UV SPECTROPHOTOMETRIC

METHOD DEVELOPMENT AND

VALIDATION FOR ESTIMATION OF
ANTHELMINTIC DRUG

A Thesis submitted to
Rajiv Gandhi Proudyogiki Vishwavidyalaya Bhopal (M.P.) In the
partial fulfillment of the requirement for the Degree of

In
Pharmaceutical chemistry
Submitted By Supervised By

Mr. Subodh Kumar Mr. Prashant Vinode

(Asso. Professor)

(Enrolment No. 0602PY22MP13)

SAGAR INSTITUTE OF PHARMACEUTICAL

SCIENCES

NH-26, NARSINGHPUR ROAD, SIRONJA, SAGAR (M.P)

470228

2023-2024
 SAGAR INSTITUTE OF PHARMACEUTICAL
SCIENCES, SAGAR (M.P.)
Approved by AICTE,PCI New Delhi, DTE Bhopal and Govt. of
M.P.(Affiliated to Rajiv Gandhi Proudyogiki Vishwavidyalaya Bhopal)

EXAMINER CERTIFICATE

This is to certified that the M. Pharm. Research Work and Colloquium final thesis

viva of Mr./Ms enrollment no. _

of M. Pharm. IV Sem. MPC403P is held on . The external examiner appointed

by RGPV, Bhopal forthesis viva is _ examine the candidate.

Name and Signature Name and Signature

Internal examiner External examiner

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Analytical absorption spectroscopy in ultraviolet and visible region of electromagnetic spectrum

is widely used for quantitative analysis in the field of pharmaceutical and biomedical analysis.
Although, there is steady increase in the use of other advance physical method of analysis of
analysis such as GLC, HPLC, HPTLC, and AAS is active, highest specificity, precision and
accuracy and one may be tempted to suggest the use of simple colorimetric methods based on
color reaction has diminished in importance and application. However, it is relevant to note that
even the latest edition to various compendia contains identification tests for different drug
substances based on the color reaction of one or more specific functional groups present in drug
molecule. Selective chemical reactions provide the method to manipulate a sample to reveal the
information desired. They also provide the means to increase the response of an analyte to
functional group or increase the selectivity of analysis by targeting certain components of the
sample to response to the selected reagents. The monochromator is used to disperse the radiation
according to the wavelength. The essential elements of a monochromator are an entrance slip,
a dispersing element and an exit slit. The entrance slit sharply defines the incoming beam of
heterochromatic radiation. Quartz and fused silica prism which are transparent throughout the
entire UV range are widely used in UV spectrophotometers. The precision of an analytical
method is the degree of agreement among individual test results when the method is repeated to
multiple samplings of a homogeneous sample. The precision of an analytical procedure is usually
expressed as the standard deviation or relative standard deviation (coefficient of variation) of a
series of measurements. Retrospective validation is the analysis of accumulated results from past
production batches manufactured under identical conditions to assess the consistency of a
process. It includes trend analysis on test results and a close examination of all recorded process
deviations and their relevant investigation reports. This type of validation is applied to established
products who are considered stable where prospective validation programs cannot be justified.
Working standard solution of mebendazole was made from the stock solution. The drug
mebendazole 25mg of IP transfer to 50ml volumetric flask add 10ml of formic acid and dissolve,
dilute with methanol to volume and mix. The solvent system to give concentration rate of
2,4,6,8,10 and 12µg/ml. The mebendazole. Accurately weighed 100 mg of pure Mebendazole
drug and transferred into a 100 ml volumetric flask. A small quantity of Phosphate buffer 6.8 was
added to ensure complete solubilization of the drug, and finally, volume was made up to the mark
with the same buffer solution. The solution was sonicated for 5 min to dissolve and remove air
completely.
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 SAGAR INSTITUTE OF PHARMACEUTICAL
SCIENCES, SAGAR (M.P.)
Approved by AICTE,PCI New Delhi, DTE Bhopal and Govt. of
M.P.(Affiliated to Rajiv Gandhi Proudyogiki Vishwavidyalaya
Bhopal)

PLAGIARISM CERTIFICATE

This is to certified that Thesis entitled_____________________________

___________________________________________________________________________________________________
___________________________________________________________________________________________________
____________________________________ were plagiarized by student through software and
the matched result was found to be ____%. The thesis was healthy and does not
contain any copyright material. The thesis was prepared and submitted under
my supervision.

