervals. Extract with six 2 0 m L
Aloxiprin quantities of dichloromethane, filter the combined extracts
through a layer o f anhydrous sodium sulfate, wash with 3 0 m l .
o f dichloromethane and dilute the combined filtrate and
9014-67-9
washings to 2 0 0 m L with dichloromethane. Dilute 2 0 mT. o f
A ctio n a n d u se the solution to 5 0 m L with dichloromethane and measure the
Salicylate; non-selective cyclo-oxygenase inhibitor; absorbance of the resulting solution at the maximum at
antipyretiq analgesic; anti-inflammatory. 3 0 8 nm , Appendix IIB . Calculate the content of
taking 2 9 3 as the value of A (l% , 1 cm) at the
P re p a ra tio n
maximum at 3 0 8 nm .
Aloxiprin Tablets
L oss on d ry in g
D E F IN IT IO N W hen dried to constant weight over phosphorus pentoxide at a
Aloxiprin is a polymeric condensation product of aluminium pressure not exceeding 0 .7 kPa, loses n o t more than 2.0% of
oxide and O-acetylsalicylic add. It contains n o t less than its w dght. Use 1 g.
7.5% and not m ore than 8.5% o f aluminium, Al, and not ASSAY
less than 79.0% and n o t more than 87.4% o f total salicylates, For ahimm mm
calculated as O-acetylsalicylic ad d , CgHsOi, both calculated Ignite 2 g in a tared silica crudble, heat gently until the
with reference to the dried substance. organic m atter is destroyed and then ignite to constant
C H A R A C T E R IS T IC S w dght at 1000°. Each g of residue is equivalent to 0 .5 2 9 2 g
A fine, white or slightly pink powder. of Al.
Practically insoluble in water, practically insoluble in ethanol For total salicylates
(96%) and in ether. T o 0 .2 5 g add 5 0 m L of 1m sodium hydroxide and boil gendy
until dissolved. Cool, add 50 m L of water, adjust the pH to
ID E N T IF IC A T IO N
between 2 .4 0 and 2 .5 0 with 1m hydrochloric acid and dilute to
A. Boil 1 g with 2 0 m L o f 2 m hydrochloric add, cool, filter
5 0 0 m L with water. T o 5 m L add 4 m L of iron(m) chloride
and reserve the filtrate. Dissolve the residue in 10 m L of
solution, allow to stand for 3 0 minutes, dilute to 5 0 m L w ith
0 .1 m sodium hydroxide and neutralise with 1m acetic add.
water and measure the absorbance of the resulting solution at
1 m L of the resulting solution yidds reaction A characteristic
the maximum at 5 3 0 nm , Appendix II B, using in the
o f salicylates, Appendix VI.
reference cell a solution prepared by diluting 4 m L of iron(m)
B. T h e filtrate reserved in test A yields the reaction chloride solution to 5 0 m L with water. Calculate the content of
characteristic o f aluminium salts, Appendix VI. total salicylates as C ^ H ^ from the absorbance obtained by
repeating the procedure using 4 m L of a 0.05% w/v solution
 1-104 Alprazolam 2016

of salicyUc add in place o f the solution being examined and Reference solution (b) Dissolve 10 m g o f alprazolam CRS and
b eg in n in g at the words ‘add 4 m L o f ừon(w) chloride 10 m g o f midazolam CRS in methanol R and dilute to 10 m L
solution..’. Each g o f salicylic a d d is equivalent to 1.305 g of with the same solvent.
C 9Ỉ Ỉ 8O 4. Plate TLC silica gel GF2 5 4 plate R.
Mobile phase glacial acetic acid R, water R, methanol R, ethyl
acetate R (2:15:20:80 VIV/VIV).
Application 5 |iL.
Alprazolam ★ ★ Development Over a path o f 12 cm.
★ ★
(Ph. Eur. monograph 1065) ***** Drying In. air.
Detection Examine in ultraviolet light at 254 nm.
H3C ^ w System suitability: reference solution (b):
— the chromatogram shows 2 clearly separately spots.
Results T h e principal spot in the chrom atogram obtained with
the test solution is similar in position and size to the principa]
spot in the chromatogram obtained with reference
solution (a).
TESTS
Related substances
Liquid chromatography (2.2.29).
C17H13aN4 308.8 28981-97-7
Buffer solution Dissolve 7.7 g of ammonium acetate R in
1000 m L o f water R and adjust to p H 4.2 with glacial acetic
Action and use
Benzodiazepine. add R.
Test solution Dissolve 0.100 g o f the substance to be
PhEir __________________________________________________________ examined in dimethylformamide R and dilute to 10.0 m L with
DEFINITION the same solvent.
8-Chloro-1-m ethyl-6-phenyl-4/i- [ 1,2,4] triazolo [4,3-a] Reference solution (a) Dissolve 2 m g o f alprazolam CRS and
[1,4] benzodiazepine. 2 m g o f triazolam CRS in dimethylformamide R and dilute to
100.0 m L with the same solvent.
Content
99.0 per cent to 101.0 per cent (dried substance). Reference solution (b) Dilute 5.0 m L o f the test solution to
100.0 m l. with dimethylformamide R. Dilute 0.5 m L o f this
CHARACTERS solution to 10.0 m L with dimethylformamide R.
Appearance
Cdumn:
W hite or almost white, crystallin e powder.
— size. I = 0.25 m, 0 = 4.6 mm;
Solubility — stationary phase: phenylsüyl silica gelfor chromatography R1
Practically insoluble in water, freely soluble in methylene (5 nm).
chloride, sparingly soluble in acetone and in ethanol Mobile phase:
(96 per cent). — mobile phase A: buffer solution, methanol R (44:56 VIV)\
It shows polymorphism (5.9). — mobile phase B: buffer solution, methanol R (5:95 V!V)\
IDENTIFICATION — temperature:. 40 °C;
First identification B.
Second identification A , C. lim e Mobile phaae A Mobile phase B
(min) (percent V/V) (ptT cent V7V)
A. Dissolve the substance to be examined in the smallest
0 -1 5 98 2
necessary quantity o f ethyl acetate R and evaporate to dryness
on a water-bath. Thoroughly mix 5.0 m g o f the substance to 15 -3 5 98 -> 1 2 + 99
be examined with 5.0 mg of alprazolam CRS. T he melting 3 5 -4 0 1 99
point (2.2.14) o f the mixture does n o t differ by more than
2 °C from the melting point o f the substance to be
examined. Flow rate 2 mL/min.
B. Infrared absorption spectrophotometry (2.2.24). Detection Spectrophotom eter at 254 nm.
Preparation Discs. Irgecdon 10 |iL; inject dtmethylformamide R a sa blank.
Comparison alprazolam CRS. Retention time Triazolam = about 9 min;
If the spectra obtained in the solid state show differences, alprazolam = about 10 min.
dissolve the substance to be examined and the reference System suitability: reference solution (a):
substance separately in the m in im u m volume of ethyl — resolution: minim um 1.5 between the peaks due to
acetate R, evaporate to dryness on a water-bath and record triazolam and alprazolam.
new spectra using the residues. Limits:
C. Thin-layer chromatography (2.2.27). — totah not more than the area o f the principal peak in the
Test solution Dissolve 10 m g o f the substance to be exam in ed chrom atogram obtained with reference solution (b)
in methanol R and dilute to 10 m L w ith the same solvent. (0.25 per cent);
— disregard lima: 0.2 times the area o f the principal peak in
Reference solution (a) Dissolve 10 m g o f alprazolam CRS in
the chromatogram obtained with reference solution (b)
methanol R and dilute to 10 m L with the same solvent.
(0.05 per cent).
2016 Alprazolam 1-105

L oss o n d ry in g (2.2.32)
M axim um 0.5 per cent, determined on 1.000 g by drying in
an oven at 105 °C.
S u lfa te d a s h (2.4.14)
Maximum 0.1 per cent, determined on 1.0 g.
A SSA Y
Dissolve 0.140 g in 50 m L of a mixture o f 2 volumes of
acetic anhydride R and 3 volumes of anhydrous acetic acid R.
Titrate with 0.1 M perchloric acid, determining the end-point G. 7-chloro- l-methyl-5-phenyl[ 1,2,4]triazolo [4,3-a] quinolin-
potentiometrically (2.2.20). Titrate to the 2nd point of 4-amine,
inflexion.
1 m L of 0.1 M perchloric add is equivalent to 15.44 mg
o f C 17H 13CIN 4.
STO R A G E
Protected from light.
IM P U R IT IE S

N y CH3
H . bis[[4-(2-ben2oyl-4-chlorophenyl)-5-methyl-4H'-lj2}4-
and enantiomer triazol-3-yl] methyl] amine.
OH

A. (4R5)-3-amino-6-chloro-2-methyl-4-phenyl-3,4-
dihydroquinazolin-4-olj
and enantiomer

I. [5-chloro-2-[3- [[(6i?5)-8-chloro- 6-hydroxy-1-methyl-6 -

phenyl-4H- [ 1,2,4] triazolo [4,3-a] [ 1,4]benzodiazepin-5 (6H)-yI]
methyl] -5-m ethyl-4ii-1,2,4-triazol-4-yl] phenyl]
phenylmethanone,
B. R = C H 2O H : [5-chloro-2-[3-(hydroxymethyl)-5-methyl-
4 H - 1j2j4-triazol-4-yl] phenyl] phenyimethanone,
C. R = H : [5-chloro-2-[3-methyl-4/f-1,2,4-triazol-4-yl]
phenyl] phenylmethanone,
F. R = C H 2C1: [5-chloro-2-[3-(chloromethyl)-5-methyl-4H-
1,2 }4-triazol-4-yl] phenyl] phenylmethanone,

J. 2,17-dichloro-6,13-dimethyl-l 8b, 19a-diphenyl-

8b, 19adihydro-1OH, 18b H- [ 1,2,4] triazolo [4 " ',3 '" : 1 ",2 "]
quinolo [3 ",4 " :4 ',5 '] oxazolo [3 ',2 '-d\-1,2,4-triazolo [4,3-a]
[ 1,4] benzodiazepine.
PhEur

D . 8-chloro-1-ethenyl-6-phenyl-4/i- [1,2,4] triazolo [4,3-a]

[ 1,4] benzodiazepine,

E. (2 -amino-5-chlorophenyl)phenylmethanone,
 1-106 Alprenolol Hydrochloride 2016

Test solution (a) Dissolve 0.50 g o f die substance to be

Alprenolol Hydrochloride ***\ examined in methanol R and dilute to 10 m L w ith die same
(Ph Eur monograph 0876) * solvent.
Test solution (b) D ilute 1 m L o f test solution (a) to 50 m L
with methanol R.
Reference solution (a) Dissolve 10 m g o f alprenolol
hydrochloride CRS in methanol R and dilute to 10 m L with the
same solvent.
Reference solution (b) Dissolve 10 m g o f alprenolol
C 15H 24C1N02 285.8 13707-88-5 hydrochloride CRS and 10 m g o f oxprenold hydrochloride CRS
in methanol R and dilute to 10 m L with the same solvent.
Action and use Reference solution (c) Dilute 5 m L o f test solution (b) to
Beta-adrenoceptor antagonist. 50 mT. with methanolR
PhEur___________________________________________________________ Plate TLC silica gel G {date R.
DEFINITION Mobile phase Place 2 beakers each containing 30 m L of
ammonia R at die bottom o f die tank containing a mixture of
(2RS)-1 -[(1 -M ethylethyl)amino]-3- [2-(prop-2-enyI)phenoxy]
5 volumes o f methanol R and 95 volumes o f ethyl acetate R.
propan-2-ol hydrochloride.
Application 5 |iL.
Content
99.0 per cent to 101.0 per cent (dried substance). Development Over a p ath o f 15 cm in a tank saturated for at
least 1 h.
CHARACTERS
Drying Ax. 100 °C for 15 min.
Appearance
W hite or alm ost white, crystalline pow der o r colourless
Detection Expose to iodine vapour for up to 6 h.
crystals. System suitability: reference solution (b):
— the chrom atogram shows 2 clearly separated spots.
Solubility
Very soluble in water, freely soluble in ethanol (96 p er cent) Limits: test solution (a):
— impurity D-. any spot with an Rp value greater than th at of
and in methylene chloride.
the principal spot is not more intense than die principal
IDENTIFICATION spot in the chrom atogram obtained with reference
First identification B, D. solution (c) (0.2 per cent).
Second identification A , C, D. Related substances
A. M elting point (2.2. 14): 108 °C to 112 °C. Liquid chrom atography (2.2.29).
B. Infrared absorption spectrophotom etry (2.2.24). Test solution Dissolve 20.0 m g of die substance to be
Comparison alprenolol hydrochloride CRS. exam ine d in the mobile phase and dilute to 10.0 m L with

C. Examine the chrom atograms obtained in the test for the mobile phase.
im purity D. Reference solution (a) Dissolve 4.0 m g o f alprenolol
Detection Examine in daylight, after exposure to iodine hydrochloride CRS and 0.8 m g of 4-isopropylphenol R in the
vapour for 30 min. mobile phase and dilute to 100.0 m L with the mobile phase.

Results T he principal spot in the chrom atogram obtained with Reference solution (b) D ilute 4.0 m L of the test solution to
100.0 m L with die mobile phase. D ilute 1.0 m L o f this
test solution (b) is similar in position, colour and size to the
principal spot in the chrom atogram obtained with reference solution to 10.0 m L with the mobile phase.
solution (a). Column:
— size. I = 0.15 m , 0 = 4 mm;
D . It gives reaction (a) of chlorides (2.3.1).
— stationary phase, octylsüyl silica gel for chromatography R
TESTS (5 pm).
Solution S Mobile phase M ix 0.656 g o f sodium octanesidfonate R with
Dissolve 1.0 g in carbon dioxide-free water R and dilute to 150 mT. o f acetomtrüe R and dilute to 500 m L with
50 m L with the same solvent. phosphate buffer p H 2.8 prepared as follows: mix 1.78 g of
Appearance of solution phosphoric add R and 15.6 g o f sodium dihydrogen phosphate R
Solution S is clear (2.2.1) and n o t m ore intensely coloured and dilute to 2000 m L w ith water R
than reference solution B9 (2.2.2, Method II). Flow rate 1 mlVmin.
Acidity or alkalinity Detection Spectrophotom eter at 280 nm .
T o 10 m L o f solution S add 0.2 mT. o f methyl red solution R Equilibration W ith the mobile phase for about 1 h.
and 0.2 m L o f 0.01 M hydrochloric acid; the solution is red.
Injection 20 nL.
A dd 0.4 m L o f 0.01 M sodium hydroxide; the solution is
yellow. Run time Twice the retention time of alprenolol.
Impurity C Retention time Alprenolol = about 11 min;
M axim um 0.1 per c e n t 4-isopropylphenol = about 18 min.

Dissolve 0.25 g in ethanol (96 per cent) R and dilute to

System suitability: reference solution (a):
— resolution: m inim um 5 between the peaks due to
25 m L with the same solvent. T h e absorbance (2.2.25)
alprenolol and 4-isopropylphenol; if necessary, adjust the
measured at 297 nm is n o t greater than 0.20.
concentration o f sodium octanesulfonate and/or
Impurity D acetonitrile in the mobile phase (increase the
Thin-layer chrom atography (2.2.27).
2016 Alprostadil 1-107

concentration o f sodium octanesulfonate to increase the

retention time of alprenolol and increase the
concentration of acetonitrile to decrease the
retention times o f both compounds).
Limits:
— unspecified impurities: for each impurity, not more than D . 1,1 '-[(l-methylethyl)imino]bis[3-[2-(prop-2-enyl)
0.25 times the area of the principal peak in the phenoxy]propan- 2 -ol].
chromatogram obtained with reference solution (b)
------------------------------------------------------------------------------------------------------------------------------------------------------ P hE ir
(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 o f the principal peak in
the chromatogram obtained with reference solution (b)
(0.04 per cent).
H eav y m e ta ls (2.4.8)
M aximum 10 ppm.
Dissolve 2.0 g in 20 m L o f water R. 12 m L o f the solution
complies with test A. Prepare the reference solution using
lead standard solution (1 ppm Pb) R.
L oss o n d ry in g (2.2.31)
M aximum 0.5 per cent, determined on 1.000 g by drying
over diphosphorus pentoxide R at a pressure n ot exceeding C20H 34O 5 354.5 745-65-3
2.7 kPa.
A ctio n a n d u se
S u lfa te d a sh (2.4.14) Prostaglandin E x (PG Ei)
M aximum 0.1 per cent, determined on 1.0 g.
P h E ir __________________________________________________________________________________________
A SSA Y
Dissolve 0.400 g in 25 m L o f a m ixture of equal volumes o f D E F IN IT IO N
anhydrous ethanol R and water R. A dd 10 m L of 0.01 M 7- [( 1 2R,3R) -3-Hydroxy-2-[( 1£,35) -3-hydroxyoa - 1-enyl] -
hydrochloric add. Carry out a potentiometric titration (2.2.20), 5-oxocyclopentyl]heptanoic acid.
using 0.1 M sodium hydroxide. Read the volume added C o n te n t
between the 2 points of inflexion. 95.0 per cent to 102.5 p er cent (anhydrous substance).
1 m L of 0.1 M sodium hydroxide is equivalent to 28.58 mg CHARACTERS
of C 15H 24CINO 2. A p p e a ra n c e
STORA GE W hite or slighdy yellowish, crystalline powder.
Protected from light. S olu b ility
IM P U R IT IE S Practically insoluble in water, freely soluble in ethanol
Specified impurities C, D (96 p er cent), soluble in acetone, slightly soluble in ethyl
acetate.
Other detectable impurities (the following substances would, if
present at a sufficient level, be detected by one or other of ID E N T IF IC A T IO N
the tests in the monograph. They are limited by the general A Specific optical rotation (2.2.7): -7 0 to —60 (anhydrous
acceptance criterion for other/unspecified impurities and/or substance).
by the general monograph Substances for pharmaceutical use Immediately before use, dissolve 50 m g in ethanol
(2034). It is therefore not necessary to identify these (96per cent) R and dilute to 10.0 m l . with the same solvent.
impurities for demonstration of compliance. See also 5.10.
B. Infrared absorption spectrophotometry (2.2.24).
Control of impurities in substances for pharmaceutical use): A, B.
Comparison alprostadil CRS.
H OH C. Examine the chromatograms obtained in the assay.
0. .R1 Results T he principal peak in the chromatogram obtained
and enantiomer
with the test solution is similar in retention time and size to
R2 the principal peak in the chromatogram obtained with the
reference solution.
A. R1 = O H , R 2 = C H 2-C H = C H 2: (2ftS)-3-[2-(prop-2- TESTS
enyl) phenoxy] propan- 1, 2-diol, R e la te d su b stan c e s
C. R1 = N H -C H (C H 3) 2, R2 = C H = C H -C H 3: Liquid chromatography (2.2.29). Prepare the solutions protected
(2RS) -1 - [(1 -methylethyl)amino] -3- [2 -(prop-1-enyl) phenoxy] from light.
propan- 2-ol, Test solution Dissolve 10.0 mg o f the substance to be
exam in ed in a mixture o f equal volumes of acetonitrile R1 and
water R and dilute to 10.0 mL with the same mixture of
solvents.
ch2 Reference solution (a) Dilute 100 (xL o f the test solution to
20.0 m L with a mixture o f equal volumes of acetonitrile R1
B. 2-(prop-2-enyl)phenol, and water R.
 1-108 Alprostadil 2016

Reference solution (b) Dissolve 1.0 m g o f dinoprostone Time Mobile phase A Mobile phase B
impurity C CRS (alprostadil impurity H ) and 1.0 m g o f the (min)____________ (percent V7V)________ (per cent V7V)
substance to be examined in a mixture o f equal volumes of 0 -5 0 100 0
acetomtrile R1 and water R and dilute to 20.0 m L with the 50-51 100 + 0 0+100
sam e mixture o f solvents.
51 -61 0 100
Reference solution (c) In order to prepare impurities A and B
in situ, dissolve 1 mg o f the substance to be examined in 61 -62 0+100 100 + 0
100 pL o f 1 M sodium hydroxide (the solution becomes 6 2 -7 2 100 0
brownish-red), wait for 3 min and add 100 |iL o f a 112 g/L
solution o f phosphoric add R (yellowish-white opalescent
solution); dilute to 5.0 m L with a mixture of equal volumes Relative retention W ith reference to alprostadil (retention
o f acetomtrUe R1 and water R. time = about 7 min): impurity A = about 2.4;
S y ste m A impurity B = about 2.6.
Column: System suitability:
— size: I = 0.25 m , 0 = 4.0 mm; — resolution: m in im u m 1 .5 between the peaks due to
— stationary phase: base-deactivated octylsUyl silica gel for impurity A and impurity B in the chrom atogram obtained
chromatography R (4 pm) with a pore size of 6 nm ; w ith reference solution (c).
— temperature: 35 °C. C arry out the test according to system A and B.
Mobile phase: Limits:
— mobile phase A: dissolve 3.9 g of sodium dihydrogen — correction factors: for the calculation of content, multiply
phosphate R in water R and dilute to 1.0 L with the same the peak areas o f the impurities listed in Table 1488.-1 by
solvent; adjust to p H 2.5 with a 2.9 g/L solution of the corresponding correction factor;
phosphoric add R (approximately 600 m L is required);
to 740 m L o f the buffer solution add 260 m L o f Table 1488.-1.
acetomtrUe R1;
Imparity Relative retention Relative retention Correction factor
— mobile phase B: dissolve 3.9 g o f sodium dihydrogen (system A) (system B)
phosphate R in water R and dilute to 1.0 L with the same impurity G 0.80 - 0.7
solvent; adjust to p H 2.5 with a 2.9 g/L solution o f
impurity F 0.88 - 0.8
phosphoric add R (approximately 600 m L is required);
to 200 m L o f the buffer solution add 800 m L of impurity D 0.90 - 1.0
acetomtrUe R1; impurity H 0.96 - 0.7

impurity E 1.10 - 0.7

Time Mobile phase A Mobile phase B
impurity C 1.36 1.9
(min) (per cent V7V) (per cent V7V)
0 - 75 100 0 impurity K 1.85 0.06

75 - 76 100-»0 0+100 impurity A 232 0.7

7 6 -8 6 0 100 impurity B 2.45 1.5

8 6 -8 7 0+100 100 + 0 impurity I 4.00 1.0

87 - 102 100 0 impurity J 5.89 1.0

Flow rate 1 mL/m in. — impurity A: n o t more than 3 times the area of the
principal peak in the chrom atogram obtained with
Detection Spectrophotom eter at 200 nm .
reference solution (a) (1.5 per cent);
Injection 20 |iL. — impurity B: n o t more than the area o f the principal peak
Retention time Alprostadil = about 63 m in . in the chrom atogram obtained with reference solution (a)
System suitability: (0.5 per cent);
— resolution: m in im u m 1.5 between the peaks due to — any other impurity: n o t m ore than 1.8 times the area o f the
im purity H and alprostadil in the chromatogram obtained principal peak in the chrom atogram obtained with
with reference solution (b). reference solution (a) (0.9 per cent), and n o t more than 1
S y ste m B such peak has an area greater than the area o f the
U se the same conditions as for system A with the following principal peak in the chrom atogram obtained with
mobile phase and elution programme: reference solution (a) (0.5 per cent). Evaluate impurities
— mobile phase A: dissolve 3.9 g of sodium dihydrogen appearing at relative retentions less than 1.2 by system A
phosphate R in water R and dilute to 1.0 L with the same and impurities appearing at relative retentions greater
solvent; adjust to p H 2.5 with a 2.9 g/L solution o f than 1.2 by system B;
phosphoric add R (approximately 600 m L is required); — total: not more than 3 times the area o f the principal peak
to 600 m L o f the buffer solution add 400 m L of in the chromatogram obtained with reference solution (a)
acetomtrUe R l; (1.5 per cent);
— mobile phase B: use mobile phase B as described under — disregard limit. 0.1 times the area of the principal peak in
system A; the chromatogram obtained with reference solution (a)
(0.05 per cent).
W a te r ( 2.5.32)
M axim um 0.5 per cent, determ ined on 50 mg.
2016 Alprostadil 1-109

ASSAY
Liquid chromatography (2.2.29) as described in the test for
related substances, system A. Prepare the solutions protected
from Ught.
Test solution Dissolve 10.0 m g of the substance to be H OH
examined in a mixture of equal volumes of acetonitrile R1 and
water R and dilute to 25.0 m L with the same mixture of E. 7-[(li?,2J?,35)-3-hydroxy-2-[(l£,35)-3-hydroxyoct-l-
solvents. Dilute 3.0 m L of the solution to 20.0 m L with a enyl]-5-oxocyclopentyl]heptanoic acid
mixture o f equal volumes of acetonitrile R1 and water R. (11-epiprostaglandin E i),
Reference solution Dissolve 5.0 mg of alprostadil CRS in a
mixture of equal volumes of acetonitrile R1 and water R and
dilute to 25.0 m L with the same mixture o f solvents. Dilute
6.0 m L o f the solution to 20.0 m L with a mixture of
equal volumes o f acetonitrile R1 and water R.
HO"
Injection 20 ^L. H OH
Calculate the percentage content of C 20H 34O 5 taking into
account the assigned content of alprostadil CRS.
F . 7-[(l S,2R,3R)-3-hydroxy-2-[(1 £ ,3 6 )-3-hydroxyoct-1-
ST O R A G E enyI]-5-oxocyclopentyl]heptanoic a d d
At a tem perature o f 2 °C to 8 °C. ( 8-epiprostaglandin E i),
IM P U R IT IE S

H ’oh
H OH
G . (5Z)-7- [(1 i?,2i?3^)-3-hydroxy-2-[(l£,35)-3-hydroxyoct-
A 7- [(1 R,2S)-2- [(1 £,3.S)-3-hydroxyoct-l -enyI]-5- l-enyI]-5-oxocydopentyl]hept-5-enoic a d d
oxocyclopent-3-enyI]heptanoic acid (prostaglandin A 0 , (dinoprostone),

O
CO2H

CH3

H OH H OH

B. 7- [2- [(1£,3.S)-3-hydroxyoct-1-enyl] -5-oxocydopent-1- H . (5£)-7-[(l.R,22?,3.R)-3-hydroxy-2-[(l£,3.S)-3-hydroxyoct-

enyl]heptanoic a d d (prostaglandin B i), l-enyI]-5-oxocydopentyl]hept-5-enoic ad d
((5£)-prostaglandin E 2),

H OH
C. 7-[(li?,2i?,3i?)-3-hydroxy-2-[(l£)-3-oxooct-l-enyI]-5-
oxocydopentyl]heptanoic a d d (15-oxoprostaglandin E j), I. ethyl 7-[(1 i?,2i?,3i?)-3-hydroxy-2- [(1 £ ,3 5 )-3-hydroxyoct-
l-enyI]-5-oxocydopentyi]heptanoate (prostaglandin E i,
ethyl ester),

H OH

D . 7-[(li?,2i?,3i?)-3-hydroxy-2-[(l£*3.R)-3-hydroxyoct-l- H OH
enyl]-5-oxocyclopentyl]heptanoic a d d
(15-epiprostagJandin E i),
J. 1-methylethyl 7-[(li?,2i?,3i?)-3-hydroxy-2-[(l£,36)-3-
hydroxyoct-l-enyl]-5-oxocyclopentyl]heptanoate
(prostaglandin E i, isopropyl ester),
 1-110 Alteplase for Injection 2016

If alteplase is stored in bulk form, stability (m aintenance o f

potency) in the intended storage conditions m ust be
demonstrated.
T he production, purification and product consistency are
checked by a num ber of analytical m ethods described below,
carried out routinely as in-process controls.
K. triphenylphosphine oxide. P ro te in c o n te n t
PhEur T he protein concentration of alteplase solutions is
determ ined by measuring the absorbance (2.2.25) o f the
protein solution at 280 nm and at 320 nm , using formulation
buffer as the compensation liquid. If dilution o f alteplase
samples is necessary, the samples are diluted in formulation
★ ★
Alteplase for Injection ★ ★ buffer. F or the calculation of the alteplase concentration, the
***** absorbance value (A2so —-^320) is divided by the specific
(Ph E ut monograph 1170) absorption coefficient for alteplase o f 1.9.
P o te n c y
SYQVICRDEK TQMIYQQHQS WLRPVLRSNR VEYCWCNSGR
T h e potency of alteplase is determ ined in an in vitro clot-lysis
I------------- 1 = i ------------------- '
AQCHSVPVKS CSEPRCFNGG TCQQALYFSD FVCQCPEGFA assay as described under Assay. T he specific activity o f bulk
,____________ 1 " i_l
GKCCEIDTRA TCYEDQGISY RGTWSTAESG AECTNWNSSA alteplase is approximately 580 000 IU per milligram o f
'------------------------------ '“ I alteplase.
LAQKPYSGRR PDAIRLGLGN HNYCRNPDRD SKPWCYVFKA

GKYSSEFCST PACSEGNSDC YFGNGSAYRG THSLTESGAS iV -te rm in a l se q u e n ce

TV-terminal sequencing is applied to determ ine the correct
CLPWNSMILI ?sj^AAQALGLGKHN
GKVYTAQNPS YCRNPDGDAK
.PWCHVLKNRR
1—i r N-terminal sequence and to determine semiquantitatively
LTWEYCDVPS CSTCGLRQYS QPQFR
additional cleavage sites in the alteplase molecule, for
IKGGL example at position AA 275-276 o r at position AA 27-28.
FADIASHPWQ AAIFAKHRRS PGERFLCGGI LISSC W ILSA T he AT-terminal sequence m ust conform with the sequence o f
1
AHCFQERFPP HHLTVILGRT YRWPGEEEQ KFEVEKYIVH hum an tissue plasminogen activator.
KEFDDDTYDN DIALLQLK5D SSRCAQESSV VRTVCLPPAD Is o e le c tric fo cu sin g
LQLPDWTECE LSGYGKHEAL SPFYSERLKE AHVRLYPSSR T he consistency in the microheterogeneity o f gjycosylation of
VTDNMLCAGD TRSGGPQANL
the alteplase molecule can be dem onstrated by isoelectric
CTSQHLLNRT HDACQGDSGG
■ focusing (IEF). A complex banding pattern with 10 major
PLVCLNDGRM TLVGIISWGL GCGQKDVPGV YTKVTNYLDW
and several minor bands in the p H range 6.5-8.5 is observed.
Denaturing conditions are applied to achieve a good
separation o f differently charged variants o f alteplase.
C 2736H 4174N 91 4O 824S 45 (non-glycosylated protein) 105857-23-6 T he broad charge distribution observed is due to a
population o f molecules, which differ in the fine structure o f
A c tio n a n d u se
Tissue-type plasm inogen activator; fibrinolytic. biantenary and triantenary complex-type carbohydrate
residues, with different degrees o f substitution with sialic
PhEur___________________________________________________________
acids. T h e banding pattern o f alteplase test samples m ust be
D E F IN IT IO N consistent w ith the pattern of alteplase reference standard.
Alteplase for injection is a sterile, fieeze-dried preparation o f S in g le -c h a in a lte p la se c o n te n t
alteplase, a tissue plasminogen activator produced by T h e alteplase produced by C H O (Chinese ham ster ovary)
recom binant D N A technology. It has a potency of n o t less cells in serum-free medium is predom inantly single-chain
than 500 000 IU per milligram of protein. alteplase. T h e 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 die generation of plasmin under reducing conditions as described under Single-chain
and to the degradation of fibrin clots or blood coagulates. content (see Tests). T h e single-chain alteplase content in
Alteplase consists of 527 amino acids with a calculated bulk samples m ust be higher than 60 p er cent.
relative molecular mass of 59 050 w ithout consideration o f T ry p tic -p e p tid e m a p p in g
the carbohydrate moieties attached at positions Asn 117, T he primary structure o f the alteplase molecule is verified by
Asn 184 and Asn 448. T he total relative molecular mass is tryptic-peptide mapping as described under Identification B.
approximately 65 000. Alteplase is cleaved by plasmin T h e reduced and carboxymethylated molecule is cleaved by
betw een amino-acids 275 and 276 into a two-chain form trypsin into about 50 peptides, which are separated by
(A chain and B chain) that are connected by a disulfide bridge reverse-phase liquid chromatography. A characteristic
betw een Cys 264 and Cys 395. T he single-chain form and chrom atogram (fingerprint) is obtained. T h e identity of the
the two-chain form show comparable fibrinolytic activity in tryptic-peptide map o f a given alteplase sample with the
vitro. profile o f a well-characterised reference standard is an
P R O D U C T IO N indirect confirmation of the amino-acid sequence, becam e
even single amino-acid exchanges in individual peptides can
Alteplase is produced by recom binant D NA synthesis in cell
be detected by this sensitive technique. In addition, complex
culture; the fermentation takes place in serum-free m edium.
peaks o f the glycopeptides can be isolated from the tryptic-
T he purification process is designed to remove efficiendy peptide m ap and separated in a second dimension, either by
potential impurities, such as antibiotics, D N A and protein reverse-phase liquid chrom atography under modified
contam inants derived both from the h ost cell and from the conditions o r by capillary electrophoresis. By this two­
production m edium , and potential viral contaminants.
2016 Alteplase for Injection 1-111

dimensional separation of glycopeptide variants, lot-to-lot and reference standard and using a relative molecular mass of
consistency of the microheterogeneity o f glycosylation can be 180.2 for mannose and a relative molecular mass of 59 050
demonstrated. for the alteplase protein moiety. T h e neutral sugar content of
T he tryptic-peptide map o f alteplase samples must be the alteplase samples m ust be in the range o f 70 to
consistent with the tryptic-peptide map of alteplase reference 130 per cent compared to alteplase reference standard, which
standard. contains about 12 moles of neutral sugar per mole of
alteplase.
Monomer content
T h e m onom er content of alteplase is m easured by gel- CHARACTERS
perm eation liquid chromatography under non-reduced W hite or slightly yellow powder or solid friable mass.
conditions as described under M onomer content (see Tests). Reconstitute the preparation as stated on the label immediately
T he m onom er content of alteplase bulk samples must be before carrying out the Identification, Tests (except those for
higher than 95 per cent solubility and water) and Assay.
Type I/Type II alteplase content IDENTIFICATION
C H O cells produce 2 glycosylation variants o f alteplase. A. T he 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 II alteplase is only
glycosylated at positions Asn 117 and Asn 448. Test solution Dilute the preparation to be examined with
water R to obtain a solution containing about 1 mg of
T he ratio o f Type I/Type II alteplase is constant in the range
alteplase per millilitre. Dialyse about 2.5 m L o f the solution
o f 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 g/L of urea R,
Type II. T h e content of alteplase Type I and Type II can be
44 g/L o f tris(hydroxymethyl) aminomethane R and 1.5 g/L of
determ ined by a densitometric scan o f SDS-PAGE (sodium
sodium edetate R and adjusted to p H 8.6, using a mem brane
dodecyl sulfate polyacrylamide gel electrophoresis) gel.
with a cut-oflf point corresponding to a relative molecular
Plasmin-treated samples of alteplase, which are reduced and
mass o f 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 II
millilitre 10 |iL o f a 156 g/L solution of dithiothreitol R. Allow
alteplase A-chain (AA 1-275) and alteplase B-chain
to stand for 4 h, cool in iced water and add per millilitre of
(AA 276-527). T h e ratio o f Type I/Type II alteplase is
solution 25 pL o f a freshly prepared 190 g/L solution of
determ ined from a calibration curve, which is obtained by a
iodoacetic add R. Allow to stand in the dark for 30 min.
densitometric scan of defined mixtures o f purified Type I
A dd per millilitre 50 |oL of dithiothreitol solution to stop the
alteplase and T ype II alteplase standards.
reaction. Dialyse for 24 h against an 8 g/L solution of
SDS-PAGE ammonium hydrogen carbonate R. Add 1 part of trypsin for
SD S-PAG E (silver staining) is used to demonstrate purity of peptide mapping R to 100 parts o f the protein and allow to
the alteplase bulk material and the integrity o f the alteplase stand for 6 h to 8 h. Repeat the addition of trypsin and allow
molecule. F or 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 m ust occur in SDS-PAGE gels at a loading am ount
suitable reference standard instead of the preparation to be
o f 2.5 |xg alteplase protein per lane and a limit of detection of
examined.
5 ng per protein (BSA) band.
T he chromatographic procedure may be carried out using:
Bacterial endotoxins (2.6.14) — a column 0.1 m long and 4.6 mm in internal diameter
Less than 1 IU p er milligram of alteplase. packed with octadecylsüyl silica gelfor chromatography R
Sialic adds (5 jim to 10 pm);
Proceed using a suitable validated m ethod devdoped Mobile phase A 8 g/L solution of sodium dikydrogen
according to general chapter 2.2.59. Glycan analysis of phosphate R , adjusted to p H 2.85 with phosphoric
glycoproteins. T h e sialic ad d s content for the test samples acid R, filtered and degassed;
m ust 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 acetomtrUe R in mobile phase A;
sialic a d d s 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 1 mL/min. After injection of the solution, increase the
assay buffer, containing 34.8 g/L of arginine R, 0.1 g/L 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 m inute until the ratio of mobile phase A to mobile phase B is
acid R, to a protein concentration of 50 [ig/m L Prepare the 60:40, then increase the proportion of mobile phase B at a
following concentrations o f mannose in the same assay buffer rate o f 1.33 per cent per minute until the ratio o f mobile
for a calibration curve: 20, 30, 40, 50 and 60 (ig/mL. Pipette phase A to mobile phase B is 20:80 and then continue
2 m L of alteplase samples and reference standard, as well as elution with this mixture for a further 10 min. Record the
2 m L o f each mannose concentration in duplicate in reagent chromatogram for the reference solution: the test is not valid
tubes. A dd 50 jaL of phenol R, followed by 5 m L of sulfuric unless the resolution of peaks 6 (peptides 268-275) and 7
acid R, in each reagent tube. Incubate the mixture for 30 m in (peptides 1-7) is at least 1.5; whf and are n o t more than
at room tem perature. M easure the absorbance at 492 nm for 0.4 min. Inject about 100 |iL o f 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. T he neutral sugar content is comparison with the chromatograms of the reference
expressed in moles of neutral sugar per mole of alteplase, solution. There should n o t be any additional significant peaks
taking into account the dilution factor for alteplase samples o r shoulders, a significant peak or shoulder being defined as
 1-112 Alteplase for Injection 2016

one with an area response equal to or greater than 5 p er cent — a colum n 0.6 m long and 7.5 m m in internal diam eter
of peak 19 (peptides 278-296); no significant peak is missing. packed with silica-based, rigid, hydrophilic gel with
A type chromatogram for identification of the peaks cited is spherical particles 10 pm to 13 pm in diam eter, suitable
shown in Figure 1170.-1. for size-exclusion chromatography;
— as mobile phase at a flow rate o f 0.5 m L/m in a solution
TESTS
c o n tainin g 30 g/L of sodium dihydrogen phosphate R and
A p p e a ra n c e o f so lu tio n
1 g/L o f sodium dodecyl sulfate R, adjusted to p H 6.8 with
T he reconstituted preparation is clear (2.2. I) and n o t more
dilute sodium hydroxide solution R;
intensely coloured than reference solution Y7 (2.2.2,
— as detector a spectrophotom eter set at 214 nm .
Method II).
Inject about 50 pL o f the test solution and record the
p H (2.2.3)
chrom atogram. T he chrom atogram shows 2 m ajor peaks
7.1 to 7.5. corresponding to single-chain and two-chain alteplase.
S o lu b ility Calculate the relative am ount o f single-chain alteplase from
A dd the volume o f the liquid stated on the label. the peak area values.
T h e preparation dissolves completely within 2 m in at 20 °C T h e test is n o t valid unless: the num ber o f theoretical plates
to 25 °C. calculated on the basis o f the single-chain alteplase peak is at
P r o te in c o n te n t least 1000. T h e content of single-chain alteplase is n o t less
Prepare a solution of the substance to be examined with an than 60 per cent o f the total am ount o f alteplase-related
accurately known concentration of about 1 g/L. Using a substances found.
34.8 g/L solution of arginine R adjusted to p H 7.3 with M o n o m e r c o n te n t
phosphoric acid R, dilute an accurately measured volume o f Examine by liquid chromatography (2.2.29).
the solution o f 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 m g per millilitre.
0.5 to 1.0 (test solution). M easure the absorbance (2.2.25) o f
the solution at the maximum at about 280 nm and at T h e chromatographic procedure may be carried out using:
320 nm using the arginine solution as the compensation — a colum n 0.6 m long and 7.5 m m in internal diam eter
liquid. Calculate the protein content in the portion o f packed with silica-based rigid, hydrophilic gel with
alteplase taken from the following expression: spherical particles 10 pm to 13 pm in diameter, suitable
for size-exclusion chromatography;
— as mobile phase at a flow rate o f 0.5 m L/m in a solution
V (-^280 ~ -<4.320)
containing 30 g/L of sodium dihydrogen phosphate R and
1.9
1 g/L o f sodium dodecyl sulfate R, adjusted to p H 6.8 with
dilute sodium hydroxide solution R;
in which V is the volume o f th e test solution, A 2 S0 is the
— as detector a spectrophotom eter set at 214 nm .
absorbance at the maximum at about 280 nm and A 32q is the
absorbance at 320 nm. Inject the test solution and record the chromatogram.
T h e test is n o t valid unless the num ber o f theoretical plates
Single-chain c o n te n t
calculated for the alteplase m onom er peak is at least 1000.
Examine by liquid chrom atography (2.2.29).
M easure the response for all peaks, i.e. peaks corresponding
Test solution Dissolve the preparation to be examined in to alteplase species o f different molecular masses. Calculate
water R to obtain a solution containing about 1 m g o f the relative content of m onom er from the area values of these
alteplase per millilitre. Place about 1 m L o f the solution in a peaks. T h e m onom er content for alteplase m ust be at least
tube, add 3 m L of a 3 g/L solution o f dxthiotkreitd R in the 95 p er c e n t
mobile phase, place a cap on the tube and heat at about
80 °C for 3 m in to 5 min.
T h e chromatographic procedure may be carried out using:
2016 Altizide 1-113

W a te r (2.5.12) valid unless the correlation coefficient is —0.9900 to

N o t more than 4.0 per cent, determined by the semi-micro —1.0000. From the line equation and the clot-lysis time for
determ ination o f water. the test solution, calculate the logarithm o f the activity U&
B a c te ria l en d o to x in s (2.6.14) from the following equation:
Less than 1 IU per milligram of protein.
,o g u A = l O e g t A
S te rility (2. 6.1) 0
It complies with the test for sterility.
Calculate the alteplase activity in International U nits per
A SSA Y millilitre from the following expression:
T h e potency of alteplase is determined by comparing its
ability to activate plasminogen to form plasmin with the same D xU a
capacity o f a reference preparation calibrated in International
U nits. T he formation o f plasmin is measured by the in which D is the dilution factor for the test solution.
determ ination o f the lysis time of a fibrin clot in given Calculate the specific activity in the portion of the substance
conditions. to be examined from the following expression:
T h e International U nit is the activity of a stated quantity of
the International Standard o f alteplase. T he equivalence in Ua
International U nits of the International Standard is stated by P
the World H ealth Organization.
Solvent buffer A solution containing 1.38 g/L of sodium in which P is the concentration of protein obtained in the test
dihydrogen phosphate monohydrate R, 7.10 g/L of anhydrous for protein con ten t
disodium hydrogen phosphate R, 0.20 g/L o f sodium azide R and T h e estimated potency is not less than 90 per cent and not
0.10 g/L of polysorbate 80 R. m ore than 110 p er cent of the stated potency.
Human thrombin solution A solution o f human thrombin R STORA GE
containing 33 IU /m L in solvent buffer. Store in a colourless, glass container, under vacuum or under
Human fibrinogen solution A 2 g/L solution o ffibrinogen R in an inert gas, protected from light, at a tem perature of 2 °C to
solvent buffer. 30 °C.
Human plasminogen solution A 1 g/L solution of human LA B E LL IN G
plasminogen R in solvent buffer. The label states:
Test solutions U sing a solution of the substance to be — the num ber o f International Units per container;
examined containing 1 g/L, prepare serial dilutions using — the am ount o f protein per container;
solvent buffer, for example 1:5000, 1:10 000, 1:20 000. — the name and volume of the liquid to be used for
Reference solutions Using a solution of a suitable reference reconstitution.
standard having an accurately known concentration of about ---------------------------------------------------------------------- -------------- -- PhEur
1 g/L (580 000 IU o f alteplase per millilitre), prepare 5 serial
dilutions using water R to obtain reference solutions having
known concentrations in the range 9.0 IU /m L to 145 IU /m L.
T o each o f a set o f labelled glass test-tubes, add 0.5 m L of Altizide *****
hum an throm bin solution. Allocate each test and reference *, *
solution to a separate tube and add to each tube 0.5 m L o f (Ph Eur monograph 2185) *
the solution allocated to it. T o each o f a second set of
o o
labelled glass tubes, add 20 |iL of hum an plasminogen
solution, and 1 m L of hum an fibrinogen solution, mix and H’ N ' S y T S' r and enanUomer
store on ice. Beginning with the reference/thrombin mixture C l N ^s
containing the lowest num ber of International Units per H H
millilitre, record the time and separately add 200 pL o f each
o f the thrombin mixtures to the test tubes containing the C 11H 14QN30 4S3 383.9 5588-16-9
plasminogen-fibrinogen mixture. Using a vortex mixer,
intermittently mix the contents of each tube for a total of A c tio n a n d u se
15 s and carefully place in a rack in a circulating water-bath Thiazide diuretic.
at 37 °C. A visibly turbid clot forms within 30 s and bubbles
PhEur___ ______________________________________________________
subsequently form within die clot. Record the clot-lysis time
as the time between the first addition of alteplase solution D E F IN IT IO N
and the m om ent when the last bubble rises to the surface. (3RS) -6-Chloro-3- [(prop-2-enylsulfanyl) methyl] -3,4-dihydro-
U sing a least-squares fit, determine the equation o f the line 2/f-l,2,4-benzothiadiazine-7-sulfonamide 1,1-dioxide.
using the logarithms o f the concentrations of the reference C o n te n t
preparation in International Units per millilitre versus the 97.5 per cent to 102.0 per cent (anhydrous substance).
logarithms of the values of their clot-lysis times in seconds,
according to the following equation: CHA RACTERS
A p p e a ra n c e
logf = o + 6 (logl7s) W hite or almost white powder.
S o lu b ility
in which t is the clot-lysis time, Us the activity in Practically insoluble in water, soluble in methanol, practically
International U nits per millilitre of the reference preparation, insoluble in methylene chloride.
b is the slope and a the ^ in te rc ep t o f the line. T he test is not It shows polymorphism (5.9).
 1-114 Altizide 2016

ID E N T IF IC A T IO N Detection Spectrophotom eter at 270 nm .

Infrared absorption spectrophotom etry (2.2.24). Injection 5 pL.
Comparison altizide CRS. Run time Twice the retention tim e o f altizide.
If the spectra obtained show differences, dissolve 50 m g of Relative retention W ith reference to altizide (retention
the substance to be examined and 50 m g of the reference time = about 25 min): im purity A = about 0.15;
substance separately in 2 m L o f acetone R and evaporate the furosemide = about 1.05.
solvent. Precipitate by adding 1 m L o f methylene chloride R. System suitability: reference solution (c):
Evaporate to dryness and record new spectra using the — resolution: minimum 1.0 between die peaks due to altizide
residues. and furosemide.
TESTS Limits:
Im p u rity B — impurity A: not more than 3 times die area o f the
Thin-layer chrom atography (2.2.27). principal peak in the chrom atogram obtained with
Test solution Dissolve 0.200 g o f the substance to be reference solution (a) (0.3 p er cent);
examined in acetone R and dilute to 2.0 m L with the same — unspecified impurities: for each impurity, n o t m ore than the
solvent. area o f the principal peak in the chrom atogram obtained
with reference solution (a) (0.10 per cent);
Reference solution (a) Dissolve 10.0 m g o f altizide
— total: not more than 5 times the area o f the principal peak
impurity B CRS in acetone R and dilute to 25.0 m L with the
in the chromatogram obtained with reference solution (a)
same solvent.
(0.5 per cent);
Reference solution (b) T o 1.0 m L of reference solution (a) add — disregard limit. 0.5 times the area o f the principal peak in
1.0 m L o f the test solution. the chromatogram obtained w ith reference solution (a)
Reference solution (c) Dilute 5.0 mL o f reference solution (a) (0.05 per cent).
to 10.0 m L with acetone R. W a te r (2.5.32)
Plate TLC silica gel F2 5 4 plate R. Maximum 0.5 per cent, determ ined on 50.0 mg.
Mobile phase acetone R, methylene chloride R (25:75 VIV). S u lfa te d a s h (2.4.14)
Application 10 pL o f the test solution and reference M aximum 0.1 per cent, determ ined on 1.0 g.
solutions (b) and (c).
ASSAY
Development Over 2/3 o f 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 o f equal volumes o f a 10 g/L Test solution Dissolve 25.0 m g o f the substance to be
solution of potassium permanganate R and a 50 g/L solution of examined in 2 mL o f acetomtrile R and dilute to 25.0 m L
sodium carbonate R, prepared immediately before use. Allow with the mobile phase.
to stand for 30 m in and examine in daylight. Reference sdution Dissolve 25.0 m g o f altizide CRS in 2 m L of
System suitability: reference solution (b): acetomtrile R and dilute to 25.0 m L with die mobile phase.
— the chrom atogram shows 2 clearly separated spots. Calculate the percentage content o f C iiH 14Q N 304 S 3 from
Limit Any spot due to im purity 6 is n o t more intense than the declared content o f altizide CRS.
the principal spot in the chrom atogram obtained with
IM P U R IT IE S
reference solution (c) (0.2 per cent).
Specified impurities A, B
R e la te d su b s ta n c e s
liq u id chrom atography (2.2.29). Prepare the solutions
immediately before use, except reference sdution (b).
Test solution Dissolve 50 mg o f the substance to be examined
in 5 m L o f acetomtrUe R and dilute to 25 m L with the mobile
phase.
Reference solution (a) D ilute 1.0 m L o f the test solution to A. 4-am ino-6-chlorobenzene-l,3-disulfonam ide,
100.0 m L with the mobile phase. D ilute 1.0 m L of this
solution to 10.0 m L w ith the mobile phase. OCH,
Reference solution (b) In order to produce im purity A in situ,
dissolve 50 m g o f the substance to be examined in 5 m L o f
acetomtrile R and dilute to 25 m L with water R. Allow to
stand for 30 min. B. 3-[(2,2-dimethoxyethyl)sulfanyl]prop-1-ene.
Reference solution (c) Dissolve 4 mg o f furosemide CRS in ___________________________________________________________ PtiEur
2 m l . of acetomtrUe R, add 2 m L o f the test solution and
dilute to 100 m L with the mobile phase.
Column:
— size: I = 0.15 m , 0 = 3.9 mm;
— stationary phase: end-capped octadecylsüyl silica gelfor
chromatography R (5 pm);
— temperature: 30 °C.
Mobile phase acetomtrile R, water R previously adjusted to
p H 2.0 with perchloric add R (25:75 VIV).
Flow rate 0.7 mL/m in.
2016 Aluminium Chloride 1-115

Alum ***** Aluminium Chloride Hexahydrate ★ ★

★ ★
*★ ★* *****
Potash Alum; Aluminium Potassium Sulphate; * (Ph Eur monograph 0971)
Aluminium Potassium Sulfate A1C13,6H20 241.4 7784-13-6
(Ph Eur monograph 0006)
A c tio n a n d u se
A1K(S04)2j 12H20 474.4 7784-24-9
A stringent
A ction a n d u se P re p a r a tio n
Astringent. Aluminium Chloride Solution

PhEur__________________________________________________________ PhEtr.

D E F IN IT IO N D E F IN IT IO N
C o n ten t C o n te n t
99.0 per cent to 100.5 per cent of A IK C S O ^ 12H 20 . 95.0 p er cent to 101.0 per cent.
CHARACTERS CHARACTERS
A p p e a ra n c e A p p e a ra n c e
Granular powder or colourless, transparent, crystalline W hite or slightly yellow, crystalline powder or colourless
masses. crystals, deliquescent
S olubility S o lu b ility
Freely soluble in water, very soluble in boiling water, soluble Very soluble in water, freely soluble in ethanol (96 per cent),
in glycerol, practically insoluble in ethanol (96 per cent). soluble in glycerol.
ID E N T IF IC A T IO N ID E N T IF IC A T IO N
A. Solution S (see Tests) gives the reactions o f sulfates A. Dilute 0.1 m L of solution S2 (see Tests) to 2 m L with
(2.3.1). water R. T he solution gives reaction (a) o f chlorides (2.3.1).
B. Solution S gives the reaction of aluminium (2.3.1). B. Dilute 0.3 m L o f solution S2 to 2 m L with water R.
C. Shake 10 m L o f solution S with 0.5 g o f sodium hydrogen T he solution gives the reaction o f aluminium (2.3.1).
carbonate R and filter. T he filtrate gives reaction (a) o f TESTS
potassium (2.3.1). S o lu tio n S I
TESTS Dissolve 10.0 g in distilled water R and dilute to 100 m L w ith
S o lu tio n S the same solvent.
Dissolve 2.5 g in water R and dilute to 50 m L with the same S o lu tio n S 2
solvent Dilute 50 m L o f solution SI to 100 m L with water R.
A p p e a ra n c e o f so lu tio n A p p e a ra n c e o f so lu tio n
Solution S is clear (2.2.1) and colourless (2.2.2, Method IT). Solution S2 is clear (2.2.1) and no t more intensely coloured
p H (2.2.3) than reference solution B 7 (2.2.2, Method II).
3.0 to 3.5. S u lfates (2.4.13)
Dissolve 1.0 g in carbon dioxide-free water R and dilute to Maximum 100 ppm , determined on solution S I.
10 m L with die same solvent. Ir o n (2.4.9)
A m m o n iu m (2.4.1) Maximum 10 ppm , determined on solution S i.
Maximum 0.2 per cent. A lkali a n d alk alin e -e a rth m e ta ls
T o 1 m l, o f solution S add 4 m L of water R. Dilute 0.5 m L Maximum 0.5 per c e n t
o f this solution to 14 m L with water R. T o 20 m L o f solution S2 add 100 m L o f water R and heat to
Iron ( 2. 4. 9) boiling. T o the hot solution add 0.2 m l. of methyl red
Maximum 100 ppm. solution R. Add dilute ammonia R1 until the colour of the
Dilute 2 m L o f solution S to 10 m L with water R. U se in this indicator changes to yellow and dilute to 150 m L with
test 0.3 m L o f thiogfycoUic add R. water R. H eat to boiling and filter. Evaporate 75 m L of the
filtrate to dryness on a water-bath and ignite to constant
H eavy m e ta ls (2.4.8)
mass. T h e residue weighs a maximum of 2.5 mg.
M aximum 20 ppm.
H eav y m e ta ls (2.4.8)
12 m L o f solution S complies with test A. Prepare the
Maximum 20 ppm.
reference solution using lead standard solution (1 ppm Pb) R.
12 m L of solution SI complies w ith test A. Prepare the
ASSAY reference solution using lead standard solution (2 ppm Pb) R.
Dissolve 0.900 g in 20 m L of water R and carry out the
W a te r (2.5.12)
complexometric titration o f aluminium (2.5.11).
42.0 p e r cent to 48.0 per cent, determined on 50.0 mg.
1 m L of 0.1 M sodium edetate is equivalent to 47.44 mg
ofA IK (S 04 ) 2, 12H 20 . A SSAY
Dissolve 0.500 g in 25.0 m L of water R. Carry out the
__________________________________________________________ PhEur
complexometric titration o f aluminium (2.5.11). Titrate with
0.1 M zinc sulfate until the colour o f the indicator changes
from greyish-green to pink. Carry out a blank titration.
1 m L of 0.1 M sodium edetate is equivalent to 24.14 mg
o f A1C13,6H 20 .
 1-116 Aluminium Glycinate 2016