Student Name Internal Examiner

Mr. Subodh Kumar Mr. Prashant Vinode

(Asso.Professor)
 SAGAR INSTITUTE OF PHARMACEUTICAL
SCIENCES, SAGAR (M.P.)
Approved by AICTE,PCI New Delhi, DTE Bhopal and Govt. of M.P.
(Affiliated to Rajiv Gandhi Proudyogiki Vishwavidyalaya Bhopal)

Certificate
This is to certify that the thesis entitled “UV SPECROPHOTOMETRIC
METHOD DEVELOPMENT AND VALIDATION FOR ESTIMATION
OF ANTHELMINTIC DRUG” submitted in partial fulfillment of the
requirement for the degree of Master of Pharmacy in Pharmaceutical
Chemistry to Sagar Institute of Pharmaceutical Sciences, Sagar M.P.,
affiliated to Rajiv Gandhi Proudyogiki Vishwavidyalaya, Bhopal (M.P)
and work carried out by Mr. Subodh Kumar, Enrollment No.
0602PY22MP13 during the academic session 2023-2024. His work is
satisfactory and not submitted anywhere for the award of any other
degree.

I hereby forwarded his Thesis..

SUPERVISED BY

Mr. Prashant Vinode

(Asso. Professor)

Sagar Institute of Pharmaceutical Sciences, Sagar M.P.

 SAGAR INSTITUTE OF PHARMACEUTICALSCIENCES,
SAGAR (M.P.)
Approved by AICTE, PCI New Delhi, DTE Bhopal and Govt. of M.P.
(Affiliated to Rajiv Gandhi Proudyogiki Vishwavidyalaya Bhopal)

Forwarding Letter
This is to certify that the thesis entitled “UV
SPECROPHOTOMETRIC METHOD DEVELOPMENT AND VALIDATION
FOR ESTIMATION OF ANTHELMINTIC DRUG” submitted in partial
fulfillment of the requirement for the degree of Master of Pharmacy
in Pharmaceutical Chemistry from Sagar Institute of Pharmaceutical
Sciences, Sagar and work carried out by Mr. Subodh Kumar,
Enrollment No. 0602PY22MP13 under the guidance and supervision of
Mr. Prashant Vinode during theacademic session 2023-2024.
I hereby forwarded his thesis..

Forwarded by

Mr. Prashant Vinode (Asso. Professor)

Sagar Institute of Pharmaceutical Sciences, Sagar M.P.

 SAGAR INSTITUTE OF PHARMACEUTICAL
SCIENCES, SAGAR (M.P.)
Approved by AICTE, PCI New Delhi, DTE Bhopal and Govt. of M.P.
(Affiliated to Rajiv Gandhi Proudyogiki Vishwavidyalaya Bhopal)

Declaration

I hereby declare that the work incorporated in the thesis

entitled “UV SPECROPHOTOMETRIC METHOD DEVELOPMENT
AND VALIDATION FOR ESTIMATION OF ANTHELMINTIC DRUG”
embodies my own work except the guidance and suggestions received
from my supervisor Mr. Prashant Vinode, Sagar Institute of
Pharmaceutical Sciences, Sagar (M.P.).
I further declare that to the best of my knowledge the thesis
does not contain any part of any work which neither has been
submitted for the award of degree in any University not has been
published anywhere without proper citation.
I also declare that a check for plagiarism has been carried out on
this dissertation and is found within the acceptable limit and report of
which is enclosed herewith.