STORAGE A ppendix VH. U s z lead standard solution (1 ppm Pb) to

In an airtight container. prepare the standard (20 ppm ).
PhEur Mercuric salts
Dissolve 2.0 g in 10 m L o f 1m sulfuric add, transfer to a
separating funnel with the aid o f water, dilute to about
50 m L w ith water and add 50 m L o f 0.5m sulfuric acid.
Add 100 m L o f water, 2 g o f hydroxylamine hydrochloride.,
Aluminium Glycinate 1 m L o f 0.05m disodium edetate and 1 m L o f glacial acetic
acid. A dd 5 m L o f chloroform, shake, allow to separate and
discard the chloroform layer. T itrate the aqueous layer with a
solution o f dithizone in chloroform containing 8 \ig per m L
,xH20 until the chloroform layer rem ain s green. After each addition,
shake vigorously, allow the layers to separate and discard the
chloroform layer. Repeat the operation using a solution
prepared by d ilu tin g 1 mT. o f mercury standard solution
C2H6A1N04}xH20 135.1 41354-48-7 (5 ppm Hg) to 100 m L with 0.5m sulfuric add and beginning
at the words ‘Add 100 m L of water . . . T h e volume o f the
Action and use dithizone solution required by the substance being examined
Antacid. does n o t exceed that required by the mercury standard
solution.
DEFINITION
Chloride
Aluminium Glycinate is a basic alu m in iu m monoglycinate,
Dissolve 1.0 g in 10 m L o f 2m nitric add and dilute to
partly hydrated. It contains n o t less than 34.5% and not
100 m L w ith water. 15 m L o f the resulting solution complies
more than 38.5% of AI2O 3 and not less than 9.9% and not
with the limit test for chlorides, Appendix VH (330 ppm).
more than 10.8% of N , both calculated with reference to the
dried substance. Loss on drying
W hen dried to constant w dght at 130°, loses n o t more than
CHARACTERISTICS 12.0% o f its w dght. U se 1 g.
A white or alm ost white powder.
ASSAY
Practically insoluble in water and in organic solvents.
It dissolves in dilute mineral a d d s and in aqueous solutions For Al203
Dissolve 0.25 g in a m ixture o f 3 m L o f 1m hydrochloric add
of the alkali hydroxides.
and 50 m L o f water, add 50 m L o f 0. 05m disodium edetate VS
IDENTIFICATION and neutralise with 1m sodium hydroxide using methyl red
A. A dd 0.1 g to 10 m L o f a solution prepared by dissolving solution as indicator. H eat the solution to boiling, allow to
0.84 g o f citric add in 8 m L o f 1m sodium hydroxide and stand for 10 m in u tes on a water bath, cool rapidly, add
diluting to 20 m L with water. Add 0.5 m L of a 0.1% w/v about 50 m g o f xylenol orange triturate and 5 g o f hexamine
solution of ninhydrin in methanol and warm. A purple colour and titrate the excess o f disodium edetate with 0. 05m lead
is produced. nitrate VS until the solution becomes red. Each m L o f 0. 05m
B. Suspend 1 g in 25 m L o f 0.5m hydrochloric add and heat disodium edetate VS is equivalent to 2.549 m g o f A120 3.
gently until a clear solution is produced. Reserve h alf o f the
solution. T o 2 m L o f the solution add 0.15 m L o f liquefied
phenol, shake and add carefully w ithout shaking 5 m L of
dilute sodium hypochlorite solution. A blue colour is produced. ★.* * * ★
C. T h e solution reserved in test B yields the reaction
Hydrated Aluminium Hydroxide tor ★ ★
characteristic o f aluminium salts, A ppendix VI. Adsorption *****
TESTS (Ph Eur monograph 1664)
Acidity or alkalinity [A10(0H)],nH20
p H o f a suspension o f 1 g in 25 m L o f carbon dioxide-free PhEur_____________
water, 6.5 to 7.5, Appendix V L.
DEFINITION
Neutralising capacity
Shake 0.2 g vigorously with 25 m L o f 0. 1m hydrochloric add
Content
90.0 p er cent to 110.0 per cent o f the content o f aluminium
for 5 m inutes and allow to stand for 5 minutes. T he p H o f
stated on the labd.
the mixture is greater than 3.0, Appendix V L.
NOTE: shake the gel vigorously for at least 30 s immediately
Arsenic
before examining.
Dissolve 2.0 g in 18 m L o f brominated hydrochloric acid and
32 m L of water. 25 m L o f the resulting solution complies CHARACTERS
with the limit test for arsenic, Appendix VII (1 ppm). Appearance
Heavy m etals W hite or alm ost white, translucent, viscous, colloidal g d .
Dissolve 1.5 g in 20 m L o f 2m hydrochloric add and 10 m L of A supernatant may be formed upon standing.
water, add 0.5 m L o f nitric acid and boil for about Solubility
30 seconds. Cool, add 2 g o f ammonium chloride and 2 g o f A clear or almost d e a r solution is obtained with alkali
ammonium tkiocyanate and extract w ith two 10 mT. q u an tities hydroxide solutions and mineral adds.
o f a mixture o f equal parts o f isoamyl alcohol and ether. IDENTIFICATION
T o the aqueous layer add 2 g o f citric acid. 12 m L o f the
Solution S (see Tests) gives the reaction o f aluminium.
resulting solution complies w ith limit test A for heavy metals,
2016 Aluminium Hydroxide 1-117

T o 10 m L o f solution S add about 0.5 m L o f dilute Place 5 g in a test-tube immersed in ice-water, add 0.4 m L
hydrochloric add R and about 0.5 m L of thioacetamide of a 100 g/L solution of potassium chloride R, 0.1 mT. of
reagent R. N o precipitate is formed. A dd dropwise 5 m L of diphenylandne solution R and, dropwise with sh a k in g , 5 m L of
dilute sodium hydroxide solution R. Allow to stand for 1 h. sulfuric acid R. Transfer the tube to a water-bath at 50 °C.
A gelatinous white precipitate is formed which dissolves upon After 15 min, any blue colour in the solution is no t more
addition o f 5 mT. of dilute sodium hydroxide solution R. intense than that in a standard prepared a t the same time
Gradually add 5 m L of ammonium chloride solution R and and in the same manner using 5 m L of nitrate standard
allow to stand for 30 min. T he gelatinous white precipitate is solution (100 ppm NO?) R.
re-formed. S u lfates (2.4.13)
TESTS M aximum 0.5 p er cent.
S o lu tio n S Dilute 2 m L o f solution S to 20 m L with water R.
A dd 1 g to 4 m L of hydrochloric acid R. H eat at 60 °C for A m m o n iu m (2. 4.1, Method B)
1 h, cool, dilute to 50 m L with distilled water R and filter if M aximum 50 ppm , determined on 1.0 g.
necessary.
Prepare the standard using 0.5 m L of ammonium standard
p H (2.2.5) solution (100 ppm NH 4) R.
5.5 to 8.5.
A rse n ic (2.4.2, Method A)
A d so rp tio n p o w e r M aximum 1 ppm , determined on 1 g.
Dilute the substance to be examined with distilled water R to
Ir o n (2.4.9)
obtain an aluminium concentration o f 5 mg/mL. Prepare
M aximum 15 ppm , determined on 0.67 g.
bovine albumin R solutions with the following concentrations
o f bovine albumin: 0.5 mg/mL, 1 mg/mL, 2 mg/mL, H eav y m e ta ls (2.4.8)
3 mg/mL, 5 m g/m L and 10 mg/mL. I f necessary, adjust the M aximum 20 ppm .
gel and the bovine albumin R solutions to p H 6.0 with dilute Dissolve 2.0 g in 10 m L o f dilute nitric acid R and dilute to
hydrochloric acid R or dilute sodium hydroxide solution R. 20 m L with water R. T h e solution complies with test A.
F or adsorption, mix 1 part o f the diluted gel with 4 parts o f Prepare the reference solution using lead standard solution
each of the solutions o f bovine albumin R and allow to stand (2 ppm Pb) R.
at room tem perature for 1 h. During this time shake the B a c te ria l en d o to x in s (2.6.14)
mixture vigorously at least 5 times. Centrifuge or filter Less than 5 IU of endotoxin per m illigram of alu m in iu m , if
through a non-protein-retaining filter. Immediately determine intended for use in the manufacture of an adsorbed product
the protein content (2.5.33, Method 2) o f either the w ithout a further appropriate procedure for the removal of
supernatant or the filtrate. bacterial endotoxins.
It complies with the test if no bovine alb u m in is detectable in A SSAY
the supernatant or filtrate of the 2 mg/mL bovine albumin R Dissolve 2.50 g in 10 m L o f hydrochloric acid R, heating for
solution (maximum level of adsorption) and in the 30 min at 100 °C on a water-bath. Cool and dilute to 20 m L
supernatant or filtrate o f bovine albumin R solutions o f lower with water R. T o 10 m L o f the solution, add concentrated
concentrations. Solutions containing 3 mg/mL, 5 m g/m L and ammonia R until a precipitate is obtained. Add the smallest
10 mg/mL bovine albumin R may show bovine albumin in the quantity o f hydrochloric acid R needed to dissolve the.
supernatant or filtrate, proportional to the am ount o f bovine precipitate and dilute to 20 m L with water R. Carry out the
albumin in the solutions. complexometric titration o f alu m in ium (2.5.11). Carry out a
S e d im e n ta tio n blank titration.
If necessary, adjust the substance to be examined to p H 6.0
STORA GE
using dilute hydrochloric add R or dilute sodium hydroxide
At a temperature not exceeding 30 °C. D o not allow to
solution R. D ilute with distilled water R to obtain an
freeze. If the substance is sterile, store in a sterile, airtight,
aluminium concentration of approximately 5 mg/mL. I f the
tam per-proof container.
aluminium content of the substance to be e x am in e d is lower
than 5 mg/m L, adjust to p H 6.0 and dilute with a 9 g/L LABELLING
solution o f sodium chloride R to obtain an aluminium T h e label states the declared content o f alu m in iu m .
concentration o f about 1 mg/mL. After shaking for at least -------------------:____________________________________________ PhEur
30 s, place 25 m L of the preparation in a 25 m L graduated
cylinder and allow to stand for 24 h.
It complies with the test if the volume o f the clear
supernatant is less than 5 m L for die gel with an aluminium
content o f about 5 mg/mL.
Dried Aluminium Hydroxide *****
*★ ★*
It complies with the test if the volume o f the clear (Hydrated Aluminium Oxide, *
supernatant is less than 20 m L for the gel with an aluminium Ph Eur monograph 0311)
content o f about 1 mg/mL.
A ctio n a n d u se
C h lo rid e s (2.4.4)
Antacid.
Maximum 0.33 per cent.
P re p a ra tio n s
Dissolve 0.5 g in 10 m L of dilute nitric add R and dilute to
Aluminium Hydroxide Oral Suspension
500 m L w ith water R.
Chewable Aluminium Hydroxide Tablets
N itra te s
Chewable Compound M agnesium Trisilicate Tablets
M axim um 100 ppm.
Co-magaldrox Oral Suspension
Co-magaldrox Tablets
 1-118 Aluminium Magnesium Silicate 2016

PhEir ___________________________________ :_______________________ ASSAY

D E F IN IT IO N Dissolve 0.800 g in 10 m L of hydrochloric add R l, heating on
a water-bath. Cool and dilute to 50.0 m L w ith water R
C o n te n t
T o 10.0 m L o f the solution add dilute ammonia R1 until a
47.0 per cent to 60.0 per cent o f A120 3 (Mt 102.0).
predpitate begins to appear. Add the smallest quantity of
CHARACTERS dilute hydrochloric add R needed to dissolve the predpitate
A p p e a ra n c e and dilute to 20 m L with water R. Carry out the
W hite or almost white, amorphous powder. complexometric titration o f aluminium (2.5.11).
S o lu b ility 1 m ĩ, o f 0.1 M sodium edetate is equivalent to 5.098 mg
Practically insoluble in water. It dissolves in dilute mineral o f AI2O3.
adds and in solutions o f alkali hydroxides.
STORA GE
ID E N T IF IC A T IO N In an airtight container, at a tem perature not
Solution S (see Tests) gives the reaction o f aluminium exceeding 30 °c.
(2.3.1). F U N C T IO N A L IT Y -R E L A T E D C H A R A C T E R IS T IC S
TESTS This section provides information on characteristics that are
S o lu tio n S recognised as bang relevant control parameters for one or more
Dissolve 2.5 g in 15 m L of hydrochloric add R, heating on a functions of the substance when used as an excipient (see chapter
water-bath. Dilute to 100 m L with distQled water R. 5.15). This section ừ a non-mandatory part of the monograph
A p p e a ra n c e o f so lu tio n and ừ ừ not necessary to verify the characteristics to demonstrate
Solution S is n o t more opalescent than reference compliance. Control of these characteristics can however contribute
suspension II (2.2.1) and not more intensely coloured than to the quality of a medicinal product by improving the consistency
reference solution GY6 (2.2.2, Method II). of the manufacturing process and the performance of the medicinal
product during use. Where control methods are cited, they are
A lkaline im p u ritie s
recognised as being suitable for the purpose, but other methods can
Shake 1.0 g with 20 m L of carbon dioxide-free water R for
also be used Wherever results for a particular characteristic are
1 m in and filter. T o 10 m L o f the filtrate add 0.1 m L of
reported, the control method must be indicated
phenolphthalein solution R. Any pink colour disappears on the
addition of 0.3 m L o f 0.1 M hydrochloric add.
The f(Mowing characteristics may be relevantfor hydrated
aluminium oxide used as adsorbent
N e u tra lisin g c a p a c ity
P a rtic le -siz e d is trib u tio n (2.9.31)
Cany out the test at 37 °C. Disperse 0.5 g in 100 m L of
water R, heat, add 100.0 m L o f 0.1 M hydrochloric add, Specific su rfa c e a r e a (2.9.26)
previously heated, and stir continuously; the p H (2.2.3) of __________________________________________________________ Pi) Ell
the solution after 10 min, 15 min and 20 min is not less than
1.8, 2.3 and 3.0 respectively and is at no time greater than
4.5. Add 10.0 mL of 0.5 M hydrochloric add, previously
heated, stir continuously for 1 h and titrate with 0.1 M
sodium hydroxide to p H 3.5; n o t more than 35.0 m L o f 0.1 M Aluminium Magnesium Silicate *****
_ *. .*
sodium hydroxide is required. (Ph. Ear. monograph 1388) *
C h lo rid e s (2.4.4)
M axim um 1 p e r cent. A ctio n a n d u se
E xdpient.
Dissolve 0.1 g with heating in 10 m L o f dilute nitric add R
and dilute to 100 m L with water R. Dilute 5 m L of the PhEir___________________________________________________________
solution to 15 m L with water R.
D E F IN IT IO N
S u lfa te s (2.4.13)
M ixture of partides with colloidal particle size o f
M axim um 1 per cent.
montmorillonite and saponite, free from grit and non-
D ilute 4 m L o f solution S to 100 m L with distHkd water R. swellable ore.
A rse n ic (2.4.2, Method A) C o n te n t
M axim um 4 ppm , determ ined on 10 m L o f solution S. — aluminium (Al; A r 26.98): 95.0 p er cent to 105.0 per cent
H e a v y m e ta ls (2.4.8) o f the value stated on the labd;
M axim um 60 ppm. — magnesium (Mg; A T 24.30): 95.0 per cent to
Neutralise 20 m L o f solution S with concentrated ammonia R, 105.0 per cent o f the value stated on the lab d .
using metanU yellow solution R as an external indicator. Filter, CHARACTERS
if necessary, and dilute to 30 m L with water R. 12 m L o f the A p p e a ra n c e
solution complies with test A. Prepare the reference solution Almost white powder, granules o r plates.
using 10 m l- o f lead standard solution (1 ppm Pb) R.
S o lu b ility
M ic ro b ia l c o n ta m in a tio n Practically insoluble in water and in organic solvents.
T A M C : acceptance criterion 103 C FU /g (2.6.12).
It swells in water to produce a colloidal dispersion.
T Y M C : acceptance criterion 102 CFU/g (2.6.12).
ID E N T IF IC A T IO N
Absence o f bile-tolerant gram-negative bacteria (2.6.13).
A. Fuse 1 g with 2 g of anhydrous sodium carbonate R. W arm
Absence o f Escherichia colt (2.6.13). the residue with water R and filter. Addify the filtrate with
hydrochloric add R and evaporate to dryness on a water-bath.
0.25 g o f the residue gives the reaction of silicates (2.3.1).
2016 Aluminium Magnesium Silicate 1-119

B. Dissolve the remainder o f the residue obtained in L oss o n d ry in g (2.2.32)

identification test A in a mixture o f 5 m L o f dilute hydrochloric M aximum 8.0 per cent, determined on 1.000 g by drying in
acid R and 10 m L o f water R. Filter and add ammonium an oven at 105 °C.
chloride buffer solution pH 10.0 R A white, gelatinous M ic ro b ia l c o n ta m in a tio n
precipitate is formed. Centrifuge and keep the supernatant TA M C: acceptance criterion 103 CFU/g (2.6.12).
for identification C. Dissolve the remaining precipitate in
TY M C: acceptance criterion 102 CFU/g (2.6.12).
dilute hydrochloric acid R T h e solution gives the reaction of
aluminium (2.3.T). Absence o f Escherichia coli (2.6. II).
C. T he supernatant obtained after centrifugation in A SSAY
identification test B gives the reaction of magnesium (2.3.1). A lu m in iu m
Atomic absorption spectrometry (2.2.23, Method I).
TESTS
p H (2.2.3) Test solution In a platinum crucible mix 0.200 g with 1.0 g of
9.0 to 10.0. lithium metaborate R. H eat slowly at first and ignite at
1000-1200 °C for 15 min. Allow to cool, then place the
Disperse 5.0 g in 100 m L o f carbon dioxide-free water R.
crucible in a 100 m L beaker containing 25 m L o f dilute nitric
A rsen ic (2.4.2, Method A) acid R and add an additional 50 m L of dilute nitric add R,
Maximum 3 ppm. filling and submerging the crucible. Place a
Transfer 16.6 g to a 250 m L beaker containing 100 m L of polytetrafluoroethylene-coated magnetic stirring b ar 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. Pour the contents into a 250 m L
insoluble m atter to settle and decant the supernatant through beaker and remove the crucible. W arm the solution and
a rapid-flow filter paper into a 250 m L volumetric flask, transfer through a rapid-flow filter paper into a 250 m L
retaining as m uch sediment as possible in the beaker. T o the volumetric flask, wash the filter and beaker with water R and
residue in the beaker add 25 m L o f hot dilute hydrochloric dilute to 250.0 m L with water R (solution A). T o 20.0 m L of
acid R, stir, heat to boiling, allow the insoluble m atter to solution A add 20 m L o f a 10 g/L solution of sodium
settle and decant the supernatant through the filter into the chloride R and dilute to 100.0 m L with water R.
volumetric flask. Repeat the extraction with 4 additional Reference solutions Dissolve, with gentle heating, 1.000 g of
quantities, each of 25 m l , of hot dilute hydrochloric acid R, aluminium R in a mixture of 10 m L of hydrochloric acid R and
decanting each supernatant through the filter into the 10 m L o f water R. Allow to cool, then dilute to 1000.0 m L
volumetric flask. A t the last extraction, transfer as m uch of with water R (1 mg of aluminium per millilitre). Into 3
the insoluble m atter as possible onto the filter. Allow the identical volumetric flasks, each containing 0.20 g o f sodium
combined filtrates to cool to room temperature and dilute to chloride R, introduce 2.0 mL, 5.0 m L and 10.0 m L of this
250.0 m L with dilute hydrochloric acid R. Dilute 5.0 m L of solution respectively, and dilute to 100.0 m L w ith water R.
this solution to 25.0 m L with dilute hydrochloric acid R.
Source Aluminium hollow-cathode lamp.
Lead Wavelength 309 nm.
Maximum 15 ppm.
Atomisation device Oxidising acetylene-nitrous oxide flame.
Atomic absorption spectrometry (2.2.23, Method I).
M a g n e siu m
Test solution Transfer 10.0 g to a 250 m L beaker containing Atomic absorption spectrometry (2.2.23, Method I).
100 m L of dilute hydrochloric acid R. Mix, cover with a watch
glass and boil for 15 min. Allow to cool to room tem perature Test solution Dilute 25.0 m L of solution A, prepared in the
assay for aluminium, to 50.0 m L with water R. T o 5.0 m L of
and allow the insoluble m atter to setde. D ecant the
supernatant through a rapid-flow filter paper into a 400 mL this solution add 20.0 m L of lanthanum nitrate solution R and
dilute to 100.0 m L with water R.
beaker. T o the insoluble m atter in the 250 m L beaker add
25 m L of hot water R. Stir, allow the insoluble m atter to Reference solutions Place 1.000 g of magnesium R in a 250 m L
setde and decant the supernatant through the filter into the beaker containing 20 m L o f water R and carefully add 20 m L
400 m L beaker. Repeat the extraction with 2 additional o f hydrochloric add R} warming if necessary to dissolve.
quantities, each o f 25 mL, o f water R, decanting each time Transfer the solution to a volumetric flask and dilute to
the supernatant through the filter into the 400 m L beaker. 1000.0 m L with water R (1 mg of magnesium p er millilitre).
Wash the filter with 25 m L o f hot water R, collecting this D ilute 5.0 m L of this solution to 250.0 m L with water R.
filtrate in the 400 m L beaker. Concentrate the combined Into 4 identical volumetric flasks, introduce 5.0 mL,
filtrates to about 20 m L by gently boiling. I f a precipitate 10.0 m L, 15.0 m L and 20.0 mL of the solution respectively.
appears, add about 0.1 m L o f nitric acid R, heat to boiling T o each flask add 20.0 m L of lanthanum nitrate solution R
and allow to cool to room temperature. Filter the and dilute to 100.0 m L with water R.
concentrated extracts through a rapid-flow filter paper into a Source Magnesium hollow-cathode lamp.
50 m L volumetric flask. Transfer the remaining contents of Wavelength 285 nm.
the 400 m L beaker through the filter paper and into the flask
Atomisation device Reducing air-acetylene flame.
w ith water R. D ilute this solution to 50.0 m L with water R
L A B E L L IN G
Reference solutions Prepare the reference solutions using lead
standard solution (10 ppm Pb) R, diluted as necessary with T h e label states the content of aluminium and magnesium.
water R. __________________________________________________________PhEur
Source Lead hollow-cathode lamp.
Wavelength 217 nm
Atomisation device Oxidising air-acetylene flame.
 1-120 Aluminium Phosphate 2016

***** 15 min. M easure the absorbance (2.2.25) at 730 nm.

Dried Aluminium Phosphate ★ ★ Calculate the content of soluble phosphates from a
(Aluminium Phosphate, Hydrated, ***** calibration curve prepared using reference solutions (a),
Ph Eur monograph 1598) (b) and (c) after treatm en t
A1P04jxH20 122.0 7784-30-7 Sulfates (2.4.13)
M axim um 0.6 p er c e n t
(anhydrous substance)
D ilute 8 m L o f solution S to 100 m L with distilled water R.
A c tio n a n d u se A rse n ic (2.4.2)
Antacid. M aximum 1 ppm.

PhEur.
1.0 g complies with limit test A.
H e a v y m e ta ls (2.4.8)
D E F IN IT IO N
M axim um 20 ppm.
C o n te n t
94.0 per cent to 102.0 per cent of A1P0 4 (Mt 122.0) (ignited Dissolve 1.0 g in dilute hydrochloric add R and dilute to
substance). 20 m L with the same ad d . 12 m L o f the solution complies
with test A. Prepare the reference solution using lead standard
CHARACTERS solution (1 ppm Pb) R.
A p p e a ra n c e
L o ss o n ig n itio n
W hite or alm ost white powder.
10.0 p er cent to 20.0 per cent, determ ined on 1.000 g at
S o lu b ility 800 ± 50 °C.
Very slightly soluble in water, practically insoluble in ethanol
N e u tra lisin g c a p a c ity
(96 per cent). It dissolves in dilute solutions o f mineral ad d s
A dd 0.50 g to 30 m L of 0.1 M hydrochloric add previously
and alkali hydroxides.
heated to 37 °C and maintain at this tem perature for 15 min
ID E N T IF IC A T IO N while stirring. T h e p H (2.2.3) o f the mixture after 15 m in at
A. Solution S (see Tests) gives reaction (b) of phosphates 37 °C is 2.0 to 2.5.
(2.3.1). A SSA Y
B. Solution S gives the reaction o f aluminium (2.3.1). Dissolve 0.400 g in 10 m L o f dilute hydrochloric add R and
TESTS dilute to 100.0 m L with water R. T o 10.0 m L o f the
S o lu tio n S solution, add 10.0 m L o f 0.1 M sodium edetate and 30 m L of
Dissolve 2.00 g in dilute hydrochloric acid R and dilute to a mixture o f equal volumes o f ammonium acetate solution R
100 m L with the same ad d . and dilute acetic add R Boil for 3 min, then cool. A dd 25 m L
o f ethanol (96 per cent) R and 1 m L o f a freshly prepared
A p p e a ra n c e o f so lu tio n
0.25 g/L solution o f dithizone R in alcohol R. T itrate the
Solution S is clear (2.2.1) and colourless (2.2.2, Method II).
excess of sodium edetate with 0.1 M zinc sulfate until the
p H (2.2.3) colour changes to pink.
5.5 to 7.2 1 m L o f 0.1 M sodium edetate is equivalent to 12.20 m g o f
Shake 4.0 g with carbon dioxide-free water R and dilute to AIPO 4.
100 m L with the same solvent.
STORA GE
C h lo rid e s (2.4.4)
In an airtight container.
M axim um 1.3 per c e n t
PhEur
Dissolve 50.0 m g in 10 m L o f dilute nitric add R and dilute
to 200 m L with water R.
S o lu b le p h o sp h a te s
M axim um 1.0 per cent, calculated as PO 43-. ,* * * ★
★
Test solution Stir 5.0 g with 150 m L o f water R for 2 h. Filter Aluminium Phosphate Gel ★ ★
and wash the filter with 50 m L o f water R. Com bine the (Ph Eur monograph 2166) * * * * *
filtrate and the washings and dilute to 250.0 m L with
water R D ilute 10.0 m L o f this solution to 100.0 m L with A c tio n a n d u se
water R. A ntad d ; vacdne adjuvant
Reference solution (a) Dissolve 2.86 g o f potassium dihydrogen
PhEur-----------------------------------------------------------------------------------------
phosphate R in water R and dilute to 100 m L with th e same
solvent D E F IN IT IO N
Reference solution (b) Dilute 1 m L o f reference solution (a) to H ydrated A1P0 4 in g d form.
5 m L with water R. C o n te n t
Reference solution (c) Dilute 3 m L o f reference solution (a) to 19.0 per cent to 21.0 per cent o f AIPO 4.
5 m L with water R.
CHARACTERS
T reat each solution as follows. T o 5.0 m L add 4 m L o f dilute A p p e a ra n c e
sulfuric add R, 1 m L of ammonium mdybdate solution R, 5 m L Gel.
o f water R and 2 m L o f a solution containing 0.10 g of
S o lu b ility
4-methylaminophenol sulfate R, 0.5 g o f anhydrous sodium
Practically insoluble in water, in ethanol (96 per cent) and in
sulfite R and 20.0 g o f sodium metabisulfite R in 100 m L of
methylene chloride. It dissolves in dilute solutions o f mineral
water R Shake and allow to stand for 15 min. Dilute to
ad d s.
25.0 m L with water R and allow to stand for a further
 2016 Aluminium Powder 1-121

ID E N T IF IC A T IO N Dissolve 4.0 g in dilute hydrochloric add R and dilute to

A. Solution S (see Tests) gives reaction (b) of phosphates 20 m L with the same ad d . 12 m L of the solution complies
(2.3.1). with test A. Prepare the reference solution using lead standard
B. Solution S gives the reaction of aluminium (2.3.1). solution (2 ppm Pb) R.
C. It complies with the assay. A cid n e u tra lisin g c a p acity
Add 2.0 g to 30 m L of 0.1 M hydrochloric acid heated to
TESTS 37 °C and maintain at 37 °C while shaking. Determine the
S o lu tio n S p H after 15 min. The p H (2.2.3) of the mixture is 2.0 to 2.5.
Dissolve 2.00 g in dilute hydrochloric acid R and dilute to
R e sid u e o n ig n itio n
100 m L with the same add.
19.0 per cent to 23.0 per c e n t
p H (2.2.3)
H eat 0.500 g at 50 °C for 5 hours, then ignite at
6.0 to 8.0.
500 ± 50 °C until constant mass.
. P e ro x id e s
M ic ro b ia l c o n tam in a tio n
M aximum 150 ppm , expressed as hydrogen peroxide.
TA M C: acceptance criterion 103 CFU/g (2.6.12).
Test solution Dissolve with heating 1,0 g of the substance to
TY M C: acceptance criterion 102 CFU/g (2.6.12).
be examined in 5 m L o f dilute hydrochloric acid R, then add
5 m L of water R and 2 m L of dmanadxum pentoxide solution in Absence o f bile-tolerant gram-negative bacteria (2.6.13).
sulfuric acid R. Absence of Escherichia coli (2.6.13).
Reference solution D ilute 1.0 m L of dilute hydrogen peroxide A SSAY
solution R to 200.0 m L with water R. To 1 m L of this Dissolve with heating 0.300 g in 5 m L of dilute hydrochloric
solution add 9 m L o f water R and 2 m L o f divanadium acid R. A dd 45 m L of water R, 10.0 mT, of 0.1 M sodium
pentoxide solution in sulfuric acid R. edetate and 30 m L of a mixture of equal volumes of
T he test solution is no t more intensely coloured than the ammonium acetate solution R and dilute acetic add R. H eat to
reference solution. boiling and maintain boiling for 3 min. Cool, then add
C h lo rid es (2.4.4) 25 m L o f ethanol (96 per cent) R. Titrate with 0.1 M zinc
M aximum 500 ppm . sulfate, determining the end-point potentiometrically (2.2.20).
Dissolve 1.3 g in 5 mT. o f dilute nitric acid R and dilute to 1 m L of 0.1 M zinc sulfate is equivalent to 12.2 m g of A1P04.
200 mT, w ith water R. STORAGE
Soluble p h o sp h a te s In an airtight container.
Maximum 0.5 per cent, expressed as P 0 4. _________________________________________________________ PhEur
Test solution Centrifuge 10.0 g until a clear supernatant is
obtained. T o 2.00 m L of the supernatant add 20.0 m L o f a
10.3 g/L solution of hydrochloric acid R and dilute to
100.0 m L with water R. T o 10.0 m L of this solution add
10.0 mT, o f nitro-mofybdovanadic reagent R and dilute to Aluminium Powder
50.0 m L with water R. Allow to stand protected from light A1 26.98 7429-90-5
for 15 min.
Reference solution Add 10.0 m L of nitro-mofybdovanadic A c tio n a n d u se
reagent R to 10.0 m L o f a 143 mg/L solution of potassium Topical protective.
dihydrogen phosphate R and dilute to 50.0 m L with water R. P re p a r a tio n
Allow to stand protected from light for 15 min. C om pound Aluminium Paste
Measure the absorbances (2.2.25) of the 2 solutions at
D E F IN IT IO N
400 nm . T h e absorbance of the test solution is not greater
than that o f the reference solution. Aluminium Powder consists mainly o f metallic aluminium in
the form o f very small flakes, usually with an appreciable
S u lfates (2.4.13) proportion of aluminium oxide; it is lubricated with stearic
Maximum 0.2 per cent. a d d to prevent oxidation. It contains not less than 86.0% of
Dilute 25 m L of solution S to 100 m L with distilled water R. Al, calculated with reference to the substance freed from
Soluble a lu m in iu m lubricant and volatile matter.
Maximum 50 ppm. C H A R A C T E R IS T IC S
T o 16.0 g add 50 m L o f water R. H eat to boiling for 5 min. A silvery grey powder.
Cool and centrifuge. Separate the supernatant W ash the Practically insoluble in water and in ethanol (96%).
residue with 20 m L of water R and centrifuge. Separate the It dissolves in dilute adds and in aqueous solutions of alkali
supernatant and add to the first supernatant. To the hydroxides, with the evolution of hydrogen.
combined supernatants add 5 m L of hydrochloric acid R and
20 m L o f water R. Introduce all o f this solution into a ID E N T IF IC A T IO N
500 m L conical flask and carry out the complexometric A solution in 2m hydrochloric add yields the reaction
titration o f aluminium (2.5.11) using 0.01 M sodium edetate. characteristic of aluminium salts, Appendix VI.
A rsen ic (2.4.2, Method A) TESTS
M aximum 1 ppm , determined on 1.0 g. S u rfa c e -c o v e rin g po w er
H eavy m e ta ls (2.4.8) N o t less than 4000 cm2 per g w hen determined by the
M aximum 10 ppm. following method. Fill with water a shallow trough measuring
approximately 60 cm x 12 cm x 1.5 cm, fitted with a
movable partition so constructed that it is a sliding fit and
 1-122 Aluminium Sodium Silicate 2016

can be used to divide th e trough into two rectangular areas. A SSAY

Place the movable partition n ear one end and sprinkle 50 mg Transfer 0 .2 g, previously freed from lubricant by successive
o f the substance being examined on the surface o f the liquid washing with acetone and drying, to a three-necked 5 0 0 m L
confined in the smaller area. U sing a glass rod, spread the flask fitted with a 150 m L dropping funnel, an inlet tube
pow der evenly over the liquid surface until an unbroken film connected to a cylinder o f carbon dioxide and an outlet tube
covers die entire surface. M ove the partition so as to increase dipping into a water trap. Add 6 0 m L of water and disperse
the area confined and again spread the powder to cover the the substance being examined; replace the air by carbon
increased surface. Continue this process and d eterm in e the dioxide and add 100 m L o f a solution containing 56 g of
maximum unbroken surface area obtained. T h e surface- ammonium iron(m) sulfate and 7 .5 m L o f sulfuric add in water.
covering pow er is the area covered p er g of the pow der at the While m aintain in g an atm osphere o f carbon dioxide in the
breaking point of the film. flask, heat to boiling, boil for 5 minutes after the sample lias
Ir o n dissolved, cool rapidly to 2 0 ° and dilute to 2 5 0 m L with
Dissolve 10 m g in 20 m L of 2m hydrochloric acid and dilute to water. T o 5 0 mT. add 15 m L of orthophosphoric add and
100 m L with water. 10 m L o f the resulting solution complies titrate with 0 .0 2 m potassium permanganate KS. Each m L o f
with the limit test for iron, Appendix VII (1.0%). 0 .0 2 m potassium permanganate KS is equivalent to 0 .8 9 9 4 mg
o f Al.
Lead
Use two solutions prepared in the following manner.
For solution (1 ) boil 0 .4 0 g with 2 0 mT. of 2m hydrochloric
add and 10 m L of water until effervescence ceases, add
0 .5 m L o f nitric add, boil for 3 0 seconds and cool; add 2 g o f Aluminium Sodium Silicate *****
ammonium chloride and 2 g o f ammonium tfnocyanate, extract *+ +*
with three 10 m L quantities o f a m ix ture of equal volumes of (Ph Eur monograph 1676) *
amyl alcohol and ether, discard the extracts and add 2 g of PhEur___________________________________________________________
citric add. F o r solution (2) dissolve 2 g o f citric add in 10 m L
D E F IN IT IO N
of 2 m hydrochloric add and add 4 m L o f lead standard solution
(10 ppm Pb). Make solutions (1 ) and (2 ) alkaline with Silicic a d d aluminium sodium salt o f synthetic origin.
5 m ammonia and to each add 1 m L o f potassium cyanide solution C o n te n t
PbT. T he solutions should be not m ore than faintly — aluminium (Al; 26.98): 2.7 per cent to 7.9 per cent (dried
opalescent. If the colours o f the solutions differ, equalise by substance);
the addition o f about 0 .2 m L o f a highly diluted solution of — sodium (Na; 22.99): 3.7 per cent to 6.3 p er cent (dried
bu rn t sugar o r other non-reactive substance. Dilute each substance).
solution to 5 0 m L with water, add 0.1 m L of a 10% w/v CHARACTERS
solution o f sodium sulfide to each and mix thoroughly.
A p p e a ra n c e
T he colour produced in solution (1) is not more intense than
W hite o r alm ost white, fine, light, am orphous powder.
that produced in solution (2), when viewed against a white
background (100 ppm ). S o lu b ility
Practically insoluble in w ater and in organic solvents.
O th e r m e ta ls
Dissolve 2 g in 4 0 m L of 2 m hydrochloric add. D ilute 2 0 m L ID E N T IF IC A T IO N
o f the solution to 100 m L with water, make alkaline to litmus A. T ransfer 1.0 g to a 100 m L beaker and add 10 m L o f
paper by the addition o f 5 m ammonia, boil and filter. dilute hydrochloric acid R. Mix, cover with a w atch glass and
Evaporate the filtrate to dryness, add 0.05 m L o f sulfuric acid boil for 15 m in. Allow to cool to room tem perature, mix and
and ignite. T h e residue weighs not m ore than 2 mg. centrifuge the solution. 2 m L of the supernatant gives the
reaction o f aluminium {2.3.1).
L u b ric a n t
T o 2 g add 100 m L o f hot water, cover and add, drop wise, B. 2 m L o f the supernatant obtained in identification test A
sufficient o f a mixture o f equal volumes o f hydrochloric add gives reaction (a) of sodium {2.3.1).
and water to dissolve the metal alm ost completely. H eat to C. 0.2 g gives the reaction o f silicates {2.3.1).
complete dissolution, cool, filter through a hardened filter
TESTS
paper and wash the vessel and filter paper thoroughly with
p H {2.2.3)
water, dry b o th the vessel and paper at room tem perature.
9.5 to 11.5.
Extract the paper with three 100-mL quantities o f boiling,
freshly distilled acetone, using the original vessel to contain Disperse 5.0 g in 100 m L o f carbon dioxide-free water R.
the solvent and then wash th e paper with five 10-mL A rse n ic {2.4.2, Method A)
quantities o f freshly distilled acetone. Evaporate the combined M axim um 3 ppm.
filtrate and washings to dryness u sin g a rotary evaporator. Transfer 8.3 g to a 250 m L beaker containing 50 m L o f
T h e residue, after drying at 105° for 30 m inute s and allowing dilute hydrochloric acid R. M ix, cover with a watch glass and
to cool, weighs 10 to 60 mg. boil gently, with occasional stirring, for 15 min. Centrifuge,
W hen die basin containing the residue is floated in a beaker and decant the supernatant through a rapid-flow filter paper
of water suitably stirred and heated, the residue melts into a 250 m L volumetric flask. T o the residue in the beaker,
between 4 0 ° and 6 0 °. T h e residue is alm ost completely add 25 m L o f hot dilute hydrochloric acid R, stir, centrifuge,
soluble, w ith effervescence, in hot dilute sodium carbonate and decant the supernatant through the sam e filter into the
sdudon. volumetric flask. Repeat the extraction w ith 3 additional
V olatile m a t te r quantities, each o f 25 m L, o f ho t dilute hydrochloric add R,
filtering each supernatant through this filter into the
W hen heated to constant weight at 105°, loses no t m ore than
0.5% of its weight. U se 1 g. volumetric flask. Allow the combined filtrates to cool to room
tem perature and dilute to 250.0 m L with dilute hydrochloric
2016 Aluminium Stearate 1-123

add R. D ilute 10.0 m L of the solution to 25.0 m L with 1.0 m L of lanthanum chloride solution R and dilute to
water R. 50.0 m L with water R.
Lead Reference solutions Into 5 separate 50 m L volumetric flasks,
M axim um 5 ppm . introduce respectively 1.0 mL, 2.5 m l , 5.0 ml-, 7.5 m L and
10.0 m L o f aluminium standard solution (100 ppm Al) R, add
Atomic absorption spectrometry (2.2.23, Method I).
1 m L of lanthanum chloride solution R and 10 m L o f the blank
Test solution T ransfer 5.0 g to a 250 m L beaker containing solution, and dilute to 50.0 m L with water R.
50 m L o f dilute hydrochloric add R Mix, cover with a watch
glass an d boil for 15 min. Allow to cool to room Source Aluminium hollow-cathode lamp.
temperature. Centrifuge, and decant the supernatant through Wavelength 309.3 nm .
a rapid-flow filter paper into a 250 m L beaker. T o the Atomisation device Acetylene-nitrous oxide flame.
insoluble m atter add 25 m L of hot water R. Stir vigorously, Sodium
centrifuge, and decant the supernatant through the same Atomic emission spectrometry (2.2.22, Method i).
filter into the beaker. Repeat the extraction with 2 additional
Test solution T o 2.0 m L o f solution A, prepared in the assay
quantities, each o f 25 mL, o f hot water R, decanting each
o f aluminium, add 1 m L of a 12.5 g/L solution o f caesium
supernatant through the filter into the beaker. W ash the filter
chloride R and dilute to 20.0 m L with water R.
with 25 m L o f h o t water R, collecting die filtrate in the
beaker. C oncentrate the combined filtrates by gently boiling Reference solutions Into 5 separate 200 m L volumetric flasks,
to about 15 mT. Add about 0.05 m L o f heavy metal-free nitric each containing 10 m L o f a 12.5 g/L solution of caesium
add R, heat to boiling and allow to cool to room chloride R, introduce respectively 1.0 m l, 2.0 m l , 4.0 m l,
temperature. Filter the concentrated extracts through a rapid- 6.0 m L and 10.0 m L o f sodium standard solution
flow filter paper into a 25 m L volumetric flask. Transfer the (200 ppm Na) R and dilute to 200.0 m L with water R.
rem aining contents o f the beaker through the filter paper and Wavelettgth 589.0 nm .
into die volum etric flask with water R and dilute to 25.0 m L __________________________________________________________ PhEur
with the sam e solvent.
Reference solutions Into 4 separate 100 m L volumetric flasks,
introduce respectively 3.0 mL, 5.0 mL, 10.0 m L and
15.0 m L o f lead standard solution (10 ppm Pb) R, add
0.20 mT. o f heavy metal-free nitric add R and dilute to Aluminium Stearate *****
100.0 m L w ith water R.
(Ph Eur monograph 1663) *
Source L ead hollow-cathode lamp.
PhEur__________________________________________________________
Wavelength 217.0 nm.
D E F IN IT IO N
Atomisation device Air-acetylene flame.
Aluminium salts o f a mixture of solid organic a d d s consisting
L oss o n d ry in g (2.2.32) mainly o f variable proportions o f aluminium stearate and
M axim um 8.0 per cent, determ ined on 1.000 g by drying in aluminium palmitate. T h e organic adds are obtained from
an oven at 105 °C for 4 h. sources o f vegetable o r animal origin.
L oss o n ig n itio n C o n te n t
5.0 per cent to 11.0 per cent (dried substance), determ ined — aluminium (Al; A t 26.98): 3.0 per cent to 9.0 per cent
on 1.000 g by ignition in a platinum crucible to constant (dried substance);
mass a t 1000 ± 25 °C. — stearic add in the fatty add fraction: m inim um
M ic ro b ia l c o n ta m in a tio n 40.0 per cent;
T A M C : acceptance criterion 103 CFU /g (2.6.12). — sum of stearic acid and palmitic add in the fatty add fraction:
T Y M C : acceptance criterion 102 C FU /g (2.6.12). m inim um 90.0 per cent.

Absence o f Escherichia coli (2.6.13). CHARACTERS

ASSAY A p p e a ra n c e
Alum inium W hite or almost white, very fine, light powder.
Atomic absorption spectrometry (2.2.23, Method I). S olubility
A dd mixture A dd 50 m L o f nitric add R to 500 m L of Practically insoluble in water and in anhydrous ethanol.
water R. Dissolve in this solution 17 g of tartaric add R and ID E N T IF IC A T IO N
dilute to 1000 m L with water R First identification C, D
Blank solution Dissolve 1.4 g o f anhydrous lithium metaborate R Second identification A , B, D
in 60 m l. o f th e acid mixture and dilute to 200 m L with
A. Freezing point (2.2.18): minimum 53 °C, determined on
water R. the residue obtained in the preparation o f solution S
Test solution In a platinum crucible mix 0.200 g with 1.4 g o f (see Tests).
anhydrous lithium metaborate R. H eat slowly at first and ignite B. A d d value (2.5.1): 195 to 210.
at 1100 ± 25 °C for 15 m in. Cool, then place the crucible
Dissolve 0.200 g o f the residue obtained in the preparation of
in a 100 m L beaker containing 60 m L o f the a d d mixture.
solution S in 25 m L o f the prescribed mixture o f solvents.
Place a polytetrafluoroethylene-coated magnetic stirring bar
in the crucible and stir gently with a magnetic stirrer for C. Examine the chromatograms obtained in the assay of
16 h. T ran sfer the contents o f the crucible into a 200 m L stearic a d d and palmitic add.
volum etric flask. W ash the crucible, the magnetic stirring b ar Results T he 2 principal peaks in the chromatogram obtained
and the beaker with water R and dilute to 200.0 m l. with the with the test solution are similar in retention time to the
same solvent (solution A). T o 10.0 m L o f this solution, add 2 prindpal peaks in the chromatogram obtained with the
reference solution.
 1-124 Aluminium Stearate 2016

D. 1 m L o f solution S gives the reaction of aluminium Reference solution Prepare a solution containing
(2.3.1). T he addition of 0.5 m L o f dilute hydrochloric add R 0.00165 ng/mL o f cadmium nitrate tetrahydrate R in the blank
described in the general m ethod is omitted. solution (equivalent to 0.006 pg/m L o f Cd).
TESTS D ilute 1.0 m L o f the test solution to 10.0 m L with the blank
S o lu tio n S solution. Prepare mixtures of this solution, the reference
T o 5.0 g add 50 m L o f peroxide-free ether R, 20 m L o f dilute solution and the blank solution in the following proportions:
nitric add R and 20 m L of distilled water R and heat gently (1.0:0:1.0 V/V/V), (1.0:0.25:0.75 V/V/V),
under a reflux condenser until dissolution is complete. Allow (1.0:0.5:0.5 V/V/V), (1.0:0.75:0.25 V/V/V). T o each mixture
to cool. In a separating funnel, separate the aqueous layer add 50 )iL o f the modifier solution and mix. These solutions
and shake the ether layer with 2 quantities, each o f 4 mT, o f contain respectively 0 jxg, 0.0015 jig, 0.0030 ng and
distilled water R. Com bine the aqueous layers, wash with 0.0045 ng o f cadmium per millilitre from the reference
15 m L of peroxide-free ether R and dilute to 50.0 m L with solution. K eep the remaining test solution for use in the test
distilled water R (solution S). Evaporate the ether layer to for lead and nickel.
dryness and dry the residue at 100-105 °C. Keep the residue Source Cadm ium hollow-cathode lamp.
for identification tests A and B. Wavelength 228.8 nm .
A cid ity o r alk a lin ity Atomisation device Furnace.
T o 1.0 g add 20 m L o f carbon dioxide-free water R and boil Platform Pyrolytically coated with integrated tube.
for 1 min with continuous sh ak ing. Cool and filter.
Operating conditions U se the tem perature programme
T o 10 m L o f the filtrate add 0.05 m L of bromothymol blue
recom m ended for cadmium by the GFAA manufacturer.
solution R4. N o t m ore than 0.05 m L o f 0.1 M hydrochloric A n example o f tem perature parameters for GFAA analysis of
add or 0.1 M sodium hydroxide is required to change the
cadm ium is shown below.
colour of the indicator.
C h lo rid es (2.4.4)
Stage Final temperature Ramp time Hold time
M aximum 0.1 per cent.
CC) (s) (s)
Dilute 0.5 m L o f solution S to 15 m L with water R. Drying 110 10 20
S u lfates (2.4.13)
Ashing 600 10 30
M aximum 0.5 per cent.
Atomisation 1800 0 5
Dilute 0.3 m L o f solution S to 15 m L with distilled water R.
C a d m iu m
M axim um 3 ppm . L ead
Atomic absorption spectrometry (2.2.23, Method II). M axim um 10 ppm .
For the preparation of all aqueous solutions and for the rinsing of Atomic absorption spectrometry (2.2.23, MethodII).
glassware before use, employ water that has been passed through a For the preparation of aU aqueous solutions and for the rinsing of
strong-acid, strong-base, mixed-bed ion-exchange resin before use. glassware before use, employ water that has been passed through a
Select all reagents to have as low a content of cadmium, lead and strong-acid, strong-base, mixed-bed ion-exchange resin before use.
nickel as practicable and store all reagent solutions in containers of Select all reagents to have as low a content of cadmium, lead and
borosUicate glass. Clean glassware before use by soaking in warm nickel as practicable and store aU reagent solutions in containers of
8 M nitric acid for 30 min and by rinsing with deionised water. borosUicate glass. Clean glassware before use by soaking in warm
Blank solution D ilute 25 m L o f cadmium- and lead-free nitric 8 M nitric add for 30 min and by rinsing with deionised water.
acid R to 100.0 m L w ith water R. Blank sdution U se the solution described in the test for
Modifier solution Dissolve 20 g o f ammonium dihydrogen cadmium.
phosphate R and 1 g o f magnesium nitrate R in water R and Modifier solution U se the solution described in the test for
dilute to 100 m L with the same solvent. Alternatively, use an cadmium.
appropriate matrix modifier as recommended by the graphite Test solution U se the solution described in the test for
furnace atom ic absorption (GFAA) spectrometer cadmium.
manufacturer.
Reference sdution Prepare a solution of 0.100 |ig/mL of Pb by
Test solution Place 0.100 g of the substance to be examined in suitable dilutions o f lead standard solution (100 ppm Pb) R
a polytetrafluoroethylene digestion bom b and add 2.5 m L o f with the blank solution.
cadmium- and lead-free nitric acid R. Close and seal th e bom b
Prepare mixtures o f the test solution, the reference solution
according to the m anufacturer's operating instructions. When
and the blank solution in the following proportions:
using a digestion bomb, be thoroughly familiar with the safety and
(1.0:0:1.0 V/V/V), (1.0:0.5:0.5 V/V/V), (1.0:1.0:0 V/V/V).
operating instructions. Carefully follow the bomb manufacturer's
T o each mixture add 50 pL o f the modifier solution and mix.
instructions regarding care and maintenance of these digestion
These solutions contain respectively 0 |ig5 0.025 |ig and
bombs. Do not use metal-jacketed bombs or liners that have been
0.05 jig o f lead p er millilitre from the reference solution.
used with hydrochloric acid due to contamination from corrosion of
the metal jacket by hydrochloric add. H eat the bom b in an oven Source L ead hollow-cathode lamp.
at 170 °C for 3 h. Cool the bom b slowly in air to room Wavelength 283.3 nm .
tem perature accord in g to the bom b m anufacturer's Atomisation device Furnace.
instructions. Place the bom b in a fume cupboard and open Platform Pyrolytically coated with integrated tube.
carefully as corrosive gases may be expelled. Dissolve die
Operating conditions Use the tem perature programme
residue in water R and dilute to 10.0 m L with the same
recom m ended for lead by the GFAA manufacturer.
solvent.
A n example of tem perature parameters for GFAA analysis of
lead is shown below.
2016 Aluminium Stearate 1-125

Stage Final temperature Ramp time Hold time solution for 30 m in under reflux on a w ater-bath, swirling
_________________CQ___________ &__________ isi__ frequently. Allow to cool. Add 100 m L o f water R and adjust
Drying 110 10 20 to about p H 1 by adding approximately 12 m L of dilute
Ashing 450 10 30 sodium hydroxide solution R. Add 20.0 m L o f 0.1 M sodium
edetate and adjust to between pH 5 and p H 6 by the addition
Atomisation 2000 0 5
of sodium acetate R. Add 70 mg of xylenol orange triturate R
and titrate immediately and quickly with 0.1 M zinc sulfate
until the colour changes from yellow to pinkish-violet.
Nickel
M axim um 5 ppm . 1 m L of 0.1 M sodium edetate is equivalent to 2.698 mg of
Atomic absorption spectrometry (2.2.23, Method II). Al.

For the preparation of all aqueous solutions and for the rinsing of S te a ric a c id a n d p a lm itic acid
glassware before me, employ water that has been passed through a Gas chromatography (2.2.28): use the normalisation
strong-acid, strong-base, mixed-bed ion-exchange resin before use. procedure.
Select al1 reagents to have as low a content of cadmium, lead and Test solution In a conical flask fitted with a reflux condenser,
nickel as practicable and store al1 reagent solutions in containers of dissolve 0.100 g o f the substance to be examined in 5 m L o f
borosUicate glass. Clean glassware before use by soaking in warm boron trifliioride-methanol solution R. Boil under a reflux
8 M nitric acid for 30 mm and by rinsing with deionised water. condenser for 10 min. Add 4 m L of heptane R through the
Blank solution U se the solution described in the test for condenser and boil again under a reflux condenser for
cadmium. 10 min. Allow to cool. A dd 20 m L of saturated sodium
chloride solution R. Shake and allow the layers to separate.
Modifier solution Dissolve 20 g of ammonium dihydrogen
D ry the organic layer over 0.1 g of anhydrous sodium sulfate R
phosphate R in water R and dilute to 100 m L with the same
previously washed with heptane R. D ilute 1.0 m L of the
solvent. Alternatively, use an appropriate matrix modifier as
solution to 10.0 m L with heptane R.
recom m ended by the GFAA spectrometer manufacturer.
Reference solution Prepare the reference solution in the same
Test solution U se the solution described in the test for
m anner as the test solution using 50.0 m g of palmitic
cadmium.
acid CRS and 50.0 mg of stearic acid CRS instead o f the
Reference solution Prepare a solution of 0.050 ng/mL of N i by substance to be examined.
suitable dilutions o f a 0.2477 |ig/mL solution of nickel nitrate
Column:
hexahydrate R with the blank solution.
— material: fused silica;
Prepare mixtures o f the test solution, the reference solution — size. I = 30 m , 0 = 0.32 mm;
and the blank solution in the following proportions: — stationary phase: macrogol 20 000 R (film thickness
(1.0:0:1.0 V/V/V), (1.0:0.5:0.5 VIVIV), (1.0:1.0:0 VIVIV). 0.5 tun).
T o each mixture add 50 (iL of the modifier solution and mix.
Carrier gas helium for chromatography R.
T hese solutions contain respectively 0 ng, 0.0125 fog and
0.025 ng o f nickel per millilitre from the reference solution. Flow rate 2.4 ml 7min.
Source Nickel hollow-cathode lamp. Temperature:
Wavelength 232.0 nm.
Time Temperature.
Atomisation device Furnace.
(min) rc)
Platform Pyrolytically coated with integrated tube. Column 0 -2 70
Operating conditions Use the temperature programme
2 -3 6 70 240
recom m ended for nickel by the GFAA manufacturer.
A n example o f tem perature parameters for GFAA analysis of 36-41 240
nickel is shown below. Injection port 220
Detector 260
Stage Final temperature Ramp time Hold time
CC) (») (s)
Drying 110 10 20 Detection Flam e ionisation.
Ashing 1000 20 30 Injection 1 (iL.
Atomisation 2300 0 5 Relative retention W ith reference to methyl stearate: methyl
palmitate = about 0.9.
System suitability: reference solution:
L oss o n d ry in g ( 2.2.32) — resolution: minimum 5.0 between the peaks due to methyl
M axim um 6.0 per cent, determined on 1.000 g by drying in palmitate and methyl stearate;
an oven at 105 °C. — repeatability: maximum relative standard deviation of
M ic ro b ia l c o n ta m in a tio n 3.0 per cent for the areas of the peaks due to methyl
T A M C : acceptance criterion 103 C FU/g (2.6.12). palmitate and methyl stearate after 6 injections; maximum
relative standard deviation of 1.0 per cent for the ratio of
T Y M C : acceptance criterion 102 CFU/g (2.6.12).
the areas of the peaks due to methyl palm itate to the
Absence of Escherichia colt (2.6.13). areas of the peaks due to methyl stearate after
Absence of Salmonella (2.6.13). 6 injections.
A SSA Y __________________________________________________________ PhEur
A lu m in iu m
T o 0.250 g in a 250 m L conical flask add 20 m L of
methanol R and, slowly, 2 m L o f sulfuric acid R. H eat the
 1-126 Aluminium Sulfate 2016

★ ★ STO RA G E
Aluminium Sulfate ★ ★ In an airtight container.
Aluminium Sulphate *****
PhEur
(Ph Eur monograph 0165)
A12(S04)3îxH20 342.1

(anhydrous substance)
★ ★
Alverine Citrate ★ ★
P re p a r a tio n *****
Aluminium Acetate E ar D rops
(Ph Eur monograph 2156)
Ph E n _______________________________________ CO j H
ch3
D E F IN IT IO N CO2H
C o n te n t OH
51.0 per cent to 59.0 per cent of A12(S 04 )3. COjH
It contains a variable quantity of water of crystallisation.
CHARACTERS C 26H 35NO 7 473.6 5560-59-8
A p p e a ra n c e
A c tio n a n d u se
Colourless, lustrous crystals or crystalline masses.
Sm ooth muscle relaxant; antispasmodic.
S o lu b ility
P re p a r a tio n
Soluble in cold water, freely soluble in hot water, practically
Alverine Capsules
insoluble in ethanol (96 per cent).
ID E N T IF IC A T IO N PhEur_____________________________________