Supervised By Submitted By
Mr. Prashant Vinode Mr. Subodh Kumar
(Asso. Professor) (0602PY22MP13)

Forwarded by
Dr. Yuvraj Singh Dangi
(Principal)
 ACKNOWLEDGEMENT
To acknowledge one’s own dept is not an easy task. I feel deeply
obliged to express my profound gratitude and indebtedness to my diligent
project guide Mr. Prashant vinode (Associate Professor, SIPS, Sagar), his
ever inspiring guidance, keen interest; patient supervision and optimism
helped me to solve various problems encountered during the course of this
work. Her earnest desire for my every success will always be my pride and
my devotion and respect for her will be forever.

I express my feelings for Dr. Y. S. Dangi (Principal, SIPS, Sagar)

bottom from my heart, for his support, guidance and encouragements during
the journey for this path.

I express my regards to my parents for their love, care and whole

hearted support all through.

I heartly express my sincere thanks to my all teachers of SIPS, for their

support including Dr. Varsha Kashaw, Mr. Mukesh Patel, Mr. Akash Sharma,
and thanks to my seniors Mr. Luvkush Vishwakarma and my friend Mr.
Romit Jain for ever encouragement.

Above all, I am greatly indebted to the almighty, for showering with

his/her infinite bounties graces and mercies upon me and for being my
constant companion.

Date: Mr. Subodh Kumar

Place: - SAGAR 0602PY22MP13
 CONTENTS
PAGE
S.NO. TITLES
NO.

1. Introduction 01-26

2. Review of literature 27-31

3. Research envisaged & Plan 32-33

of work
4. Drug profile 34-35

5. Experimental work 36-38

6. Result and Discussion 39-51

7. Summary and Conclusion 52-53

8. References 54-59
 CHAPTER -1
INTRODUCTION
1. INTRODUCTION:

1.1 Anthelmintic

Anthelmintic Drugs-used against invasion of parasitic worms (Helminthiasis) Roundworms,

pinworms, whipworms, hookworms and tapeworms. Broad spectrum anthelmintics are

effective against parasitic flat worms and nematodes. However, the majority of drugs are more

limited in their action e.g., praziquantel, a drug used in the treatment of schistosomiasis and

thought to act by disrupting calcium homeostasis, has no activity against nematodes.

Anthelmintics are drugs that are used to treat infections with parasitic worms. This includes

both flat worms, e.g., flukes and tapeworms and round worms, i.e., nematodes. It is extremely

hard to eradicate helminthiases because of the close association between these diseases and

poverty. They are of huge importance for human tropical medicine and for veterinary

medicine. Helminth infections resulting to diseases such as ascariasis, hookworm infection

and schistosomiasis constitute the bulk of the 13 diseases classified as neglected tropical

diseases (NTDs) by the WHO. Despite the prevalence of parasitic worms, anthelmintic drug

discovery is the poor relation of the pharmaceutical industry. The search for novel

anthelmintics has traditionally involved two approaches, the empirical and the selective

methods. The simple reason is that the nations which suffer most from these tropical diseases

have little money to invest in drug discovery or therapy.

Helminth infections are one of the most prevalent diseases in developing and developed

countries. Globally, an estimated 2 billion people are infected by intestinal nematodes.

Anthelmintic drugs are used for the control of parasitic infection caused by helminths. The

demand for new and effective anthelmintic is immense, as the chemically drugs currently

employed in the control of helminth is expensive and most of them lose their efficacy in 20

Sagar Institute of Pharmaceutical Sciences, Sagar - 470 228 (M.P.) 1

years due to the problem of resistance. It is extremely hard to eradicate helminthiases because

of the close association between these diseases and poverty. The clinical development of

these common and ubiquitous infections is such that they are generally neglected until they

become manifest. They are more frequent in hot climates and in places with poor sanitary

conditions, the presence of large water tanks and carriers of parasites, and contaminated food

and water. This does not mean, however, that good economic conditions constitute a

complete safeguard against such infections. Moreover, individuals from more affluent

countries might well acquire such infections during travel to more endemic regions. Until

such time as effective vaccines can be discovered, antihelminthic chemotherapy is the only

effective, practical, and inexpensive way of keeping such infections under control. Helminth

infections resulting to diseases such as ascariasis, hookworm infection and schistosomiasis

constitute the bulk of the 13 diseases classified as neglected tropical diseases (NTDs) by the