A. Solution S (see Tests) gives reaction (a) o f sulfates (2.3.1). D E F IN IT IO N

B. Solution S gives the reaction of alu m in iu m (2.3.1). N-Ethyl-3-phenyl-N-(3-phenylpropyl)propan-1-amine
TESTS dihydrogen 2-hydroxypropane-l,2,3-tricarboxylate.
S o lu tio n S C o n te n t
Dissolve 2.5 g in water R and dilute to 50 m L with the same 99.0 per cent to 101.0 per cent (dried substance).
solvent. CHARACTERS
A p p e a ra n c e o f so lu tio n A p p e a ra n c e
Solution S is not m ore opalescent than reference W hite or almost white, crystalline powder.
suspension IU (2.2.1) and is colourless (2.2.2, Method II). S o lu b ility
p H (2.2.3) Slightly soluble in water and in methylene chloride, sparingly
2.5 to 4.0. soluble in ethanol (96 per cent).
Dissolve 0.5 g in carbon dioxide-free water R and dilute to mp
25 m L with the same solvent. A bout 104 °C.
A lk ali a n d a lk a lin e -e a rth m e ta ls ID E N T IF IC A T IO N
M axim um 0.4 per c e n t Infrared absorption spectrophotometry (2.2.24).
T o 20 m L of solution S add 100 m L of water R, heat and Comparison alverine citrate CRS.
add 0.1 m L of methyl red solution R. A dd dilute ammonia R l
until the colour o f the indicator changes to yellow. Dilute to TESTS
150 m L with water R, heat to boiling and filter. Evaporate p H (2.2.3)
75 m L of the filtrate to dryness on a water-bath and ignite. 3.5 to 4.5.
T he residue weighs a maximum o f 2 mg. Dissolve 0.250 g in carbon dioxide-free water R and dilute to
A m m onium (2.4.1) 50.0 m L with the same solvent.
M aximum 500 ppm. R e la te d su b sta n c e s
D ilute 0.4 m L of solution S to 14 m L with water R. G as chromatography (2.2.28): use the normalisation
procedure. Use freshly prepared solutions.
Ir o n (2.4.9)
M axim um 100 ppm. Test solution Dissolve 0.250 g of die substance to be
examined in water R and dilute to 20 m L w ith the same
D ilute 2 m L o f solution S to 10 m L with water R.
solvent A dd 2 m L of concentrated ammonia R and shake with
U se 0.3 m L o f thiogfycoUic add R in this test.
3 quantities, each of 15 m L, o f methylene chloride R. T o the
H eav y m e ta ls (2.4.8) com bined lower layers add anhydrous sodium sulfate R, shake,
M axim um 50 ppm . filter, and evaporate the filtrate at a tem perature not
D ilute 8 m L o f solution S to 20 m L with water R. 12 m L o f exceeding 30 °C, using a rotary evaporator. T ake up the
the solution complies with test A Prepare the reference residue with methylene chloride R and dilute to 10.0 m L with
solution using lead standard solution (1 ppm Pb) R. the same solvent.
A SSA Y Reference solution (a) Dissolve 5 m g o f alverine
Dissolve 0.500 g in 20 m L o f water R. Carry out the impurity D CRS (impurity D citrate) in 5 m L o f water R, add
complexometric titration o f aluminium (2.5.11). 1 m L o f concentrated ammonia R and shake with 3 quantities,
each of 5 m L, o f methylene chloride R. T o the combined lower
1 m L o f 0.1 M sodium edetate is equivalent to 17.11 mg
layers add anhydrous sodium sulfate R, shake, filter, and
ofAl2(S04)3. evaporate the filtrate at a tem perature no t exceeding 30 °C,
2016 Amantadine Hydrochloride 1-127

using a rotary evaporator. T ake up the residue with methylene ASSAY

chloride R, add 0.2 m L of the test solution and dilute to Dissolve 0.375 g in 50 m L of anhydrous acetic acid R. T itrate
2 m L with methylene chloride R. w ith 0.1 M perchloric acid, determ in in g the end-point
Reference solution (b) Dilute 1.0 m L o f the test solution to potentiometrically (2.2.20).
100.0 m l, with methylene chloride R. Dilute 1.0 m L of this 1 m L o f 0.1 M perchloric add is equivalent to 47.36 mg
solution to 20.0 m L with methylene chloride R. o f C 26H 35N O 7.
Reference solution (c) Dissolve the contents o f a vial of alverine STORAGE
for peak identification CRS (containing impurities C and E) in Protected from light.
1 m l, of methylene chloride R.
IMPURITIES
Column:
— material: fused silica;
Specified impurities: A, B, C, D , E.
— sizer. I = 25 m , 0 = 0.32 mm;
— stationary phase: poly(dimethyl) (diphenyl) sUoxane R (film
thickness 0.45 (im).
Carrier gas helium for chromatography R.
Flow rate 2.2 mL/min. A. R = Cl: 1-chloro-3-phenylpropane,
Split ratio 1:11. B. R = O H : 3-phenylpropan-l-ol,
Temperature: C. R = N H -C 2H 5: N-ethyl-3-phenylpropan-1 -amine.

Time Temperature
(min) CC)
Column 0 -7 120

7 -1 3 120 -» 240 D . iV-(3-cyclohexylpropyl)-N-ethyl-3-phenylpropan-l-amine,

13-21 240

21-24 240 -» 290

24-39 290

Injection port 290

Detector 290

E. 3-phenyl-AT^ST-bis(3-phenylpropyl)propan-l-amine.
Detection Flame ionisation.
PtiEur
Irtjection 1 pL.
Identification of impurities Use the chromatogram supplied
with alverine for peak identification CRS and the
chromatogram obtained with reference solution (c) to identify
the peaks due to impurities C and E. Amantadine Hydrochloride ***\
Relative retention W ith reference to alverine (retention
time = about 16 m in): impurity A = about 0.28; (Ph Em monograph 0463) *
impurity B = about 0.29; impurity C = about 0.46;
impurity D = about 0.97; impurity E = about 1.7.
System suitability: reference solution (a):
— resolution: m inim um 3.0 between the peaks due to
impurity D and alverine.
C 10H 18CIN 187.7 665-66-7
Limits:
— impurities A , B: for each impurity, maximum 0.1 per cent; Action and use
— impurity C: maxim um 0.2 per cent; Viral replication inhibitor (influenza A); dopamine receptor
— impurities D, E: for each impurity, maximum 0.3 per cent; agonist; treatm ent of influenza and Parkinson’s disease.
— unspecified impurities: for each impurity, maximum
0.10 per cent; Preparations
Amantadine Capsules
— total: maximum 1.0 per cent;
— disregard limit, the area o f the principal peak in the A m antadine Oral Solution
chrom atogram obtained with reference solution (b)
PhEir _______________________ :___________________________________
(0.05 per cent).
H eav y m e ta ls (2.4.8)
DEFINITION
Maximum 20 ppm . Tricyclo[3.3.1. l 3,7]decan-l-am ine hydrochloride.

0.5 g complies with test G. Prepare the reference solution Content

using 1 m L of lead standard solution (10 ppm Pb) R. 98.5 per cent to 101.0 per cent (anhydrous substance).

L oss o n d ry in g (2.2.32) CHARACTERS

Maximum 0.5 per cent, determined on 1.000 g by drying in Appearance
an oven at 80 °C for 2 h. W hite or almost white, crystalline powder.
S u lfa te d a sh (2.4.14) .Solubility
M axim um 0.1 per cent, determined on 1.0 g. Freely soluble in water and in ethanol (96 per cent).
 1-128 Amantadine Hydrochloride 2016

It sublimes on hearing. Temperature:

IDENTIFICATION
First identification A, D. Time Temperature
(min) CC)
Second identification B, C, D.
Column 0 -5 70
A. Infrared absorption spectrophotometry (2.2.24).
5 -2 3 70 ->250
Comparison amantadine hydrochloride CRS.
2 3 -4 0 250
B. T o 0.1 g add 1 m L o f pyridine R, mbc and add 0.1 m l. of
acetic anhydride R. H e a t to boiling for about 10 s. Pour the Injection port 220
hot solution into 10 m L o f dilute hydrochloric acid R, cool to Detector 300
5 °C and filter. T he precipitate, washed with water R and
dried m vacuo at 60 °C for 1 h, melts (2.2.14) at 147 °C to
151 °C. Detection Flam e ionisation.
C. Dissolve 0.2 g in 1 m L o f 0.1 M hydrochloric acid. Injection 1 [iL.
Add 1 m L o f a 500 g/L solution of sodium nitrite R A white Relative retention W ith reference to am antadine (retention
precipitate is formed. tim e = about 14 min): internal standard = about 0.8.
D . 1 m L of solution S (see Tests) gives reaction (a) of System suitability: reference solution:
chlorides (2.3.1). — resolution: minimum 5.0 between the peaks due to the
TESTS internal standard and amantadine.
S o lu tio n S Limits:
Dissolve 2.5 g in carbon dioxide-free water R and dilute to — unspecified impurities: calculate the ratio (R{) of the area of
25 m L with the same solvent. the peak due to amantadine to the area of the peak due
to the internal standard from the chrom atogram obtained
Appearance o f solution
with the reference solution; from the chromatogram
Solution S is clear (2.2.1) and not more intensely coloured
obtained with the test solution, calculate the ratio of the
than reference solution Y7 (2.2.2, Method II).
area o f any peak, apart from the principal peak and the
Acidity or alkalinity peak due to the internal standard, to the area of the peak
D ilute 2 m L of solution S to 10 m L with carbon dioxide-free due to the internal standard: this ratio is n o t greater than
water R. Add 0.1 m L o f methyl red solution R and 0.2 m l. of Ri (0.10 per cent);
0.01 M sodium hydroxide. T h e solution is yellow. Add 0.4 m L — total: calculate the ratio (R^) of 3 times the area of the
of 0.01 M hydrochloric add. T h e solution is red. peak due to amantadine to the area of the peak due to
Related substances the internal standard from the chrom atogram obtained
Gas chromatography (2.2.28). with the reference solution; from the chromatogram
Internal standard solution Dissolve 0.500 g o f adamantane R in obtained with the test solution, calculate the ratio of the
methylene chloride R and dilute to 10.0 m L with the same sum of the areas of any peaks, apart from the principal
solvent. peak and the peak due to the internal standard, to the
Test solution Weigh 0.5 g of the substance to be examined area o f the peak due to the internal standard: this ratio is
into a centrifuge tube. Add 9 m L of methylene chloride R and n o t greater than R2 (0.3 per cent);
— disregard limit calculate the ratio (R3) o f 0.5 times the
10 m L of a 210 g/L solution o f sodium hydroxide R. Shake for
area of the peak due to amantadine to the area of the
10 min. Discard the upper layer. Dry the lower layer over
peak due to the internal standard from the chromatogram
anhydrous sodium sulfate R. Filter and collect the filtrate in a
volu m etric flask. Add 0.1 m L o f the internal standard obtained with the reference solution; from the
chromatogram obtained with the test solution, calculate
solution and dilute to 10.0 m L with methylene chloride R.
the ratio o f the area of any peak, apart from the principal
Reference solution Weigh 5 m g o f amantadine peak and the peak due to the internal standard, to the
hydrochloride CRS into a centrifuge tube. Add 9 m L of area of the peak due to the internal standard: disregard
methylene chloride R and 10 m L of a 210 g/L solution o f any peak with a ratio less than R3 (0.05 p er cent).
sodium hydroxide R. Shake for 10 m in. Discard the upper
layer. Dry the lower layer over anhydrous sodium sulfate R. Heavy metals (2.4.8)
Filter and collect the filtrate in a volumetric flask. M axim um 20 ppm.
A dd 1.0 m L o f the internal standard solution and dilute to 12 m L o f solution S complies with test A Prepare the
100.0 m L with methylene chloride R. reference solution using lead standard solution (2 ppm Pb) R.
Column: Water (2.5.12)
— material: fused silica; M aximum 0.5 per cent, determ ined on 2.00 g.
— size. I = 30 m , 0 = 0.53 mm; Sulfated ash (2.4.14)
— stationary phase, base-deactivated M aximum 0.1 per cent, determ ined on 1.0 g.
pdy (dimethyl) (diphenyl)süoxane R (film thickness 1 Jim).
A SSA Y
Carrier gas helium for chromatography R.
Dissolve 0.150 g in a mixture o f 5.0 m L of 0.01 M
Flow rate 4 mL/min.
hydrochloric acid and 50 m L o f ethanol (96 per cent) R. Carry
Split ratio 1:50. out a potentiometric titration (2.2.20), vising 0.1 M sodium
hydroxide. Read the volume added betw een the 2 points of
inflexion.
1 m l. of 0.1 M sodium hydroxide is equivalent to 18.77 mg of
C 10H 18C1N.
2016 Ambroxol Hydrochloride 1-129

IMPURITIES B. Infrared absorption spectrophotom etry (2.2.24).

Other detectable impurities (the following substances would, if Comparison ambroxol hydrochloride CRS.
present at a sufficient level, be detected by one or other of C. Thin-layer chromatography (2.2.27).
the tests in the monograph. They are limited by the general
Test solution Dissolve 50 m g of the substance to be examined
acceptance criterion for other/unspecified impurities and/or
in methanol R and dilute to 5 m L with the same solvent.
by the general monograph Substances for pharmaceutical use
(2034). It is therefore n o t necessary to identify these Reference solution Dissolve 50 mg o f ambroxol
impurities for demonstration of compliance. See also 5.10. hydrochloride CRS in methanol R and dilute to 5 m L with the
Control of impurities in substances for pharmaceutical use): A , B. same solvent.
Hate TLC silica gel F 254 plate R.
a Mobile phase concentrated ammonia R, propanol R, ethyl
acetate R, hexane R (1:10:20:70 V/V/V/V).
Application 10 jjL.
A. l-chlorotricydo[3.3.1.13,7]decane, Development Over 2/3 of the plate.
Drying In air.
H Detection Examine in ultraviolet light at 254 nm .
N ^ /C H 3
T Results T h e prindpal spot in the chrom atogram obtained with
o the test solution is similar in position and size to the principal
spot in the chromatogram obtained with the reference
B. N-(tricyclo[3.3.1 . l 3,7]dec-l-yl)acetamide. solution.
PhEur D . Dissolve 25 m g in 2.5 m L of water R, mix with 1.0 m L of
dilute ammonia R1 and allow to stand for 5 m in. Filter and
addify the filtrate with dilute nitric acid R. The filtrate gives
reaction (a) of chlorides (2.3.1).
*****
Ambroxol Hydrochloride ★ ★ TESTS
*+ Solution S
(Ph. Eur. monograph 1489) *★ +*
*
Dissolve 0.75 g in methanol R and dilute to 15 m L with the
same solvent.
Appearance of solution
Br Solution S is clear (2.2.1) and n o t m ore intensely coloured
HCI
than reference solution Y 6 (2.2.2, MethodII).
NH2 pH (2.2.3)
Br 4.5 to 6.0.
Dissolve 0.2 g in carbon dioxide-free water R and dilute to
Ci3H19Br2ClN20 414.6 23828-92-4
20 m L with the same solvent.
Action and use Related substances
M ucolytic expectorant. Liquid chromatography (2.2.29). Prepare the solutions
immediately before use.
PhEir.
Test solution Dissolve 50 mg of the substance to be examined
DEFINITION in water R and dilute to 50.0 m L with the same solvent.
trans-4- [(2-Amino-3,5-dibromobenzyl) amino] cydohexanol Reference solution (a) D ilute 1.0 m L o f the test solution to
hydrochloride. 100.0 m L with water R. Dilute 1.0 m L o f this solution to
Content 10.0 m L with the mobile phase.
99.0 per cent to 101.0 per cent (dried substance). Reference solution (b) In order to prepare impurity B in situ,
CHARACTERS dissolve 5 mg o f the substance to be examined in 0.2 m L of
Appearance methanol R, add 0.04 m l. of a mixture of 1 volum e of
W hite or yellowish, crystalline powder.
formaldehyde solution R and 99 volumes of water R. H eat at
60 °C for 5 min. Evaporate to dryness under a current of
Solubility nitrogen. Dissolve the residue in 5 m l. of water R and dilute
Sparingly soluble in water, soluble in methanol, practically to 20.0 m l, with the mobile phase.
insoluble in methylene chloride.
Column:
IDENTIFICATION — sizer. I = 0.25 m , 0 = 4.0 mm ;
First identification By D. — stationary phase: octadecylstfyl silica gel for chromatography R
Second identification A j C, D. (5 pm).
A. U ltraviolet and visible absorption spectrophotometry Mobile phase A mixture o f equal volumes o f acetonitrik R and
(2.2.25). a solution prepared as follows: dissolve 1.32 g o f ammonium
Test solution Dissolve 20.0 m g in 0.05 M sulfuric acid and phosphate R in 900 m L o f water R, adjust to p H 7.0 with
dilute to 100.0 m L with the same ad d . Dilute 2.0 m L o f the
phosphoric acid R and dilute to 1000 m L with water R.
solution to 10.0 m L with 0.05 M sulfuric acid. Flow rate 1 mL/min.
Spectral range 200-350 nm. Detection Spectrophotom eter at 248 nm.
Absorption maxima At 245 nm and 310 nm . Injection 20 jiL.
Absorbance ratio ^ 245/^310 = 3.2 to 3.4. Run time 3 times the retention time o f ambroxol.
 1-130 Amfetamine Sulfate 2016

.OH
Identification of impurities U se the chrom atogram obtained
with reference solution (b) to identify the peak due to
impurity B.
»-.X )
Relative retention W ith reference to ambroxol (retention C. trans-4- [[(£)-2-am ino-3,5-dibromobenzyliden] amino]
time = about 9 min): impurity B = about 0.6.
cyclohexanol.
System suitability: reference solution (b):
— resolution: minimum 4.0 between the peaks due to .OH
impurity B and ambroxol.
Limits: . - „ O
H
— unspecified impurities: for each impurity, not more than the
area of the principal peak in the chromatogram obtained
D . os-4- [(2-amino-3,5-dibromobenzyl)amino] cyclohexanol,
with reference solution (a) (0.10 per cent),
— total: not m ore than 3 times the area of the principal peak E. A r-C H = 0 : 2-amino-3,5-dibromobenzaldehyde.
in the chromatogram obtained with reference solution (a) PhEtr
(0.3 per cent),
— disregard limit. 0.5 times the area o f the principal peak in
the chromatogram obtained with reference solution (a)
(0.05 per cent). ★*★
★ ★
H eavy m e ta ls (2.4.8) Amfetamine Sulfate ★ ★
M aximum 20 ppm. Amfetamine Sulphate * * * * *

1.0 g complies with test C. Prepare the reference solution (Ph Eur monograph 0368)
using 2 m L o f lead standard solution (10 ppm Pb) R.
L oss o n d ry in g (2.2.32)
M aximum 0.5 per cent, determined on 1.000 g by drying in H2SO4 and enantiomer
an oven at 105 °C.
S u lfa te d a s h (2.4.14)
M aximum 0.1 per cent, determined on 1.0 g. C 18H 28N 2O 4S 368.5 60-13-9
ASSAY
A c tio n a n d u se
Dissolve 0.300 g in 70 m L of ethanol (96 per cent) R and add
Releases dopamine; central nervous system stimulant.
5 m L of 0.01 M hydrochloric acid. C a n y out a potentiometric
titration (2.2.20), using 0.1 M sodium hydroxide. Read PhEur__________________________________________________________
the volume added between the 2 points o f inflexion.
D E F IN IT IO N
1 m L of 0.1 M sodium hydroxide is equivalent to 41.46 mg of
Bis [(2.&S)-1-phenylpropan-2-amine] sulfate.
C 13Hj9Br2C lN 20 .
C o n te n t
STORA GE
99.0 per cent to 100.5 per cent (dried substance).
Protected from light.
CHARACTERS
IM P U R IT IE S
A p p e a ra n c e
Other detectable impurities (the following substances would, if W hite or alm ost white powder.
present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general S o lu b ility
acceptance criterion for other/unspecified impurities and/or Freely soluble in water, slightly soluble in ethanol
by the general monograph Substances for pharmaceutical use (96 per cent).
(2034). It is therefore no t necessary to identify these ID E N T IF IC A T IO N
impurities for demonstration o f compliance. See also 5.10. First identification A , B, E.
Control of impurities in substances for pharmaceutical use): A, B, Second identification A, C, D, E.
C, D, E.
A. Optical rotation (2.2.7): —0.04° to + 0.04° (measured in a
2 dm tube), determined on solution S (see Tests).
B. Infrared absorption spectrophotometry (2.2.24).
Ar- =
Preparation Mulls in liquid paraffin R.
Br Comparison Ph. Eur. reference spectrum of amfetamine sulfate.
C. T o 50 m L of solution S add 5 m L o f strong sodium
A. A r-C H 2O H : (2-amino-3,5-dibromophenyI)methanol, hydroxide solution R and 0.5 m l. o f benzoyl chloride R and
shake. Continue to add benzoyl chloride R in portions of
0.5 m L until no further precipitate is form ed Filter, wash
the precipitate with water R, reciystallise twice from a
mixture o f equal volumes o f ethanol (96 per cent) R and

w
water R, then dry at 100-105 °C. T h e crystals m elt (2.2.14)
at 131 °C to 135 °C.
Br D . T o about 2 m g add 1 m L o f sulfuric acid-formaldehyde
reagent R. A n orange colour develops and quickly becomes
B. trans-4-(6,8-dibrom o-1,4-dihydroquinazolin-3 (2H)-yl) dark-brown.
cyclohexanol, E. Solution S gives reaction (a) o f sulfates (2.3.1).
2016 Amidotrizoic Acid 1-131

TESTS B. Thin-layer chromatography (2.2.27).

S o lu tio n S Test solution Dissolve 25 m g of the substance to be examined
Dissolve 2.0 g in carbon dioxide-free water R and dilute to in a 3 p er cent V/V solution of ammonia R in methanol R and
100 m L with the same solvent. dilute to 5 m L with the same solution.
A p p e a ra n c e o f so lu tio n Reference solution Dissolve 25 m g of amidotrizoic add
Solution S is clear (2.2.1) and colourless (2 .2 .2 , Method II). dihydrate CRS in a 3 per cent V/V solution of ammonia R in
A cid ity o r alk a lin ity methanol R and dilute to 5 m L w ith the same solution.
T o 25 mL of solution S add 0.1 m L of methyl red solution R. Plate TLC silica gd GF2 5 4 plate R
N ot more than 0.1 mL o f 0.01 M hydrochloric add or 0.01 M Mobile phase anhydrous forme add R, methyl ethyl ketone R,
sodium hydroxide is required to change the colour o f the toluene R, (20:25:60 VIV/V).
indicator. Application 2 |iL.
L oss o n d ry in g (2.2.32) Development Over 2/3 o f the plate.
M aximum 1.0 per cent, determined on 1.00 g by drying in
Drying In air until the solvents have evaporated.
an oven at 105 °C.
Detection In ultraviolet light at 254 nm .
S ulfa te d a s h (2.4.14)
M aximum 0.1 per cent, determined on 1.0 g.
Results T h e principal spot in the chromatogram obtained with
the test solution is similar in position and size to the principal
ASSAY spot in the chrom atogram obtained with the reference
Dissolve 0.300 g in 30 m L o f anhydrous acetic add R. T itrate solution.
with 0.1 M perchloric acid, determining the end-point C. H eat 50 m g gently in a small porcelain dish over a naked
potentiometrically (2 . 2 . 2 0 ). flame. Violet vapour is evolved.
1 m L of 0.1 M perchloric add is equivalent to 36.85 mg
TESTS
o f C 18H 2&N2O 4S .
A p p e a ra n c e o f so lu tio n
STORAGE T h e solution is clear (2.2.1) and colourless (2.2.2,
Protected from light. Method II).
__________________________________________________________ PhEur Dissolve 1.0 g in dilute sodium hydroxide solution R and dilute
to 20 m L with the same solution.
R e la te d su b sta n c e s
Liquid chromatography (2.2.29).
★* ★
★ ★ Solvent mixture Dissolve 0.250 g o f sodium hydroxide R and
Amidotrizoic Acid Dihydrate ★ ★
0.860 g o f sodium dihydrogen phosphate R in 50 m L of water R
(Ph Eur monograph 0873) *****
and dilute to 1000 m L with the same solvent.
Test solution Dissolve 40.0 mg of the substance to be
c o 2h
examined in 10.0 m L of the solvent mixture with the aid of
ultrasound.
2 HzO
Reference sdution (a) D ilute 1.0 m L o f the test solution to
100.0 m L with the solvent mixture. Dilute 1.0 m L of this
solution to 10.0 m L with the solvent mixture.
Reference solution (b) Dilute 1.0 m L o f reference solution (a)
C 11H 9l 3N 204J2H 20 650 50978-11-5 to 10.0 m L with the solvent mixture.

A ctio n a n d u se Reference solution (c) Dissolve the contents of a vial of

Iodinated contrast medium. amidotrizoic add for system suitability CRS (impurities A, B, C
and D) in 1.0 m L of the solvent mixture!
Preparation
Column:
Meglumine Amidotrizoate Injection
— size: I = 0.25 m , 0 = 4.6 mm;
PhEur________________________________ — stationary phase: end-capped octadecylstlyl silica gel for
chromatography R (5 jim).
D E F IN IT IO N
Mobile phase Dissolve 3.4 g of tetrabutylammonium hydrogen
3,5-Bis(acetylamino)-2,4,6-triiodobenzoic acid dihydrate.
sulfate R in a mixture o f 230 m L o f acetonitrile R and 770 m L
C o n te n t o f water R.
98.5 per cent to 101.0 per cent (dried substance).
Flow rate 1.0 mL/min.
CHARACTERS Detection Spectrophotom eter at 236 nm.
A p p e a ra n c e Injection 20 (iL.
White or almost white, crystalline powder.
Run time 4 times the retention tim e o f amidotrizoic add.
S o lu b ility
Identification of impurities Use the chromatogram supplied
Very slightly soluble in water and in ethanol (96 p er cent).
with amidotrizoic acid for system suitability CRS and the
It dissolves in dilute solutions of alkali hydroxides.
chromatogram obtained with reference solution (c) to identify
ID E N T IF IC A T IO N the peaks due to impurities A, B, C and D .
First identification A Relative retention W ith reference to amidotrizoic a d d
Second identification B, C (retention time = about 5 min): impurity B = about 0.8;
A. Infrared absorption spectrophotometry (2.2.24). impurity C = about 0.9; impurity A = about 1.4;
impurity D = about 1.8.
Comparison amidotrizoic acid dihydrate CRS.
 1-132 Amidotrizoic Acid 2016

System suitability. through a sintered-glass filter (2.1.2) and wash the filter with
— resolution: m inim um 1.5 between the peaks due to several quantities o f water R. Collect the filtrate and
impurities B and C in the chromatogram obtained with washings. Add 40 m L o f dilute sulfuric acid R and titrate
reference solution (c); immediately with 0.1 M silver nitrate. D eterm ine the
— signal-to-noise ratio: minimum 25 for the principal peak in end-point potentiometrically (2.2.20), using a suitable
the chromatogram obtained with reference solution (b). electrode system such as silver/mercurous sulfate.
Limits: 1 m L of 0.1 M silver nitrate is equivalent to 20.47 m g of
— impurity B: not m ore than the area of the principal peak C 11H 9I 3N 2O 4.
in the chromatogram obtained with reference solution (a)
STO RA G E
(0.1 per cent);
Protected from light.
— impurities A , D: for each impurity, not more than the area
of the principal peak in the chromatogram obtained with IM P U R IT IE S
reference solution (b) (0.01 per cent); Specified impurities A, B, D
— unspecified impurities: for each impurity, not more than Other detectable impurities (the following substances would, if
0.5 times the area o f the principal peak in the present at a sufficient level, be detected by one or other o f
chrom atogram obtained with reference solution (a) the tests in the monograph. T hey are limited by the general
(0.05 per cent); acceptance criterion for other/unspecified impurities and/or
— total: not m ore than 1.5 times the area of the principal by the general m onograph Substances for pharmaceutical use
peak in the chromatogram obtained with reference (2034). It is therefore not necessary to identify these
solution (a) (0.15 per cent); impurities for dem onstration of compliance. See also 5.10.
— disregard limit. 0.3 times the area o f the principal peak in Control of impurities in substances for pharmaceutical use): C, E.
the chromatogram obtained with reference solution (a)
(0.03 per cent), except for the peaks due to impurities A CO2H
and D.
H a lid e s ex p ressed a s ch lo rid es (2.4.4)
M aximum 150 ppm . h2n
Dissolve 0.55 g in a mixture o f 4 m L o f dilute sodium
hydroxide solution R and 15 m L of water R. Add 6 m l. of
dilute nitric acid R and filter. A. 3-(acetylamino)-5-amino-2,4,6-triiodobenzoic acid,
F re e a ro m a tic a m in e s
Maintain the solutions and reagents in iced water, protected from c o 2h

bright light. T o 0.50 g in a 50 m L volumetric flask add

15 m L of water R. Shake and add 1 m L of dilute sodium
hydroxide solution R. Cool in iced water, add 5 m l. of a A XX1
freshly prepared 5 g/L solution o f sodium nitrite R and 12 m L I
of dilute hydrochloric acid R. Shake gently and allow to stand
for exactly 2 min after adding the hydrochloric acid. B. 3,5-bis(acetylamino)-2,4-diiodobenzoic ad d ,
Add 10 m L of a 20 g/L solution of ammonium stdfamate R.
Allow to stand for 5 min, shaking frequently, and add c o 2h
0.15 m L of a 100 g/L solution o f a-naphthol R in ethanol
(96 per cent) R. Shake and allow to stand for 5 min.
Add 3.5 m L o f buffer solution pH 10.9 R, mix and dilute to
50.0 m L 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 m anner C. 3,5-bis(acetylamino)-2,6-diiodobenzoic a d d ,
but omitting the substance to be examined, is n o t greater
than 0.30.
H eav y m e ta ls (2.4.8)
Maximum 20 ppm.
Dissolve 2.0 g in 4 m L of dilute sodium hydroxide solution R
and dilute to 20 m L w ith water R. 12 m L of the solution
complies with test A. Prepare the reference solution using
lead standard solution (2 ppm Pb) R. D . 3-(acetylamino)-5-[(iodoacetyl)amino]-2,4,6-
L oss o n d ry in g (2.2.32) triiodobenzoic ad d ,
4.5 p er cent to 7.0 per cent, determ ined on 0.500 g by
drying in an oven at 105 °C. CO2H
S u lfa te d a s h (2.4.14)
M aximum 0.1 per cent, determined on 1.0 g.
A SSA Y
T o 0.150 g in a 250 m L round-bottom ed flask add 5 m L of
strong sodium hydroxide solution R, 20 m l. o f water R, 1 g of
zinc powder R and a few glass beads. Boil under a reflux E. 3-(acetylamino)-5- (diacetylamino)-2,4,6-triiodobenzoic
condenser for 30 min. Allow to cool and rinse the condenser ad d .
with 20 m L o f water R, adding the rinsings to the flask. Filter
PhEur
2016 Amikacin 1-133

Dissolve 0.1 g in carbon dioxide-free water R and dilute to

Amikacin 10 m L with the same solvent
(Ph Eur monograph 1289) * Specific o p tic a l ro ta tio n (2.2.7)
+ 97 to + 105 (anhydrous substance).
Dissolve 0.50 g in water R and dilute to 25.0 m L with the
same solvent.
R elated su b sta n c e s
Liquid chromatography (2.2.29).
Test solution Dissolve 25 mg of the substance to be examined
in mobile phase A and dilute to 50.0 m L w ith mobile
phase A.
Reference solution (a) Dilute 1.0 m L of the test solution to
100.0 m L with mobile phase A.
585.6 37517-28-5
Reference solution (b) Dilute 1.0 m L of reference solution (a)
to 10.0 m L with mobile phase A.
A ctio n a n d u se
Aminoglycoside antibacterial. Reference solution (c) Dissolve 5 m g of amikacin for system
suitability CRS (containing impurities A, B, F and H ) in
PhEut______ ._____ ______________________________________________ mobile phase A and dilute to 10 m L with mobile phase A.
D E F IN IT IO N Reference solution (d) Dissolve 5.0 mg of amikacin
6-0-(3-Amino-3-deoxy-oc-D-glucopyranosyl)-4-0-(6-amino-6- impurity I CRS in mobile phase A and dilute to 20.0 m L with
deoxy-a-D-glucopyranosyl)-1-N- [(25)-4-amino-2- mobile phase A. Dilute 1.0 m L of the solution to 100.0 m L
hydroxybutanoyl] -2-deoxy-D-streptamine. with mobile phase A.
Antimicrobial substance obtained from kanamycin A. Column:
— sizer. I = 0.25 m , 0 = 4.6 mm;
Semi-synthetic product derived from a fermentation p ro d u ct
— stationary phase: end-capped octadecylsüyl silica gel for
C o n te n t chromatography R (5 pm);
96.5 per cent to 102.0 per cent (anhydrous substance). — temperature: 40 °C.
CHARACTERS Mobile phase:
A p p e a ra n c e — mobile phase A: a mixture prepared with carbon dioxide-free
White or almost white powder. water R, containing 1.8 g/L o f sodium octanesulfonate R,
S o lu b ility 20 g/L of anhydrous sodium sulfate R1, 1.4 per cent V/V of
Sparingly soluble in water, slightly soluble in methanol,
tetrahydrqfuran R, and 5 per cent V/V o f 0.2 M potassium
practically insoluble in acetone and in ethanol (96 per cent).
dihydrogen phosphate R previously adjusted to p H 3.0 with
dilute phosphoric acid R', degas;
ID E N T IF IC A T IO N — mobile phase B: a mixture prepared with carbon dioxide-free
A. Infrared absorption spectrophotometry (2.2.24). water R, containing 1.8 g/L o f sodium octanesulfonate R,
Comparison amikacin CRS. 28 g/L of anhydrous sodium sulfate R1, 1.4 per cent V!V of
B. Thin-layer chromatography (2.2.27). tetrahydrqfuran R, and 5 per cent V/V o f 0.2 M potassium
dihydrogen phosphate R previously adjusted to p H 3.0 with
Test solution Dissolve 25 mg of the substance to be examined
dilute phosphoric acid R ; degas;
in water R and dilute to 10 m L with the same solvent
Reference solution (a) Dissolve 25 m g o f amikacin CRS in
water R and dilute to 10 m L with the same solvent. Tune Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
Reference solution (b) Dissolve 5 mg o f kanamycin 0 -3 100 0
monosulfate CRS in 1 m L of the test solution and dilute to
10 m L with water R. 3 - 38.0 10030 0*70

Plate TLC silica gel plate R. 38.0 - 38.1 30 0 70-» 100

Mobile phase methylene chloride R, ammonia R, methanol R 38.1 - 68 0 100
(25:30:40 VfV/V).
Application 5 pL.
Flow rate 1.0 mlVmin.
Development Over 3/4 of the plate.
Post-column solution Mixture of 1 volume o f carbonate-free
Drying Luair. sodium hydroxide solution R and 24 volumes o f previously
Detection Spray w ith mnhydrin solution R1 and heat at 110 °C degassed carbon dioxide-free water R, which is added in a
for 5 min. pulseless m aim er to the column effluent using a 375 jiL
System suitability: reference solution (b): polymeric mixing coil.
— the chromatogram shows 2 clearly separated spots. Flow rate of post-column solution 0.3 mL/min.
Results T h e principal spot in the chromatogram obtained with Detection Pulsed amperometric detector or equivalent with a
the test solution is similar in position, colour and size to the gold indicator electrode, a silver-silver chloride reference
principal spot in the chromatogram obtained with reference electrode, and a stainless steel auxiliary electrode which is the
solution (a). cell body, held at respectively + 0.05 V detection, + 0.75 V
TESTS oxidation and — 0.15 V reduction potentials, with pulse
p H (2.2.3) durations according to the instrument used.
9.5 to 11.5. byection 20 pL.
 1-134 Amikacin 2016

Identification of impurities Use the chromatogram supplied Calculate the percentage content o f C 22H 43N 5O 13 taking into
with amikacin for system suitability CRS and the account the assigned content o f armkadn CRS.
chromatogram obtained with reference solution (c) to identify IM P U R IT IE S
the peaks due to impurities A, B, F and H ; use the
Spedfied impurities A, B, F, H , I
chromatogram obtained with reference solution (d) to
identify the peak due to impurity I. Other detectable impurities (the following substances would, if
present at a sufficient level, be detected by one o r other of
Relative retention W ith reference to amikacin (retention
the tests in the monograph. T hey are limited by the general
time = about 28 min): impurity I = about 0.13;
acceptance criterion for other/unspecified impurities and/or
impurity F = about 0.92; impurity B = about 0.95;
by the general monograph Substances for pharm aceutical use
impurity A = about 1.62; impurity H = about 1.95.
(2034). It is therefore not necessary to identify these
System suitability: reference solution (c): impurities for demonstration o f compliance. See also 5.10.
— peak-tchvalley ratio: minimum 5, where Hp = height above Control of impurities in substances for pharmaceutical use): C, D,
the baseline of the peak due to im purity B and E, G.
Hv = height above the baseline o f the lowest point o f the
curve separating this peak from the peak due to amikacin;
if necessary, adjust the volume of tetrahydrofuran in the
mobile phase.
Calculation ofpercentage contents:
— for im purity I, use the concentration of impurity I in
reference solution (d);
— for impurities other than I, use the concentration o f
amikacin in reference solution (a).
Limits:
— impurities A , B, F, H} I: for each impurity, maximum
0.5 per cent;
— any other impurity: for each impurity, maximum
0.5 per cent; A. 4-0-(3-amino-3-deoxy-q-D-glucopyranosyl)-6-0-(6-amino-
— total: maximum 1.5 per cent; 6 -deoxy-a-D-glucopyranosyi)-1-N- [(2S)-4-amino-2-
— reporting threshold: 0.1 per cent. hydroxybutanoyl] -2-deoxy-L-streptamine,
W a te r (2.5.12)
M aximum 8.5 per cent, determined on 0.200 g.
S u lfa te d a s h (2.4.14)
Maximum 0.5 per cent, determined on 1.0 g.
A SSAY
Liquid chromatography (2.2.29).
Test solution Dissolve 50.0 m g o f the substance to be
examined in the mobile phase and dilute to 10.0 m L with
the mobile phase.
Reference solution Dissolve 50.0 mg o f amikacin CRS in the
mobile phase and dilute to 10.0 m L with the mobile phase.
Column: B. 4-0-(3-amino-3-deoxy-ce-D-glucopyranosyl)-6-0-(6-amino-
— size. I = 0.25 m , 0 = 4.6 mm; 6-deoxy-a-D-glucopyranosyl)-1,3-N-bis [(2S)-4-amino-2-
— stationary phase, end-capped octadecylsüyl silica gelfor hydroxybutanoyl] -2-deoxy-L-streptamine,
chromatography R (5 nm);
— temperature: 40 °C.
Mobile phase A mixture prepared with carbon dioxide-free
water R, containing 1.8 g/L o f sodium octanesulfonate R,
20 g/L of anhydrous sodium sulfate R1, 5.8 per cent V/V of
acetomtrüe R l, and 5 p er cent V/V o f 0.2 M potassium
dihydrogen phosphate R previously adjusted to p H 3.0 with
dilute phosphoric add R; degas.
Flow rate 1.0 mlVmin.
Detection Spectrophotom eter at 200 nm . *
Irtjection 20 |1L. C . 4-0-(6-amino-6-deoxy-cc-D-glucopyranosyl)-6-0-[3-[[(2S)-
Run time 1.3 times the retention time o f amikacin. 4-amino-2-hydroxybutanoyi] amino] -3-deoxy-a-D-
Retention time Amikacin = about 30 min. glucopyranosyl] -2-deoxy-D-streptamine,
System suitability: reference solution:
— symmetry factor, maximum 1.5 for the peak due to
amikacin; if necessary, adjust the am ount o f acetomtrüe R l
in the mobile phase; peak splitting may be observed w hen
the retention time becomes too short;
— repeatability: maximum relative standard deviation of
1.5 per cent after 6 injections.
2016 Amikacin Sulfate 1-135

D . 6-0-(3-amino-3-deoxy-a-D-glucopyranosyl)-4-0- H . 6-0-(3-amino-3-deoxy-a-D-glucopyranosyI) -1 -N- [(2S) -4-

(6-amino-6-deoxy-a-D-glucopyranosyI)-2-deoxy-D-streptamine amino-2-hydroxybutanoyl]-4-0-(2j6-diamino-2j6-dideoxy-a-
(kanamycin), D-glucopyranosyl)-2-deoxy-D-streptamine3

HOjC NH,

H OH

I. (25)-4-amino-2-hydroxybutanoic acid.
PhEur

★ ★
E. 4-0-(3-amino-3-deoxy-a-D-glucopyranosyl)-6-0- [6-[[(25)- Amikacin Sulfate ★ ★
4-amino-2-hydroxybutanoyl] amino] -6-deoxy-a-D- *****
Amikacin Sulphate
glucopyranosyl] -2-deoxy-L-streptamine3
(Ph. Eur. monograph 1290)

F. 6 -0 - (3-amino-3-deoxy-a-D-giucopyranosyl)-4-0- [6- [(25)-

4-amino-2-hydroxybutanoyl] amino-6-deoxy-a-D- 022^ 47^ 502 x82 782 39831-55-5
glucopyranosyl] -1 -N - [(25)-4-amino-2-hydroxybutanoyl] -2-
A c tio n a n d u se
deoxy-p-stxeptamine,
Aminoglycoside antibacterial.
P re p a r a tio n
Amikacin Injection

PhEtr_____________________________

D E F IN IT IO N
6-0-(3-Amino-3-deoxy-a-D-glucopyranosyl)-4-0-(6-amino-6-
deoxy-a-i>glucopyranosyl)-l-N-[(25)-4-amino-2-
hydroxybutanoyl] -2-deoxy-D-strq)tamine sulfate.
Antimicrobial substance obtained from kanamycin A.
Semi-synthetic product derived from a fermentation product.
G . 6 -0 - (3-amino-3-deoxy-a-D-glucopyranosyl)-4-0-
(6-amino-6-deoxy-a-D-glucopyranosyl)-l-N-[(2R)-4-amino-2- C o n te n t
hydroxybutanoyl] -2-deoxy-D-streptamine, 96.5 per cent to 102.0 per cent (dried substance).
CHARACTERS
A p p e a ra n c e
White or almost white powder.
Solubility
Freely soluble in water, practically insoluble in acetone and
in ethanol (96 p er cent).
ID E N T IF IC A T IO N
A. Infrared absorption spectrophotometry (2.2.24).
Comparison amikacin sulfate CRS.
B. Thin-layer chromatography (2.2.27).
 1-136 Amikacin Sulfate 2016

Test solution Dissolve 25 mg o f the substance to be examined tetrahydrqfuran R, and 5 per cent V/V o f 0.2 M potassium
in water R and dilute to 10 m L with the same solvent. dihydrogen phosphate R previously adjusted to p H 3.0 with
Reference solution (a) Dissolve 25 m g of amikacin sulfate CRS dilute phosphoric add R; degas;
in water R and dilute to 10 m l. with the same solvent.
Reference solution (b) Dissolve 5 mg o f kanamycin Time Mobile phase A Mobile phase B
monosulfate CRS in 1 m L o f the test solution and dilute to (min) (per cent V/V) (per cent V/V)
10 m L with water R. 0-3 100 0

Plate TLC silica gel plate R 3 - 38.0 1 00*30 0*70

Mobile phase methylene chloride R, ammonia R, methanol R 38.0 - 38.1 30* 0 7 0 * 100
(25:30:40 V/V/V).
38.1 - 68 0 100
Application 5 |iL.
Development Over 3/4 of the plate.
Flow rate 1.0 mL/min.
Drying In air.
Post-column solution M ixture o f 1 volume of carbonate-free
Detection Spray with mnhydrin solution R1 and heat at 110 °C sodium hydroxide solution R and 24 volumes o f previously
for 5 min.
degassed carbon dioxide-free water R, which is added in a
System suitability: reference solution (b): pulseless m anner to the colum n effluent using a 375 pL
— the chromatogram shows 2 clearly separated spots. polymeric mixing coil.
Results The principal spot in the chromatogram obtained with Flow rate of post-column solution 0.3 mL/min.
the test solution is similar in position, colour and size to the
Detection Pulsed amperometric detector or equivalent with a
principal spot in the chromatogram obtained with reference
gold indicator electrode, a silver-silver chloride reference
solution (a).
electrode, and a stainless steel auxiliary electrode which is the
C. It gives reaction (a) of sulfates (2.3.1). cell body, held at respectively + 0.05 V detection, + 0.75 V
TESTS oxidation and — 0.15 V reduction potentials, with pulse
p H (2.2.3) durations according to the instrum ent used.
2.0 to 4.0. Injection 20 |iL.
Dissolve 0.1 g in carbon dioxide-free water R and dilute to Identification of impurities Use the chromatogram supplied
10 m L with the same solvent. with armkadn for system suitability CRS and the
S pecific o p tic a l ro ta tio n (2.2.7) chromatogram obtained with reference solution (c) to identify
+ 76 to + 84 (dried substance). the peaks due to impurities A, B, F and H ; use the
chromatogram obtained with reference solution (d) to
Dissolve 0.50 g in water R and dilute to 25.0 m L with the
identify the peak due to im purity I.
sam e solvent.
Relative retention W ith reference to amikacin (retention
R e la te d su b s ta n c e s
time = about 28 min): impurity I = about 0.13;
Liquid chrom atography (2.2.29).
impurity F = about 0.92; impurity B = about 0.95;
Test solution Dissolve 33 m g o f the substance to be examined impurity A = about 1.62; im purity H = about 1.95.
in mobile phase A and dilute to 50.0 m L with mobile System suitability: reference solution (c):
phase A — peak-to-vaUey ratio: minimum 5, where Hp = height above
Reference solution (a) Dilute 1.0 m L o f the test solution to the baseline of the peak due to im purity B and
100.0 m L w ith mobile phase A. Hv = height above the baseline o f the lowest point o f the
Reference solution (b) Dilute 1.0 m L o f reference solution (a) curve separating this peak from the peak due to amikacin;
to 10.0 m L with mobile phase A. if necessary, adjust the volume o f tetrahydrofuran in the
Reference solution (c) Dissolve 5 mg o f amikacin for system mobile phase.
suitability CRS (containing impurities A, B, F and H ) in Calculation of percentage contents:
mobile phase A and dilute to 10 m l. with mobile phase A. — for impurity I, use the concentration o f im purity I in
Reference solution (d) Dissolve 6.6 m g o f amikacin reference solution (d);
impurity I CRS in mobile phase A and dilute to 20.0 m L with — for impurities other than I, use the concentration of
mobile phase A. Dilute 1.0 m l. o f the solution to 100.0 m L amikacin sulfate in reference solution (a).
w ith mobile phase A. Limits.
Column: — impurities A , B, F, H, I: for each impurity, maximum
— size. I = 0.25 m, 0 = 4.6 mm; 0.5 p er cent;
— stationary phase: end-capped octadecyhüyl silica gel for — arty other impurity: for each impurity, m axim um
chromatography R (5 pm); 0.5 p er cent;
— temperature: 40 °C. — total: maximum 1.5 p er cent;
— reporting threshold: 0.1 p er cent.
Mobile phase:
— mobile phase A: a mixture prepared with carbon dioxide-free Sulfate
water R, containing 1.8 g/L o f sodium octanesulfonate R, 23.3 per cent to 25.8 per cent (dried substance).
20 g/L o f anhydrous sodium sulfate R l, 1.4 per cent V/V o f Dissolve 0.250 g in 100 m L o f water R and adjust the
tetrahydrofuran R, and 5 per cent V/V of 0.2 M potassium solution to p H 11 using concentrated ammonia R.
dihydrogen phosphate R previously adjusted to p H 3.0 with A dd 10.0 m L o f 0.1 M barium chloride and about 0.5 m g of
dilute phosphoric acid R; degas; phthalein purple R Titrate with 0.1 M sodium edetate adding
— mobile phase B: a mixture prepared with carbon dioxide-free 50 m l. of ethanol (96 per cent) R when the colour o f the
water R, containing 1.8 g/L o f sodium octanesulfonate R, solution begins to change and continue the titration until the
28 g/L o f anhydrous sodium sulfate R l , 1.4 per cent V/V of violet-blue colour disappears.
2016 Amikacin Sulfate 1-137

1 m l. o f 0.1 M barium chloride is equivalent to 9.606 mg of

sulfate (SO 4).
L oss o n d ry in g (2.2.32)
M aximum 13.0 p e r cent, determined on 0.500 g by drying in
an oven at 105 °C at a pressure not exceeding 0.7 kPa for
3 h.
P y ro g e n s (2.6.8)
If intended for use in the manufacture of parenteral
preparations w ithout a further appropriate procedure for the
removal of pyrogens, it complies with the test for pyrogens.
Inject per kilogram o f the rabbit’s mass 5 m L o f a solution
A. 4-0-(3-amino-3-deoxy-a-D-glucopyranosyl)-6-0-(6-amino-
containing 25 m g o f the substance to be examined in water
6 -deoxy-a-D-ghicopyranosyl)-1-N-[(2S) -4-amino-2 -
for injections R.
hydroxybutanoyl] - 2-deoxy-L-streptamine,
A SSAY
Liquid chrom atography (2.2.29).
Test solution Dissolve 50.0 mg of the substance to be
examined in the mobile phase and dilute to 10.0 m L with
the mobile phase.
Reference solution Dissolve 50.0 mg o f amikacin sulfate CRS in
the mobile phase and dilute to 10.0 m L with the mobile
phase.
Column'.
— sizer. I — 0.25 m , 0 = 4.6 mm;
— stationary phase', end-capped octadecylsUyl silica gel for
chromatography R (5 |am);
— temperature: 40 °C. B. 4-0-(3-amino-3-deoxy-a-D-ghicopyranosyl)-6-0-(6-amino-
Mobile phase A mixture prepared with carbon dioxide-free 6-deoxy-a-D-glucopyranosyl)-1,3-N-bis [(25)-4-amino-2-
water R, containing 1.8 g/L o f sodium octanesulfonate R, hydroxybutanoyl] -2-deoxy-L-streptamine,
20 g/L o f anhydrous sodium sulfate R l, 5.8 p er cent V/V of
acetonitrile R l, and 5 per cent VIV o f 0.2 M potassium
dihydrogen phosphate R previously adjusted to p H 3.0 with
dilute phosphoric acid R; degas.
Flow rate 1.0 m l7min.
Detection Spectrophotom eter at 200 nm.
Injection 20 |iL.
Rim 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 C. 4-0-(6-amino-6-deoxy-a-D-glucopyranosyl)-6-0-[3-[[(25)-
amikacin; if necessary, adjust the am ount o f acetonitrile R l 4-amino-2-hydroxybutanoyl] amino]-3-deoxy-a-D-
in the mobile phase; peak splitting may be observed when glucopyranosyl] -2-deoxy-D-streptamine,
the retention tim e becomes too short;
— repeatability: m axim um relative standard deviation of
1.5 per cent after 6 injections.
Calculate the percentage content o f C 22H 47N 5O 21S 2 taking
into account the assigned content o f amikacin sulfate CRS.
STO R A G E
If the substance is sterile, store in a sterile, airtight, tam per­
proof container.
IM P U R IT IE S
Specified impurities A, B, F, H , I
Other detectable impurities (the following substances would, if D . 6-0-(3-amino-3-deoxy-a-D-ghicopyranosyl)-4-0-
present at a sufficient level, be detected by one or other of (6-amino-6-deoxy-a-D-glucopyranosyl)-2-deoxy-D-streptamine
the tests in the m onograph. They are limited by the general (kanamycin),
acceptance criterion for other/unspecified impurities and/or
by die general m onograph Substances for pharmaceutical use
(2034). It is therefore n o t necessary to identify these
impurities for dem onstration o f compliance. See also 5.10.
Control of impurities in substances for pharmaceutical use): C, D,
E, G.
 1-138 Amiloride Hydrochloride 2016

Amiloride Hydrochloride
(Ph. Eur. monograph 0651) *

O NH
CK .N. JLN J k NH,

H2N
XX H
Y

N NH2
, HCI, 2 H ,0 .

E. 4-0-(3-amino-3-deoxy-a-D-giucopyranosyl)-6-0-[6- [ [(25)- C6HgQ2N70j2H20 302.1 17440-83^1

4-amino-2-hydroxybutanoyI]amino]-6-deoxy-a-D-
glucopyranosyfl-2-deoxy-L-streptamine, Action and use
Sodium channel blocker; potassium-sparing diuretic.
Preparations
Amiloride Tablets
Co-amilofruse Tablets
Co-amilozide Oral Solution
Co-amilozide Tablets

PhEur__________________________________________________________

DEFINITION
3,5-Diarnino-Ar-carbamirnidoyl-6-chloropyrazine-2-
F. 6-0-(3-amino-3-deoxy-a-D-glucopyranosyl)-4-0- [6- [(25)- carboxamide hydrochloride dihydrate.
4-amino-2-hydroxybutanoyl] amino-6-deoxy-a-D-
Content
glucopyranosyQ-l-N- [(25)-4-amino-2-hydroxybutanoyi]-2-
98.0 per cent to 101.0 per cent (anhydrous substance).
deoxy-D -streptam ine}
CHARACTERS
Appearance
Pale yellow or greenish-yellow powder.
Solubility
Slightly soluble in water and in anhydrous ethanol.
IDENTIFICATION
First identification A, D.
Second identification B, C, D.
A. Infrared absorption spectrophotom etry (2.2.24).
Comparison amiloride hydrochloride CRS.
G. 6-0-(3-amino-3-deoxy-a-D-glucopyranosyl)-4-0-
B. Thin-layer chromatography (2.2.27).
(6-araino-6-deoxy-a-D-glucopyranosyl)-l-7V-[(2i?l)-4-ainino-2-
hydroxybutanoyl]-2-deoxy-D-streptamine, Test solution Dissolve 40 m g o f the substance to be examined
in methanol R and dilute to 10 m L w ith the same solvent.
Reference solution Dissolve 40 m g o f amiloride
hydrochloride CRS in methanol R and dilute to 10 m L with the
same solvent.
Plate TLC silica gel plate R.
Mobile phase dilute ammonia R l, water R, dioxan R
(6:6:88 VIV/V); freshly prepared mixture.
Application 5 |iL.
Development Over 2/3 of the plate.
Drying In air.
H . 6-0-(3-amino-3-deoxy-a-D-gJucopyranosyJ)-l-N-[(25)-4- Detection Examine in ultraviolet light at 365 nm.
amino-2-hydroxybutanoyI] -4-0- (2,6-diamino-2, 6-dideoxy-a- Results T h e principal spot in the chrom atogram obtained with
D-gJucopyranosyl)-2-deoxy-D-streptaminej the test solution is similar in position, fluorescence and size
to the principal spot in the chrom atogram obtained w ith the
H° 2 C ^ ^ , N H 2 reference solution.
H OH C. Dissolve about 10 m g in 10 m L o f water R. Add 10 m L
o f a 200 g/L solution of cetrimide R} 0.25 m L of dilute sodium
I. (25)-4-amino-2-hydroxybutanoic add. hydroxide solution R and 1 m L o f bromine water R. A greenish-
yellow colour is produced. Add 2 m L o f dilute hydrochloric
___________________________________________________________ PhEur
acid R. T h e solution becomes deep yellow and shows blue
fluorescence in ultraviolet light at 365 nm.
D. It gives reaction (b) o f chlorides (2.3.1).
2016 Aminobenzoic Acid 1-139