WHO. The search for novel anthelmintics has traditionally involved two approaches, the

empirical and the selective methods. The empirical approach involves the screening of large

numbers of chemicals, quite unrelated to each other, possessing no known anthelmintic

activity, and screened in the hope that one or more of them may exhibit sufficient activity to

constitute a chemical lead. This method is most commonly used in large-scale drug

development programs. The selective approach involves biological investigation of the

activity of chemicals allied structurally to those known to possess activity against a particular

organism. The major objective of this approach is increased activity or decreased toxicity

through chemical modification of the parent compound. This approach has recently been used

in a drug development program funded by a Primate Foundation.

Sagar Institute of Pharmaceutical Sciences, Sagar - 470 228 (M.P.) 2

2. Mebendazole

Mebendazole(MBZ) is a broad spectrum anthelmintic drug of the benzimidazole class.

Chemically, MBZ is methyl-5 - benzoyl benzimidazole - 2 - carbamate. It is available in

tablets, syrup, or suspension dosage forms [1-2] . Mebendazole is commonly used to treat

pinworm, whipworm, roundworm, and hookworm infections. It has poor aqueous solubility

and oral bioavailability [3-4] . It causes many adverse effects such as anemia and liver

damage in high doses. It is a well-known anthelmintic drug, but due to its poor aqueous

solubility, low bioavailability, and high first-pass metabolism, it has not achieved the

therapeutic efficacy of the drug [5-7].Methods for the determination of Mebendazole

drug in the pharmaceutical formulation alone or combination with other drugs. In this

study, efforts were made to develop a simple, easy and economical UV spectrophotometric

method using suitable solvent for the determination of Mebendazole in the raw materials as

well as in the marketed dosage formulations. The International Conference on

Harmonization (ICH) guidelines under section Q2 (R1) was used to validate the developed

method [8-10].

It is BCS class II drug having low aqueous solubility (71.3 mg/L) and High permeability

(Log P= 2.8) ultimately leads to variable absorption of mebendazole. It undergo extensive

hepatic first pass metabolism. It has very low bioavailability (5-10%) and maximum amount

of drug is protein bound (90-95%).

Analysis of the drug is the most important aspect of formulation development. A suitable

method is essential for the estimation of bulk drug, of the drug in formulation, in dissolution

studies and in biological samples. Chemically Mebendazole is methyl (5-benzoyl-

1Hbenzimidazol-2yl) carbamate. It is white to slightly yellow, amorphous powder, almost

odourless. It is practically insoluble in water, alcohol, methylene chloride, ether, chloroform

and in dilute mineral acids whereas freely soluble in formic acids. It irreversibly block

glucose uptake in susceptible helminthes, thereby depleting glycogen stored in parasites.

Sagar Institute of Pharmaceutical Sciences, Sagar - 470 228 (M.P.) 3

It is official in Chin., Eur., Int., US, British, Indian, and Viet. pharmacopoeia. It is available

as chewable tablet, suspension and sachets in market. It is given as 100 mg twice daily for 3

consecutive days. Repeated after 2-3 weeks if needed (to kill ova developed later). There are

many reported methods for analysis of Mebendazole either alone or in combination with

other drugs in pharmaceutical dosage forms or individually in biological fluids. The

quantification of Mebendazole in human plasma by LC-MS has been reported. The

estimation of Mebendazole in bulk and in tablet dosage form was done by UV and RP-HPLC
method in alone or combination with forced degradation studies. There is a need for a

simple, rapid, cost effective and reproducible method for development of Mebendazole.

Therefore objective of the study was to develop a simple, accurate, precise, cost effective

and reproducible UV-Visible method for estimation of mebendazole as per International

Conference on Harmonization (ICH) guidelines Q2(R1).