TESTS ASSAY
Free acid Dissolve 0.200 g in a mixture o f 5.0 m L o f 0.01 M
Dissolve 1.0 g in a mixture o f 50 m L o f methanol R and hydrochloric add and 50 m L of ethanol (96 per cent) R. Carry
50 m L o f water R and titrate with 0.1 M sodium hydroxide, out a potentiom etric titration (2.2.20), using 0.1 M sodium
determ ining the end-point potentiometrically (2.2.20). hydroxide. Read the volume added between the 2 points o f
N o t m ore than 0.3 m L o f 0.1 M sodium hydroxide is required inflexion.
to reach the end-point. 1 m L of 0.1 M sodium hydroxide is equivalent to 26.61 m g
Related substances Of C6H 9CI2N 7O.
Liquid chrom atography (2.2.29). STORAGE
Test solution Dissolve 20.0 m g o f the substance to be Protected from light.
examined in a mixture of 1 volume o f acetonitrile R and
IM P U R IT IE S
3 volumes o f water R and dilute to 10.0 m L with the same
mixture o f solvents.
Other detectable impurities (the following substances would, if
present at a sufficient level, be detected by one o r other o f
Reference solution (a) Dilute 1.0 m L o f the test solution to the tests in the monograph. T hey are limited by the general
100.0 m L with a mixture o f 1 volume o f acetomtrUe R and
acceptance criterion for other/unspecified impurities and/or
3 volumes o f water R. by the general monograph Substances for pharmaceutical use
Reference solution (b) Dilute 1.0 m L o f reference solution (a) (2034). It is therefore not necessary to identify these
to 10.0 m l, with a mixture o f 1 volume o f acetonitrUe R and impurities for demonstration o f compliance. See also 5.10.
3 volumes of water R. Control of impurities in substances for pharmaceutical use): A .
Reference solution (c) Dissolve 5.0 m g o f amHoride
impurity A CRS in a mixture of 1 volume o f acetonitrUe R and
3 volumes of water R and dilute to 5.0 m L with die same ,c h 3
mixture o f solvents. Dilute 1.0 m L o f this solution to
100.0 m L with a mixture of 1 volume o f acetonitrUe R and HjN N NH2
3 volumes o f water R.
Column: A. methyl 3,5-diamino-6-chloropyrazine-2-carboxylate.
— sizer. I = 0.25 m , 0 = 4.6 mm;
PhEur
— stationary phase: octadecylsHyl silica gel for chromatography R
(5 pm ).
Mobile phase M ix 5 volumes o f tetramethylammoraum hydroxide
solution R, 250 volumes o f acetonitrUe R and 745 volumes of
* * *★
water R ; adjust to p H 7.0 with a mixture o f 1 volume of Aminobenzoic Acid *
★ ★
phosphoric add R and 9 volumes o f water R. Adjust the
(4-Aminobenzoic Add, Ph Eur monograph 1687) * * * * *
concentration o f acetonitrile in the mobile phase so that the
retention time o f im purity A is 5-6 min (an increase in the
concentration o f acetonitrile results in a shorter retention
time). A djust die concentration o f tetramethylammonium
hydroxide and o f phosphoric a d d keeping the p H at 7.0 so
that the retention time of amiloride is 9-12 min (an increase
h 2n
XT
in the concentration results in a shorter retention time for
CzHyNOa 137.1 150-13-0
amiloride).
Flow rate 1 mL/min. A ctio n a n d u se
Detection Spectrophotom eter at 254 run. Skin protective.
Irqection 20 pL. PhEtr__________ __
Run time 5 times the retention time o f amiloride.
D E F IN IT IO N
System suitability: reference solution (b):
4-Aminobenzoic acid.
— signal-to-noise ratio: m inim u m 5.0 for the peak due to
amiloride. C o n te n t
Limits: 99.0 p er cent to 101.0 per cent (anhydrous substance).
— unspecified impurities: for each impurity, n o t more than CHARACTERS
0.2 tim es the area of the peak due to impurity A in the A p p e a ra n c e
chrom atogram obtained with reference solution (c) W hite o r slightly yellow, crystalline powder.
(0.10 per cent);
S o lu b ility
— total: not m ore than the area o f the peak due to
Slightly soluble in water, freely soluble in alcohol. It dissolves
im purity A in the chromatogram obtained with reference
in dilute solutions o f alkali hydroxides.
solution (c) (0.5 per cent);
— disregard limit. 0.1 times the area of the peak due to ID E N T IF IC A T IO N
im purity A in the chromatogram obtained with reference First identification B
solution (c) (0.05 per cent). Second identification A , C
Water (2.5.12) A. M elting point (2.2.14): 186 °C to 189 °C.
11.0 per cent to 13.0 per cent, determ ined on 0.200 g. B. Infrared absorption spectrophotometry (2.2.24).
Sulfated ash (2.4.14) Comparison 4-aminobenzoic add CRS.
M axim um 0.1 p er cent, determined on 1.0. g. C. Thin-layer chromatography (2.2.27).
 1-140 Aminobenzoic Acid 2016

Test solution Dissolve 20 mg o f the substance to be examined — any other impurity: not more than 0.5 times the area o f the
in methanol R and dilute to 20 m L w ith the same solvent. peak due to im purity A in the chrom atogram obtained
Reference solution (a) Dissolve 20 m g o f 4-aminobenzoic with the reference solution (0.1 p er cent),
add CRS in methanol R and dilute to 20 m L with the same — total: n o t more than 2.5 times the area o f the peak due to
solvent. impurity A in the chrom atogram obtained with the
reference solution (0.5 p er cent),
Reference solution (b) Dissolve 10 m g o f 4-nitrobenzoic acid R
— disregard limit: 0.1 times the area o f the peak due to
in 10 m L o f reference solution (a).
impurity A in the chrom atogram obtained with the
Plate Suitable silica gel with a fluorescent indicator having an reference solution (0.02 per cent).
optimal intensity at 254 nm as the coatin g substance.
Impurity C and im purity D
Mobile phase glacial acetic add R, hexane R, methylene
Gas chromatography (2.2.28).
' chloride R (5:20:75 VIVIV).
Internal standard solution Dissolve 20.0 m g o f lauric add R in
Application 1 |iL.
methylene chloride R and dilute to 100.0 m L with the same
Development Over a path of 10 cm. solvent.
Drying In air. Test solution Dissolve 1.000 g o f the substance to be
Detection Examine in ultraviolet light at 254 nm. examined in 10.0 m L o f an 84 g/L solution o f sodium
System suitability T he chromatogram obtained with reference hydroxide R and extract with 2 quantities, each o f 10 mT, of
solution (b) shows 2 clearly separated spots. methylene chloride R. Combine and wash with 5 m L of
Results T he principal spot in the chrom atogram obtained with water R; filter through anhydrous sodium sulfate R. W ash the
the test solution is similar in position and size to the principal filter with methylene chloride R. Evaporate in a water-bath at
spot in the chromatogram obtained with reference 50-60 °C to obtain a volume o f about 1-5 mL. A dd 1.0 m L
solution (a). o f the internal standard solution and dilute to 10.0 m L with
methylene chloride R.
TESTS
Reference solution (a) Dissolve 20.0 m g o f aniline R in
A p p e a ra n c e o f so lu tio n
methylene chloride R and dilute to 100.0 m L w ith the same
The solution is clear (2.2.1) and no t m ore intensely coloured
solvent.
than reference solution B5 (2.2.2, Method II).
Reference solution (b) Dissolve 20.0 m g o f p-tohddine R in
Dissolve 1.0 g in alcohol R and dilute to 20 m L with the
methylene chloride R and dilute to 100.0 m L with the same
same solvent.
solvent.
Related substances Reference solution (c) Dilute 0.50 m L o f reference solution (a),
Liquid chromatography (2.2.29). 0.50 m L o f reference solution (b) and 10.0 m L o f the
Test solution Dissolve 25.0 mg o f the substance to be internal standard solution to 100.0 m L with methylene
examined in the mobile phase and dilute to 100.0 m L with chloride R.
the mobile phase. Column:
Reference solution Dissolve 25.0 m g o f 4-nitrobenzoic add R — material: fused silica,
and 25.0 m g o f benzocaine R in methanol R and dilute to — size. I = 30 m , 0 = 0.32 m m ,
100.0 m L with the same solvent. D ilute 1.0 m L to 50.0 m L — stationary phase. poly[methyl(95)phenyl(5)JsUoxane R (film
with the mobile phase. Dilute 1.0 m L o f this solution to thickness 0.5 nm).
10.0 m L with the mobile phase. Carrier gas helium for chromatography R.
Column: Flow rate 1.0 mL/min.
— size. I — 0.12 m , 0 = 4.0 m m ,
Split ratio 1:10.
— stationary phase: octylsUyl silica gel for chromatography R
(5 nm). Temperature:
Mobile phase Mix 20 volumes o f a mixture o f 70 volumes of
acetomtrUe R and 80 volumes o f methanol R, and 80 volumes H um Temperature
of a solution containing 1.5 g/L o f potassium dihydrogen (min) (°C)
Column 0 -4 130
phosphate R and 2.5 g/L of sodium octanesulfoncae R adjusted
to p H 2.2 with phosphoric add R. 4-6.5 130->180
Flow rate 1.0 mlVmin. 6.5 -11.5 180
Detection Spectrophotom eter at 270 nm . Injection port 280
Injection 20 |iL. 300
Detector
Run time 11 times the retention time o f 4-aminobenzoic add.
Relative retention W ith reference to 4-aminobenzoic a d d
(retention tim e = about 3 min): impurity A = about 4; Detection Flame ionisation.
impurity B = about 9. Injection 2 nL; inject the test solution and reference
Limits: solution (c).
— impurity A: not m ore than the area o f the corresponding Retention time Internal standard = about 9.5 min.
peak in the chromatogram obtained with the reference Limits:
solution (0.2 per cent), — impurity C: calculate th e ratio (K) o f the area o f the peak
— impurity B: n o t more than the area o f the corresponding due to impurity C to the area of the peak due to the
peak in the chromatogram obtained with the reference internal standard from the chromatogram obtained with
solution (0.2 per cent), reference solution (c); calculate the ratio o f the area o f the
peak due to impurity C to the area o f the peak due to the
internal standard from the chrom atogram obtained with
2016 Aminocaproic Acid 1-141

the test solution: this ratio is not greater than R CHARACTERS

(10 ppm ), A white or almost white, crystalline pow der or colourless
— impurity D: calculate the ratio (R) of the area o f the peak crystals, fredy soluble in water, slightly soluble in alcohol.
due to impurity D to the area of the peak due to the It melts at about 205 °C with decomposition.
internal standard from the chromatogram obtained with
ID E N T IF IC A T IO N
reference solution (c); calculate the ratio o f the area o f the
peak due to impurity D to the area o f the peak due to the First identification A.
internal standard from the chromatogram obtained with Second identification B, C, D.
the test solution: this ratio is not greater than R A. Examine by infrared absorption spectrophotometry
(10 ppm ). (2.2.24), comparing with the spectrum obtained with
Ir o n (2.4.9) aminocaproic acid CRS. Examine the substances prepared as
M axim um 40 ppm . discs.
Dissolve 0.250 g in 3 m L of alcohol R and dilute to 10.0 m L B. Examine the chromatograms obtained in the test for
w ith water R. ninhydrin-positive substances. T h e p rindpal spot in the
chromatogram obtained with the test solution (b) is similar in
H e a v y m e ta ls (2.4.8)
position, colour and size to the principal spot in the
M axim um 20 ppm .
chromatogram obtained with reference solution (a).
1.0 g complies with test C . Prepare the reference solution
C. Dissolve 0.5 g in 4 m L of a mixture o f equal volumes of
using 2 m L o f lead standard solution (10 ppm Pb) R.
dilute hydrochloric acid R and water R Evaporate to dryness by
W a te r (2.5.12) heating on a water-bath. Dry the residue in a desiccator.
M axim um 0.2 p er cent, determined on 1.00 g. Dissolve the residue in about 2 m L o f boiling ethanol R.
S u lfa te d a s h (2.4.14) Allow to cool and maintain at 4 °C to 8 °C for 3 h. Filter
M axim um 0.1 per cent, determined on 1.0 g. under reduced pressure. The residue washed with about
10 m L o f acetone R and dried at 60 °C for 30 min, m dts
A SSA Y
(2.2.14) at 131 °C to 133 °C.
Dissolve 0.100 g with heating in 50 m L o f carbon dioxide-free
water R. T itrate with 0.1 M sodium hydroxide determining the D . Dissolve about 5 mg in 0.5 m L o f distilled water R.
end-point potentiometrically (2.2.20). A dd 3 m L o f dimetkylformarmde R and 2 m L of ascorbic acid
solution R. H eat on a water-bath. An orange colour develops.
1 m L o f 0.1 M sodium hydroxide is equivalent to 13.71 m g o f
C 7H 7N 0 2. TESTS
S o lu tio n S
STORA GE
Dissolve 10.0 g in carbon dioxide-free water R and dilute to
Protected from light.
50.0 m L w ith the same solvent.
IM P U R IT IE S A p p e a ra n c e o f so lu tio n
Solution S is colourless (2.2.2, Method II) and remains clear
(2.2.1) on standing for 24 h.
p H (2.2.3)
T h e p H o f solution S is 7.5 to 8.0.
A. R = C 0 2H , R ' = N 0 2: 4-nitrobenzoic ad d , A b so rb a n c e (2.2.25)
B. R = C 0 - 0 - C 2H 5, R ' = N H 2: ethyl 4-aminobenzoate A. T h e absorbance o f solution S at 287 nm is not more
(benzocaine), th an 0.10 and at 450 nm is not more th an 0.03.
C. R = H , R ' = N H 2: aniline, B. Place 2.0 g in an even layer in a shallow dish 9 cm in
diameter, cover and allow to stand at 98 °C to 102 °C for
D . R = C H 3, R ' = N H 2: 4-methylaniline (p-toluidine).
72 h. Dissolve in water R and dilute to 10.0 m L with the
__________________________________________________________ PhEur same solvent. T h e absorbance o f the solution at 287 nm is
no t more than 0.15 and at 450 nm is n o t more than 0.03.
N in h y d rin -p o sitiv e su b stan c es
Examine by thin-layer chromatography (2.2.27), using a
Aminocaproic Acid i***% suitable silica gel as the coating substance.
*+ Test solution (a) Dissolve 0.10 g of the substance to be
(Ph Eur monograph 0874) * examined in water R and dilute to 10 m L with the same
solvent.
Test solution (b) Dilute 1 m L o f test solution (a) to 50 m L
w ith water R.
Q H J3N 02 131.2 60-32-2 Reference solution (a) Dissolve 10 mg o f aminocaproic
acid CRS in water R and dilute to 50 m L with the same
A c tio n a n d u s e solvent.
Antifibrinolytic.
Reference solution (b) Dilute 5 m L o f test solution (b) to
PhEur__________________________________________________________ 20 m L with water R.
D E F IN IT IO N Reference solution (c) Dissolve 10 mg o f aminocaproic acid CRS
Aminocaproic a d d contains not less than 98.5 p er cent and and 10 mg o f leucine CRS in water R an d dilute to 25 m L
not more th an the equivalent of 101.0 per cent of with the same solvent.
6-am inohexanoic ad d , calculated with reference to the dried Apply separately to the plate 5 (iL o f each solution. Allow the
substance. plate to dry in air. D evdop over a p ath of 15 cm using a
 1-142 Aminoglutethimide 2016

mixture of 20 volumes o f glacial acetic acid R, 20 volumes of C. Thin-layer chromatography (2.2.27).

water R and 60 volumes o f butanol R. Dry the plate in a Test solution Dissolve 25 m g of the substance to be examined
current of w arm air. Spray with mnhydrin solution R and heat in acetone R and dilute to 5 m L with the same solvent.
at 100 °C to 105 °C for 15 min. Any spot in the Reference solution (a) Dissolve 25 m g of
chromatogram obtained with the test solution (a), apart from aminoglutethimide CRS in acetone R and dilute to 5 m L with
the principal spot, is n o t more intense than the spot in the the same solvent.
chromatogram obtained with reference solution (b)
(0.5 per cent). T he test is not valid unless the chromatogram
Reference solution (b) Dissolve 25 mg of
aminoglutethimide CRS and 25 mg o f glutethimide CRS in
obtained with reference solution (c) shows two clearly
acetone R and dilute to 5 m L with the same solvent.
separated principal spots.
Plate TLC stHca gel F 254 plate R.
H eav y m e ta ls (2.4.8)
12 m L of solution S complies with test A for heavy metals Mobile phase glacial acetic add R, methanol R, ethyl acetate R
(10 ppm ). Prepare the reference solution using lead standard (0.5:15:85 VIVIV).
solution (2 ppm Pb) R. Application 5 |oL.
L oss o n d ry in g (2.2.32) Development Over 3/4 of the plate.
N ot more than 0.5 per cent, determined on 1.000 g by Drying In air.
drying in an oven at 105 °C. Detection Examine in ultraviolet light at 254 nm.
S u lfa te d a sh (2.4.14) System suitability: reference solution (b):
N ot more than 0.1 per cent, determined on 1.0 g. — the chromatogram shows 2 clearly separed spots.
A SSA Y Results T h e principal spot in the chromatogram obtained with
Dissolve 0.100 g in 20 m L o f anhydrous acetic acid R. Using the test solution is similar in position and size to the principal
0.1 m L of crystal violet solution R as indicator, titrate with spot in the chromatogram obtained with reference
0.1 M perchloric add until the colour changes from bluish- solution (a).
violet to bluish-green. TESTS
1 m L o f 0.1 M perchloric add is equivalent to 13.12 m g o f S o lu tio n S
C sH jaN O * Dissolve 1.0 g in methanol R and dilute to 20.0 m L with the
__________________________________________________________ PhEur same solvent.
A p p e a ra n c e o f so lu tio n
Solution S is clear (2.2.1) and not more intensely coloured
than reference solution Y7 (2.2.2, Method II).
Aminoglutethimide ***** O p tic a l ro ta tio n (2.2.7)
★. * —0.10° to + 0.10°, determined on solution S.
(Ph. Eur. monograph 1291) *
R e la te d su b sta n c e s
H Liquid chromatography (2.2.29).
O ^N ^O
Solvent mixture methanol R, acetate buffer solution pH 5.0 R
I L J \ NH2 and enantiomer (50:50 VIV).
Test solution Dissolve 0.100 g of the substance to be
H3< /
examined in the solvent mixture and dilute to 50.0 m L with
the solvent mixture.
C i 3H16N 20 2 232.3 125-84-8
Reference solution (a) Dissolve 5.0 m g o f aminoglutethimide
A c tio n a n d u se impurity A CRS in the solvent mixture and dilute to 25.0 m L
Inhibitor of adrenal corticosteroid synthesis; used in chemical with the solvent mixture.
adrenalectomy. Reference solution (b) Dilute 1.0 m L of reference solution (a)
to 10.0 m L with the solvent mixture.
PhEur__________________________________________________________
Reference solution (c) Dilute 1.0 m L o f the test solution to
D E F IN IT IO N 100.0 m L with the solvent mixture.
(3i?5)-3-(4-Aminophenyl)-3-ethylpiperidine-2j6-dione. Reference solution (d) Dilute 1.0 m L o f the test solution to
C o n te n t 10.0 m L with reference solution (a). -
98.0 per cent to 101.5 per cent (dried substance). Column:
— size. I = 0.15 m, 0 = 3.9 mm;
CHARACTERS
— stationary phase, octadecylsüyl silica gel for chromatography R
A p p e a ra n c e
(4 Jim);
W hite or slightly yellow, crystalline powder.
— temperature: 40 °C.
S olubility
Mobile phase Mix 27 volumes of methanol R and 73 volumes
Practically insoluble in water, freely soluble in acetone,
o f acetate buffer solution pH 5.0 R
soluble in methanol.
Flow rate 1.3 mL/min.
ID E N T IF IC A T IO N
Detection Spectrophotometer at 240 nm.
First identification B
Injection 10 pL of the test solution and reference
Second identification A, C solutions (b), (c) and (d).
A. Melting point (2.2.14): 150 °C to 154 °C. Run time 4 times the retention time o f aminoglutethimide.
B. Infrared absorption spectrophotometry (2.2.24).
Comparison aminoglutethimide CRS.
2016 Aminoglutethimide 1-143

Identification of impurities Use the chromatogram obtained Prepare the reference solution vising lead standard solution
with reference solution (b) to identify die peak due to (1 ppm Pb) obtained by diluting lead standard solution
impurity A. (100 ppm Pb) R with a mixture o f 5 m L o f water R and
Relative retention W ith reference to aminoglutethimide 15 m L o f acetone R
(retention time = about 9 min): impurity A = about 1.3. L oss o n d ry in g (2.2.32)
System suitability Reference solution (d): M axim um 0.5 p er cent, determined on 1.000 g by drying in
— resolution: m inim u m 2.0 between the peaks due to an oven at 105 °C.
am in oglu teth im id e and impurity A. S u lfa te d a sh (2.4.14)
Limits: M axim um 0.1 per cent, determined on 1.0 g.
— impurity A: not m ore than twice die area o f the principal
A SSA Y
peak in the chromatogram obtained w ith reference
Dissolve 0.180 g in 50 m L of anhydrous acetic acid R and
solution (b) (2.0 p er cent);
titrate with 0.1 M perchloric acid, determining the end-point
— unspecified impurities: for each impurity, n o t more than
potentiometrically (2.2.20).
0.1 times the area o f the principal peak in die
chromatogram obtained with reference solution (c) 1 m L of 0.1 M perchloric acid is equivalent to 23.23 mg
(0.10 per cent); Of C 13H 16N 2O 2.
— sum of impurities other than A: not more than the area o f IM P U R IT IE S
the principal peak in die chromatogram obtained with Specified impurities A, D .
reference solution (c) (1.0 per cent);
Other detectable impurities (the following substances would, if
— total: maximum 2.0 per cent for the sum of die contents
present at a sufficient level, be detected by one o r other o f
o f all impurities;
the tests in the monograph. T hey are limited by the general
— disregard limit: 0.05 times the area of the principal peak in
acceptance criterion for other/unspecified impurities and/or
the chromatogram obtained with reference solution (c)
by the general monograph Substances for pharmaceutical use
(0.05 per cent).
(2034). It is therefore not necessary to identify these
Impurity D impurities for demonstration o f compliance. See also 5.10.
Liquid chromatography (2.2.29). Carry out die test protected Control of impurities in substances for pharmaceutical use): B, C.
from light Use shaking, not sordcadon or heat, to dissolve the
reference substance and the substance to be examined.
Test solution Dissolve 0.100 g of the substance to be
examined in dimethyl sulfoxide R and dilute to 100.0 m L with R4 and enantiomer
the same solvent.
Reference solution Dissolve 3.0 mg of aminoglutethimide
impurity D CRS in dimethyl sulfoxide R and dilute to
100.0 m L with the same solvent. Dilute 1.0 m L of this A. R3 = N H 2) R4 = H: (3RS)-3-(3-aminophenyl)-3-
solution to 100.0 m l, with dimethyl sulfoxide R. ethylpiperidine-2,6-dione (3-aminoglutethimide),
Column: B. R3 = N O * R4 = H:
— sizer. I = 0.12 m , 0 = 4 mm; (3i?S)-3-ethyl-3-(3-nitrophenyl)piperidine-2,6-dione,
— stationary phase: octadecylsUyl silica gel for chromatography R C. R3 = H , R4 = N 0 2:
(5 pm). (3i?5)-3-ethyl-3-(4-nitrophenyl)piperidine-2,6-dione,
Mobile phase Dissolve 0.285 g of sodium edetate R in water R,
add 7.5 m L o f dilute acetic acid R and 50 m l, of 0.1 M
potassium hydroxide and dilute to 1000 m L with water R;
adjust to p H 5.0 with glacial acetic acid R’, mix 350 m L of
this solution with 650 m L of methanol R.
Flow rate 1.0 mL/min.
Detection Spectrophotometer at 328 nm.
Injection 10 |iL.
System suitability T est solution: D . 3,3'-[diazenediylbis(4,l-phenylene)]bis(3-ethylpiperidine-
— number of theoretical plates: minimum 3300, calculated for 2, 6-dione) (azoglutethimide).
the principal peak; PhEur
— mass distribution ratio: 2.0 to 5.0 for the principal peak;
— symmetry factor, m axim um 1.2 for the principal peak.
Limit:
— impurity D: n o t m ore than the area of the principal peak
in the chromatogram obtained with the reference solution
(300 ppm).
Sulfates (2.4.13)
M aximum 500 ppm .
Dilute 6 m L o f solution S to 15 m L with distilled water R.
Heavy m etals (2.4.8)
M aximum 10 ppm.
Dissolve 2.0 g in 15 m L o f acetone R and dilute to 20 m L
with water R. 12 m L of the solution complies with test B.
 1-144 Aminophylline 2016

E. W ater (see Tests).

Aminophylline ;***%
F . T h e pred p itate gives the reaction o f xanthines (2.3.1).
*★ ★*
(TheophyUine-ethyknediamine, anhydrous, *
TESTS
Ph Eur monograph 0300)
Appearance o f solution
T h e solution is n o t m ore opalescent than reference
suspension II (2.2.1) and n o t m ore intensely coloured than
reference solution GY6 (2.2.2, Method II).
Dissolve 0.5 g with gentle warming in 10 m L o f carbon
dioxide-free water R.
Related substances
Liquid chrom atography (2.2.29).
C 16H 24N 10O 4 420.4 317-34-0 Test solution Dissolve 47 m g o f the substance to be examined
in the mobile phase and dilute to 20.0 m L w ith the mobile
A c tio n a n d u se phase.
N on-sdective phosphodiesterase inhibitor; treatm ent o f
Reference solution (a) D ilute 1.0 m L o f the test solution to
reversible airways obstruction. 100.0 m L with the mobile phase. D ilute 1.0 m l . o f this
P re p a r a tio n s solution to 10.0 m l . with the mobile phase.
Aminophylline Injection Reference solution (b) Dissolve 10 m g o f theobromine R
Aminophylline Tablets (impurity G ) in the mobile phase, add 5 m L o f the test
Prolonged-release Aminophylline Tablets solution and dilute to 100 m L with the mobile phase. Dilute
5 m L o f this solution to 50 m L with the mobile phase.
Ph Eur___________________________________________________________
Column:
D E F IN IT IO N — sizer. I = 0.25 m , 0 = 4 mm;
C o n te n t — stationary phase: octadecylsUyl silica gelfor chromatography R
— theophylline (C 7H&N4O 2; 180.2): 84.0 per cent to (7 nm).
87.4 per cent (anhydrous substance); Mobile phase M ix 7 volumes o f acetomtrUefor
— ethylenedianane (C 2HgN 2; 60.1): 13.5 p er cent to chromatography R and 93 volumes o f a 1.36 g/L solution of
15.0 per cent (anhydrous substance). sodium acetate R containing 0.50 per cent V/V o f glacial acetic
CHARACTERS add R.
A p p e a ra n c e Flow rate 2.0 mL/min.
W hite or slightly yellowish powder, sometimes granular, Detection Spectrophotom eter at 272 nm .
hygroscopic. Irqection 20 jjL .
S o lu b ility Run time 3.5 times the retention time o f theophylline.
Freely soluble in water (the solution becomes cloudy through Relative retention W ith reference to theophylline (retention
absorption o f carbon dioxide), practically insoluble in tim e = about 6 min): impurity G = about 0.6.
anhydrous ethanol.
System suitability: reference solution (b):
ID E N T IF IC A T IO N — resolution: m in im u m 2.0 between the peaks due to
First identification B, C, E. impurity G and theophylline.
Second identification A , C, D, E, F. Limits:
Dissolve 1.0 g in 10 m L o f water R and add 2 m l . o f dilute — unspecified impurities: for each impurity, n o t more than the
hydrochloric acid R dropwise with shaking. Filter. U se the area o f the prindpal peak in the chrom atogram obtained
predpitate for identification tests A, B, D and F and the with reference solution (a) (0.10 per cent);
filtrate for identification test C. — total: n o t m ore than the area o f the prin d p al peak in the
chrom atogram obtained with reference solution (a)
A. M d tin g point (2.2.14): 270 °C to 274 °C, determ ined
(0.1 p er cent);
after w ash in g the predpitate with water R and drying at
— disregard Umit. 0.5 times the area o f the principal peak in
105 °C.
the chrom atogram obtained with reference solution (a)
B. Infrared absorption spectrophotom etry (2.2.24). (0.05 per cent).
Preparation P redpitate, washed with water R and dried at
Heavy m etals (2.4.8)
105 °C.
M aximum 20 ppm.
Comparison theophylline CRS. Solvent water R.
C. T o the filtrate add 0.2 m L o f benzoyl chloride R, m ake
0.500 g complies with test H . Prepare the reference solution
alkaline with dilute sodium hydroxide solution R and shake
using 1 m L o f lead standard solution (10 ppm Pb) R.
vigorously. F ilter the predpitate, wash with 10 m L o f
T h e substance predpitates after addition o f bitjfer solution
water R, dissolve in 5 m L o f h o t ethanol (96 per cent) R and
pH 3.5 R. D ilute to 100 m L with water R, the substance
add 5 m L o f water R. A predpitate is formed, which, when
re-dissolves completely.
washed and dried at 105 °C, m d ts (2.2.14) at 248 °C to
252 °C. Water (2.5.12)
M axim um 1.5 per cent, determ ined on 0.50 g.
D. H eat about 10 m g o f the predpitate with 1.0 m L o f a
360 g/L solution o f potassium hydroxide R in a water-bath at Sulfated ash (2.4.14)
90 °C for 3 m in, then add 1.0 m L o f diazodsed sulfanUic acid M aximum 0.1 per cent, determ ined on 1.0 g.
solution R. A red colour slowly devdops. C an y out a blank
te s t
2016 Aminophylline Hydrate 1-145

A SSA Y
^ n^ v n
Etfaylenediamine
Dissolve 0.250 g in 30 m L of water R. Add 0.1 m L of
bromocresol green solution R. T itrate with 0.1 M hydrochloric 0
n>< 1 H
add until a green colour is obtained. ch3

1 m L of 0.1 M hydrochloric add is equivalent to 3.005 m g of

C2HgN2‘ E. l,3-dim ethyl-7,9-dihydro-lH-purine-2,6,8(3/i)-trione,
T h eo p h y llin e
H eat 0.200 g to constant mass in an oven at 135 °C. OH
Dissolve the residue with heating in 100 m L o f water R}
allow to cool, add 20 m L of 0.1 M silver nitrate and shake.
Add 1 mT. of bromothymol blue solution R l. Titrate with 0.1 M o
X x> I
sodium hydroxide.. ch3
1 m L of 0.1 M sodium hydroxide is equivalent to 18.02 m g o f
C 7H 8N 4O 2. F. 7-(2-hydroxyethyl)-1,3-dimethyl-3,7 -dihydro-1H -purine-
2, 6-dione (etofylline),
STO RA G E
In an airtight container, protected from light
0 pi
IM P U R IT IE S 1 '
Other detectable impurities (the following substances would, if
present at a sufficient level, be detected by one or other of X V> !
the tests in the monograph. They are limited by the general
ch3
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 G. 3,7-dimethyl-3,7-dihydro-lH-purine-2,6-dione
impurities for demonstration of compliance. See also 5.10. (theobromine).
Control of impurities in substances for pharmaceutical use): A, B, PhEur
C, D, E, F, G.

0 pt
1 ' ★ ★
Aminophylline Hydrate
lx > 1
Çlheophyïïme-eàiylenediamine Hydrate,
★ ★
*****
ch3 Ph Eur monograph 0301)

A. 1,3,7-trimethyl-3,7-dihydro-lH-purine-2,6-dione
(caffeine),
, xH 20
H2N'
1 1 >
I
ch3
HN
X L //>
C 16H 24N 10O 4PCH2O 420.4 72487-55-9
CH,
(anhydrous substance)
B. 3-methyl-3 37-dîhydro-1H-purine-236-dione3
A c tio n a n d u se
Non-selective phosphodiesterase inhibitor; treatm ent of
A J. CHO
reversible airways obstruction.

O
JjC. N NH2
P re p a r a tio n
Aminophylline Injection
CH,
Aminophylline Tablets
Prolonged-release AminopyQine Tablets
C. AT-(6-am ino-l^-dim ethyl-2,4-dioxo-l,2,3,4-
tetrahydropyrimidin-5-yl)formamide, PhEur.

D E F IN IT IO N
C o n te n t
h3c x
— theophylline (C7H8N402; 180.2): 84.0 per cent to
»V>
hn ^ n
87.4 per cent (anhydrous substance);
— ethylenediamine (C 2H 8N 2; 60.1): 13.5 per cent to
CH, 15.0 per cent (anhydrous substance).
CHARACTERS
D. N-methyl-5-(methylamino)- lH-imidazole-4-carboxamide, A p p e a ra n c e
W hite or slightly yellowish powder, sometimes granular.
 1-146 Aminophylline Hydrate 2016

S o lu b ility Injection 20 pL.

Freely soluble in water (the solution becomes cloudy through Run time 3.5 times the retention time o f theophylline.
absorption o f carbon dioxide), practically insoluble in
Relative retention W ith reference to theophylline (retention
anhydrous ethanol.
tim e = about 6 min): im purity G = about 0.6.
IDENTIFICATION System suitability: reference solution (b):
First identification B, C, E. — resolution: minimum 2.0 between the peaks due to
Second identification A , C, D, E, F. im purity G and theophylline.
Dissolve 1.0 g in 10 m L of water R and add 2 m L o f dilute Limits:
hydrochloric acid R dropwise with shaking. Filter. Use the — unspecified impurities: for each impurity, n o t m ore than the
precipitate for identification tests A, B, D and F and the area of the principal peak in the chrom atogram obtained
filtrate for identification test C. with reference solution (a) (0.10 per cent);
— total: n o t more than the area o f the principal peak in the
A. Melting point (2.2.14): 270 °C to 274 °C, determined
chrom atogram obtained with reference solution (a)
after washing the precipitate with water R and drying at
(0.1 per cent);
105 °C.
— disregard limit. 0.5 times the area o f the principal peak in
B. Tnfrared absorption spectrophotom etry (2.2.24).
the chromatogram obtained with reference solution (a)
Preparation Precipitate, washed with water R and dried at (0.05 per cent).
105 °C.
H e a v y m e ta ls (2.4.8)
Comparison theophylline CRS. M aximum 20 ppm.
C. T o the filtrate add 0.2 m L o f benzoyl chloride R, make Solvent water R.
alkaline with dilute sodium hydroxide solution R and shake
0.500 g complies with test H . Prepare the reference solution
vigorously. Filter the precipitate, wash with 10 m L o f
using 1 m L o f lead standard solution (10 ppm Pb) R.
water R, dissolve in 5 m L o f h o t ethanol (96 per cent) R and
T h e substance precipitates after addition o f buffer solution
add 5 m L o f water R. A precipitate is formed, which, when
pH 3.5 R. D ilute to 100 m L with water R; the substance
washed and dried at 105 °C, melts (2.2.14) at 248 °C to
re-dissolves completely.
252 °C.
W a te r (2.5.12)
D . H eat about 10 m g o f the precipitate with 1.0 m L o f a
3.0 per cent to 8.0 per cent, determ ined on 0.50 g.
360 g/L solution of potassium hydroxide R in a water-bath at
90 °C for 3 m in, then add 1.0 m L o f diazotised stdfanUic acid S u lfa te d a s h (2.4.14)
solution R. A red colour slowly develops. Carry out a blank M aximum 0.1 per cent, determ ined on 1.0 g.
te s t A SSA Y
E. W ater (see Tests). Ethyienediamine
F. T he precipitate gives the reaction o f xanthines (2.3.1). Dissolve 0.250 g in 30 m L o f water R. A dd 0.1 m L o f
bromocresol green solution R. T itrate with 0.1 M hydrochloric
TESTS
acid until a green colour is obtained.
A p p e a ra n c e o f so lu tio n
1 mT. o f 0.1 M hydrochloric acid is equivalent to 3.005 m g of
T he solution is not m ore opalescent than reference
suspension II (2.2.T) and n o t m ore intensely coloured than C 2H8N2.
reference solution GY6 (2.2.2, Method II). Theophylline
Dissolve 0.5 g with gentle warming in 10 m l. of carbon H eat 0.200 g to constant mass in an oven at 135 °C.
dioxide-free water R Dissolve the residue with heating in 100 m L o f water R,
allow to cool, add 20 mT. o f 0.1 M stiver nitrate and shake.
R e la te d su b s ta n c e s
A dd 1 mT. o f bromothymol blue solution R l. T itrate with 0.1 M
Liquid chrom atography (2.2.29).
sodium hydroxide.
Test solution Dissolve 50 mg o f the substance to be examined 1 mT. o f 0.1 M sodium hydroxide is equivalent to 18.02 m g of
in the mobile phase and dilute to 20.0 m L with the mobile
C rH sN ^ .
phase.
Reference solution (a) D ilute 1.0 m l, o f the test solution to STORAGE
100.0 m L w ith the mobile phase. D ilute 1.0 m l. o f this In a well-filled, airtight container, protected from light.
solution to 10.0 m L w ith the mobile phase. IMPURITIES
Reference solution (b) Dissolve 10 m g o f theobromine R Other detectable impurities (the following substances would, if
(impurity G ) in the mobile phase, add 5 m L o f the test present at a sufficient level, be detected by one or other of
solution and dilute to 100 m L with the mobile phase. D ilute the tests in the m onograph. T hey are limited by the general
5 m L o f this solution to 50 m L with the mobile phase. acceptance criterion for other/unspecified impurities and/or
Column: by the general monograph Substances for pharm aceutical use
— size. I — 0.25 m, 0 = 4 mm; (2034). It is therefore no t necessary to identify these
— stationary phase: octadecylsifyl silica gel for chromatography R impurities for dem onstration o f compliance. See also 5.10.
(7 pm). Control of impurities in substances fin- pharmaceutical use): A , B,
Mobile phase M ix 7 volumes o f acetortitrile for C, D, E, F, G.
chromatography R and 93 volumes o f a 1.36 gJL solution of
sodium acetate R containing 0.50 per cent V/V of glacial acetic
add R.
Flow rate 2.0 mL/min.
Detection Spectrophotometer at 272 nm.
2016 Amiodarone Hydrochloride 1-147

i ? Hs Amiodarone Hydrochloride *****

1Y>
★ ★
H,c' N' l V ' N
(Ph. Eur. monograph 0803) * * * * *
o^ n- ^ n
ch3
H,C

A. 1,3,7-trim ethyl-3,7-dihydro-lH-purine-2,6-dione , HQ
(caffeine), N. .CH,

C25H3oCn2N 0 3 682 19774-82-4

lV > I A ctio n a n d u se
ch3
Potassium channel blocker; class ID antiarrhythmic.
P re p a ra tio n s
B. 3-methyl-3,7-dihydro-lH-purine-2,6-dione,
Amiodarone Intravenous Infusion
Amiodarone Oral Suspension
h3c^ I B . Amiodarone Tablets
N | c
PhEir.
O ^N NH2
D E F IN IT IO N
ch3
(2-Butylben2ofuran-3-yl)[4-[2-(diethylamino)ethoxy]-3j5-
diiodophenyljmethanone hydrochloride.
C. N -(6-am ino-l,3-dim ethyl-2,4-dioxo-l,2,3,4-
tetrahydropyrimidin-5-yl)formamide, C o n te n t
98.5 per cent to 101.0 p er cent (dried substance).
CHARACTERS
H,C A p p e a ra n c e
rV s W hite or almost white, fine, crystalline powder.
H N N S olu b ility
I
CH3 Very slightly soluble in water, freely soluble in methylene
chloride, soluble in methanol, sparingly soluble in ethanol
D . N-methyl-5-(methylamino)-lH-imidazole-4-carboxamide, (96 per cent).
ID E N T IF IC A T IO N
A. Infrared absorption spectrophotometry (2.2.24).
Comparison amiodarone hydrochloride CRS.
H*C'1n\ A[ ) ==0
o ^ n ^ n B. It gives reaction (b) o f chlorides (2.3.1).
I M TESTS
CH,
A p p e a ra n c e o f so lu tio n
T h e solution is clear (2.2.1) and n o t more intensely coloured
E. 1,3-dim ethyl-7,9-dihydro-lii-purine-2,6,8(3H)-trione,
than reference solution GY5 or BY5 (2.2.2, Method II).
Dissolve 1.0 g in methanol R and dilute to 20 m L with the
OH same solvent
pH (2.2.3)
1
oIA
JL >
HA > 3.2 to 3.8.
I
ch3 Dissolve 1.0 g in carbon dioxide-free water R, heating at 80 °C,
cool and dilute to 20 m L with the same solvent
F . 7-(2-hydroxyethyl)-1,3-dimethyl-3,7-dihydro-1H -purine- Im p u rity H
2,6-dione (etofylline), Thin-layer chromatography (2.2.27). Prepare the solutions
immediately before use and keep protectedfrom bright light.

X " Test solution Dissolve 0.500 g o f the substance to be

examined in methylene chloride R and dilute to 5.0 m L with

ÍJC> I
the same solvent.
Reference solution (a) Dissolve 10.0 m g of
ch3 (2-chloroethyl)diethylamine hydrochloride R (impurity H) in
methylene chloride R and dilute to .50.0 m L with the same
G . 3,7-dim ethyl-3,7-dihydro-lii-purine-2,6-dione solvent. Dilute 2.0 m L o f the solution to 20.0 m L with
(theobromine). methylene chloride R.
PhEur
Reference solution (b) Mix 2.0 m L o f the test solution and
2.0 m L o f reference solution (a).
Plate TLC silica gel F2 5 4 plate R.
 1-148 Amiodarone Hydrochloride 2016

Mobile phase anhydrous formic add R, methanol R, methylene — disregard limit. 0.25 times the area o f the peak due to
chloride R (5:10:85 VIVIV). amiodarone in the chrom atogram obtained with the
Application 50 (iL o f the test solution and reference reference solution (0.05 per cent).
solution (a); 100 pL o f reference solution (b). Io d id e s
Development Over 2/3 o f the plate. M axim um 150 ppm.
Drying In a current o f cold air. Prepare the test and reference solutions simultaneously.
Detection Spray with potassium iodobismuthate solution R l and Solution A A dd 1.50 g o f the substance to be examined to
then with dilute hydrogen peroxide solution R', exam in e 40 m L o f water R at 80 °C and shake until completely
immediately in daylight. dissolved. Cool and dilute to 50.0 m L w ith water R.
System suitability: reference solution (b): Test solution T o 15.0 m L o f solution A add 1.0 m L o f
— the spot due to im purity H is clearly visible. 0.1 M hydrochloric acid and 1.0 mT. o f 0.05 Mpotassium
Limit: iodate. Dilute to 20.0 m L with water R. Allow to stand
— impurity H: any spot with the same Rp as the spot due to protected from light for 4 h.
im purity H in the chromatogram obtained with reference Reference solution T o 15.0 m L o f solution A add 1.0 m L of
solution (b) is n o t m ore intense th an the spot in the 0.1 M hydrochloric add, 1.0 m L of an 88.2 mg/L solution o f
chrom atogram obtained w ith reference solution (a) potassium iodide R and 1.0 m L o f 0.05 Mpotassium iodate.
(0.02 per cent). D ilute to 20.0 m L with water R. Allow to stand protected
Related substances from light for 4 h.
Liquid chromatography (2.2.29). M easure the absorbances (2.2.25) o f the solutions at 420 nm ,
Buffer solution pH 49 T o 800 mT. o f water R add 3.0 m L o f using a mixture o f 15.0 m L o f solution A and 1.0 m L o f
glacial acetic add R, adjust to p H 4.9 with dilute ammonia R l 0.1 M hydrochloric acid diluted to 20.0 m L with water R as
and dilute to 1000 m L with water R. the compensation liquid. T h e absorbance o f the test solution
is n o t greater than half the absorbance o f the reference
Test solution Dissolve 0.125 g o f the substance to be
solution.
ex a m in ed in a mixture of equal volumes of acetonitrUe R and
water R and dilute to 25.0 m L with the same mixture of H e a v y m e ta ls (2.4.8)
solvents. M axim um 20 ppm .
Reference solution Dissolve 5 m g o f amiodarone 1.0 g complies with test C. Prepare the reference solution
impurity D CRS, 5 m g of amiodarone impurity E CRS and using 2 m L o f lead standard solution (10 ppm Pb) R.
5.0 m g o f amiodarone hydrochloride CRS in methanol R and L o ss o n d ry in g (2.2.32)
dilute to 25.0 m L w ith the sam e solvent. Dilute 1.0 m L o f Maximum 0.5 per cent, determ ined on 1.000 g by drying at
the solution to 20.0 m L with a mixture o f equal volumes o f 50 °C at a pressure not exceeding 0.3 kPa for 4 h.
acetonitrile R and water R. S u lfa te d a s h (2.4.14)
Column: M aximum 0.1 per cent, determ ined on 1.0 g.
— size: I = 0.15 m , 0 = 4.6 mm;
— stationary phase, end-capped octadecylsUyl silica gel for A SSA Y
chromatography R (5 pm); Dissolve 0.600 g in a mixture o f 5.0 m L o f
— temperature: 30 °C. 0.01 M hydrochloric add and 75 m L o f ethanol (96 per cent) R.
Carry out a potentiom etric titration (2.2.20), using
Mobile phase Buffer solution p H 4.9, methanol R, acetonitrile R
0.1 M sodium hydroxide. Read the volume added between the
(30:30:40 VIVIV).
2 points o f inflexion.
Flow rate 1 mL/min.
1 mT. o f 0.1 M sodium hydroxide is equivalent to 68.18 m g of
Detection Spectrophotom eter at 240 nm . C 25H 3oCU2N 0 3 .
Injection 10 (iL.
STORAGE
Run time Twice the retention time o f am iodarone. Protected from light, at a tem perature n o t exceeding 30 °C.
Relative retention W ith reference to amiodarone (retention
time = about 24 m in): im purity A = about 0.26; IM P U R IT IE S
im purity D = about 0.29; im purity E = about 0.37; Specified impurities A, B, C , D , E, F , G, H
impurity B = about 0.49; im purity C = about 0.55;
im purity G = about 0.62; im purity F = about 0.69.
System suitability: reference solution:
— resolution: m inim um 3.5 between the peaks due to
impurities D and E.
Limits:
— impurities A , B, C, D, E, F, G: for each impurity, not
m ore than the area o f the peak due to amiodarone in the
A. (2-butyIbenzofuran-3-yl) [4-[2-(diethylamino)ethoxy]
chrom atogram obtained w ith the reference solution
phenyl] methanone,
(0.2 per cent);
— unspecified impurities: for each impurity, n o t more than
0.5 times the area o f the peak due to amiodarone in the
chrom atogram obtained w ith the reference solution
(0.10 per cent);
— total: not m ore th an 2.5 times the area of the peak due to
amiodarone in the chrom atogram obtained with the
reference solution (0.5 per cent);
2016 Amisulpride 1-149

and enantiomer ★* *
Amisulpride ★ ★
★ ★
(Ph Eur monograph 1490) *****

o\w/o
h 3c .
N
H u^
' ON and enantiomer.
B. (2-butylbenzofuran-3-yI) [4-[2-(ethyiamino)ethoxy]-3,5-
diiodophenyl] methanone, h2n och3 k CH3
and enantiomer
C 17H 27N 3O 4S 369.5 71675-85-9

Action and use

D opamine receptor antagonist; neuroleptic.
Preparations
Amisulpride Oral Solution
C. (2-butylbenzofuran-3-yl) [4-[2-(diethyiamino)ethoxy]-3- Amisulpride Tablets
iodophenyl] methanone,
P h E tr ______________________________________________________________

DEFINITION
4-Amino-N- [ [(2i?5)-1-ethylpyrrolidin-2-yl] methyl] -5-
(ethylsulfonyl)-2-methoxybenzamide.
Content
99.0 per cent to 101.0 per cent (dried substance).
CHARACTERS
D . (2-butyIbenzofuran-3-yl)(4-hydroxy-3,5- Appearance
diiodophenyl)methanone, W hite or almost white, crystalline powder.
Solubility
H,c Practically insoluble in water, freely soluble in methylene
chloride, sparingly soluble in anhydrous ethanol.
mp
About 126 °C.
IDENTIFICATION
Infrared absorption spectrophotometry (2.2.24).
E. (2-butyIbenzofuran-3-yl) (4-hydroxyphenyl)methanone,
Comparison amisulpride CRS.
TESTS
Appearance o f solution
T h e solution is clear (2.2.1) and not more intensely coloured
than reference solution Y6 (2.2.2, Method II).
Dissolve 1.0 g in 3 m L of a mixture of 1 volume of acetic
acid R and 4 volumes of water Rs and dilute to 20 m L with
F. (2-butylbenzofuran-3-yl) (4-hydroxy-3-iodophenyl) water R.
methanone, Impurity A
Thin-layer chromatography (2.2.27).
Test solution Dissolve 0.20 g of th e substance to be examined
in methanol R and dilute to 10 m L with the same solvent.
Reference solution (a) Dissolve 5 mg of sulpiride
impurity A CRS (amisulpride impurity A) in methanol R and
dilute to 25 mT. with the same solvent. Dilute 2 m L of the
solution to 20 m L with methanol R.
Reference solution (b) Dilute 1 m L of the test solution to
10 m L with reference solution (a).
G. [4-[2-(diethylamino)ethoxy]-3,5-diiodophenyl] [2-[(1 jR5)- Plate TLC silica gel G plate R.
1-methoxybutyl] benzofuran-3-yl] methanone,
Mobile phase 50 per cent VIV solution of concentrated
ammonia R, anhydrous ethanol R, di-isopropyl ether R
,c h 3
r
,N_XH3
(10:25:65 VIVIV); use the upper layer obtained after shaking
the mixture.
Application 10 (iL.
H . 2-chloro-N,N-diethylethanamine (2-chlorotriethylamine, Development Over 2/3 of the plate.
(2-chloroethyl)diethyIamine). Drying In air.
 1-150 Amisulpride 2016

Detection Spray with ninhydrin solution R and heat at — reporting threshold: 0.05 per cent.
100-105 °C for 15 min. H ea v y m e ta ls (2.4.8)
Retardation factors Im purity A = about 0.2; M axim um 10 ppm.
amisulpride = about 0.5. Dissolve 4.0 g by gently heating in 5 m L o f dilute acetic
System suitability T he chrom atogram obtained with reference add R. Allow to cool and dilute to 20 m L w ith water R.
solution (b) shows 2 clearly separated spots. 12 m L o f the solution complies with test A. Prepare the
Limit. reference solution using lead standard solution (2 ppm Pb) R.
— impurity A: any spot due to impurity A is not more L o ss o n d ry in g (2.2.32)
intense than the corresponding spot in the chromatogram Maximum 0.5 per cent, determ ined on 1.000 g by drying in
obtained with reference solution (a) (0.1 per cent). an oven at 105 °C for 3 h.
R e la te d su b s ta n c e s S u lfa te d a s h (2.4.14)
liq u id chromatography (2.2.29). M axim um 0.1 per cent, determ ined on 1.0 g.
Solvent mixture acetomtrUe R l, methanol R2, mobile phase A A SSA Y
(12:16:72 V/V/V).
Dissolve 0.300 g with shaking in a mixture o f 5 m L o f acetic
Test solution Dissolve 0.10 g o f the substance to be examined anhydride R and 50 m L o f anhydrous acetic acid R. T itrate
in 16 m L o f methanol R2, add 12 m L of acetomtrUe R l and w ith 0.1 M perchloric add, determining the end-point
dilute to 100.0 m L with mobile phase A. potentiometrically (2.2.20).
Reference solution (a) Dilute 1.0 m L o f the test solution to 1 m L o f 0.1 M perchloric add is equivalent to 36.95 m g o f
100.0 m L with the solvent mixture. Dilute 1.0 m L o f this C 17H 2 7N 3O 4 S .
solution to 10.0 m L with the solvent mixture.
IM P U R IT IE S
Reference solution (b) Dissolve the contents o f a vial of
Spedfied impurities A
amisulpride for system suitabUity CRS (containing impurity B)
in 1.0 m L of the solvent mixture. Other detectable impurities (the following substances w ould, if
present at a sufficient level, be detected by one or other o f
Column:
the tests in the monograph. They are lim ited by th e general
— size: I = 0.25 m , 0 = 4.6 mm ;
acceptance criterion for other/unspecified impurities and/or
— stationary phase:, base-deactivated oaylsüyl sUica gel for
by the general monograph Substances for pharmaceutical use
chromatography R (5 nm);
— temperature: 40 °C.
(2034). It is therefore not necessary to identify these
impurities for demonstration o f compliance. See also 5.10.
Mobile phase:
Control of impurities in substances for pharmaceutical use): B, C,
— mobile phase A: dissolve 0.7 g o f sodium octanesulfonate R
D, E, F, G3 H.
in 930 m L o f water R , add 45.0 m l. o f a 5 per cent V/V
solution o f dilute sulfuric acid R, adjust to p H 2.3 with a
5 per cent V/V solution o f dilute sulfuric add R, and dilute h2n
to 1000 m L with water R; H N and enantiomer
— mobile phase B: methanol R2;
CH3
— mobile phase C: acetomtrUe R l :;

A. [(1RS) -1 -ethylpyrrolidin-2-yI] m ethanam ine,

Time Mobile phase A Mobile phase B Mobile phase C
(min) (per cent V/V/V) (per cent V/V/V) (percent V/V/V)
o
0 -1 8 72 16 12

18 - 35 72 50 16 38 12
\
and enantiomer

Flow rate 1.5 mL/min.

Detection Spectrophotom eter a t 225 nm . B . 4 - am in o - A/-[[(2i?«S)-1 -ethylpyrrolidin-2-yl] methyl]-5-
Injection 10 ¿iL. (ethylsulfonyl)-2-hydroxybenzamide,
Identification of impurities U se the chromatogram obtained
with reference solution (b) to identify the peak due to
im purity B.
and enantiomer
Relative retention W ith reference to amisulpride (retention
tim e = about 17 min): im purity B = about 1.1. H,N
System suitability: reference solution (b):
— peak-to-vaUey ratio: minimum 2.0, where Hp = height
above the baseline o f the peak due to impurity B and C . 4 - am ino - iV- [[(2ÆS)-1 -ethylpyirolidin-2-yl]methyl] -5-iodo-
Hv = height above the baseline o f the lowest point o f the 2-methoxybenzamide,
curve separating this peak from the peak due to
amisulpride.
Calculation of percentage contents Use the concentration of
amisulpride in reference solution (a).
Limits:
— unspecified impurities: for each impurity, maximum
0.10 per cent;
— total: m aximum 0.3 per cent;
2016 Amitriptyline Hydrochloride 1-151

o o Content
*o
h 3c N -^ O 99.0 per cent to 101.0 per cent (dried substance).
and enantiomer
H H N
h2n och3
CHARACTERS
CH3 Appearance
W hite or almost white powder or colourless crystals.
D . 4-am ino-N - [ [ (2RS)-1 -ethylpyrrolidin-2-yl] methyl] -2- Solubility
methoxy-5-(methylsulfonyl)benzamide, Freely soluble in water, in ethanol (96 per cent) and in
methylene chloride.
o o
IDENTIFICATION

H2N.XX. ^ OCH3
A. Infrared absorption spectrophotometry (2.2.24).
Comparison amitriptyline hydrochloride CRS.
B. 20 m g gives reaction (a) of chlorides (2.3.1).
E. 4-amino-5-(ethylsulfonyl)-2-methoxybenzoic acid.
TESTS
Appearance of solution
T he solution is clear (2.2.1) and not more intensely coloured
na QN
H and enantiomer than reference solution B7 (2.2.2, Method II).
I '0
h2n 0CH3 I Dissolve 1.25 g in water R and dilute to 25 m L with the
ch3
same solvent

F. 4-am ino-N- [[(2i?S)-1-ethyl-1-oxidopyrrolidin-2-yl] Acidity or alkalinity

methyl]-5-(ethylsulfonyl)-2-methoxybenzamide, Dissolve 0.20 g in carbon dioxide-free water R and dilute to
10 m L with the same solvent. Add 0.1 m L of methyl red
solution R and 0.2 m L o f 0.01 M sodium hydroxide.
o o
❖'! T he solution is yellow. Add 0.4 m L of 0.01 M hydrochloric
h 3c s ^CH3
N acid. T he solution is red.
H H
and enantiomer Related substances
H,N 0CH3
Liquid chromatography (2.2.29).
G . 4-amincHN-[(3i?S)-l-ethylpiperidin-3-yl]-5-(ethylsulfonyl)- Test solution Dissolve 50.0 mg o f the substance to be
2-methoxybenzamide, examined in the mobile phase and dilute to 50.0 m L with
the mobile phase.
Reference solution (a) Dissolve 5.0 m g of dibenzosuberone CRS
(impurity A) and 5.0 m g of cydobenzaprine hydrochloride CRS
and enantiomer (impurity B) in 5.0 m L o f the test solution and dilute to
H!N ' ^ A o c g3H>H ^ 100.0 m L with the mobile phase.
CH,
Reference solution (b) Dilute 1.0 m L of reference solution (a)
to 50.0 m L with the mobile phase.
H . 4-am ino-N - [ [(2i?S)-l -ethylpyrrolidin-2-yl] methyl] -5-
(ethylsulfonyl)-2-methoxy-AT-methylbenzamide. Column:
— sizer. I = 0.15 m, 0 = 4.6 mm;
PhEur
— stationary phase: end-capped polar-embedded octadecylsUyl
amorphous organosUica polymer R (5 pm);
— temperature: 40 °C.
Mobile phase Mix 35 volumes of acetonitrile R and 65 volumes
★ ★
Amitriptyline Hydrochloride ★ ★ o f a 5.23 g/L solution o f dipotassium hydrogen phosphate R
previously adjusted to p H 7.0 with phosphoric add R.
(Ph Eur monograph 0464) *****
Flow rate 1.2 mL/min.
Detection Spectrophotometer at 220 nm.
Injection 10 |iL.