2.1 MECHANISM OF ACTION:

Mebendazole acts by inhibiting the production of microtubules via binding to colchicines

bindingsite of β-tubulin and thereby blocking polymerization of tubulin dimers in the

intestinal cells of parasites.[11]. As a consequence, glucose uptake, and the digestive and

reproductive capacities parasites are interrupted, resulting in immobilization, hindrance of

egg production and death of the helminthes. Mebendazole is poorly absorbed in the digestive

tract making it an effective medication for managing intestinal helminthic infections with

very few side effects. There is a possibility for the development of resistance to

mebendazole. The mechanism of resistance to benzimidazole is most likely due to changes in

β-tubulin protein, which decreases the binding of mebendazole to β-tubulin.[12].

2.2 ADMINISTRATION:

Mebendazole is administered orally without regard to meals. It must be chewed completely

before swallowing. For patients who have difficulty taking the tablet, it can be placed in a

Sagar Institute of Pharmaceutical Sciences, Sagar - 470 228 (M.P.) 4

spoon and mixed with 2 to 3 ml of drinking water using a dosing syringe.

The pill absorbs the water and turns into a soft mass with semi-solid consistency, which is

easily swallowable.

Dosing of mebendazole for common FDA indications are listed below:

 Roundworm (Ascarislumbricoides): 100 mg twice daily (morning and night) for

threeconsecutive days.

 Hookworm (Ancylostomaduodenale): 100 mg twice daily (morning and night) for

threeconsecutive days.

 Whipworm (Trichuristrichiura): 100 mg twice daily (morning and night) for three
consecutive days.

 Pinworm (Enterobiusvermicularis): 100-mg single oral dose.

If the patient does not achieve satisfactory results after three weeks of medication, then a

second course of therapy is recommended. Dosing of mebendazole for common non-FDA

approved indications are listed below:

 Capillariasis Infection: 200 mg to be administered orally twice daily for 20 days[13][14].

 Cestodes Infection: 300 mg to be administered orally twice daily for 3 to 6 days[15].

 Filariasis: 300 mg to be taken orally daily for 28 to 45 days[16].

2.3 ADVERSE EFFECT:

 Loss of appetite

 Abdominal pain

 Diarrhea

 Nausea

 Vomiting

 Headache

 Tinnitus

Sagar Institute of Pharmaceutical Sciences, Sagar - 470 228 (M.P.) 5

Mebendazole toxicity is usually limited to gastrointestinal irritation, but there are reports of

other serious side effects including neutropenia (including agranulocytosis) and/or

thrombocytopenia, particularly in patients who have received higher dosages or had a more

prolonged treatment course than usually recommended [18].

2.4 CONTRAINDICATION:

Mebendazole is contraindicated in a person with documented hypersensitivity to

mebendazole orthe excipients. Mebendazole is contraindicated in children below the age of 1

year for the mass treatment of single or mixed gastrointestinal infestations because of the risk

of convulsion, which has been reported during postmarketing use. There is limited studied in

children below the age of

2 years. Clinicians must use the drug with attention in patients with hepatic disease or

dysfunction since the liver metabolizes the drug by the CYP450 system. Also, it should be

used carefully in a patient with biliary obstruction as the medication gets extensively

expelled via the biliary system. Avoid concomitant use of mebendazole and metronidazole

as there is a higherrisk of Stevens-Johnson syndrome/toxic epidermal necrolysis[19].

The FDA classified mebendazole as a category C drug, which states either studies in animals

have shown adverse outcomes on the fetus, and there are no available verified studies in

women. In recent studies, reports about firsttrimester exposure to mebendazole are limited;

however, researchers have not observed an increased incidence of congenital defects, while

used during the second or third trimester.[20][21] Drugs should only be an option if the

likely advantage justifies the possible risk to the fetus. Mebendazole is present in breast milk.

In a limited case series report using mebendazole during lactation, no adverse outcomes

associated with the drug occurred in nursing infants.[22] Mebendazole is considered

compatible during breastfeeding with the latest studies.

Sagar Institute of Pharmaceutical Sciences, Sagar - 470 228 (M.P.) 6

2.5 MONITRING:

Mebendazole efficacy is observable from improvement in symptoms of helminthic

infections. Periodic assessment of hematopoietic and hepatic functions is advisable during

prolonged therapy. There have been reports of neutropenia and agranulocytosis with

mebendazole use at higher doses and for more prolonged durations treatment of helminth

infections. Elderly patients and patients with comorbid conditions like liver impairment

and/or end-stage renal disease require close monitoring. It is also advisable to check for

helminth ova in feces within 3 to 4 weeks following the initial therapy of mebendazole.

2.6 TOXICITY:

In the state of overdose, gastrointestinal symptoms (e.g., nausea, diarrhea, vomiting, and

abdominal pain) may occur. Severe toxicity is not typically an issue. In instances of toxicity,

induce vomiting and purging using activated charcoal if recent ingestion has occurred, and

only if the patient can protect their airway. There is no specifiantidote for mebendazole

overdose.[23]

Treatment is generally supportive. Use fluids and electrolytes in symptomatic patients who

develop significant diarrhea and/or vomiting.

2.7 USES:

 Pinworm

 Whipworm

 Roundworm

 Hookworm infections

Sagar Institute of Pharmaceutical Sciences, Sagar - 470 228 (M.P.) 7

3. UV-VISIBLE SPECTROSCOPY

The technique of UV-visible spectrophotometry is one of the most frequently employed in

pharmaceutical analysis. It involves the measurement of the amount of ultraviolet (190-

380nm) or visible (380-800nm) radiation absorbed by a substance in solution. Instruments

which measure the ratio,or a function of the ratio,of the intensity of two beams of light in

UV-visible region,are called UV-visible spectrophotometer[24].

A spectrophotometer or a photometer that can measure the intensity of light as a function of

its wavelength. Single beam and double beam are the two major classes of

spectrophotometer.The relative light intensity of the beam measured by the single beam

spectrophotometer before and after a test sample is inserted and the double beam

spectrometer competes the light intensity between its two light paths, the relative light

intensity of the produced beam before and after a test sample is the one path and the

instruments are easier and more stable whereas the single beam instruments are optically

simpler and more compact, which can also have larger dynamic range. Linear range of

absorption and spectral bandwidth measurement are the important features of

spectrophotometers[25].

The amount of absorption depends on the wavelengths of radiation and the structure of the

compound. The absorption of radiation is due to the subtraction of energy from the radition

beam when electrons in orbital of lower energy are excited into orbital of higher energy.

Since this is electron transition phenomenon. UV is sometimes called electronic

spectroscopy. In case of organic molecules,the electronic transition could be ascribed to aπ

electrons transition from the ground state to an excited state ((π* or n*). There are four types

of absorption bands that occur due to the electronic transition of a molecule:

A. R-Bands: Compound with C=O or NO2groups, λ max<100

B. K-Bands: Compound in conjugated system, λ max>10,000

C. B-Bands: Due to aromatic and hetro aromatic (Benzenoid bands) system λ max<2000

Sagar Institute of Pharmaceutical Sciences, Sagar - 470 228 (M.P.) 8

D. E-Bands: Compound in aromatic system,(Ethylene bands),λmax2000 to 14,000

3.1 Principle of UV-Visible Spectroscopy:

The Principle of UV-Visible Spectroscopy is based on the absorption of ultraviolet

light or visible light by chemical compounds, which results in the production of distinct
spectra. Spectroscopy is based on the interaction between light and matter. When the matter
absorbs the light, it undergoes excitation and de-excitation, resulting in the production of a
spectrum. When matter absorbs ultraviolet radiation, the electrons present in it undergo
excitation. This causes them to jump from a ground state (an energy state with a relatively
small amount of energy associated with it) to an excited state (an energy state with a
relatively large amount of energy associated with it). It is important to note that the
difference in the energies of the ground state and the excited state of the electron is always
equal to the amount of ultraviolet radiation or visible radiation absorbed by
it.Instrumentation of a UV-Visible Spectrophotometer
The principle of measurement for UV-Visible spectroscopy or UV-Visible

spectrophotometer is relatively straightforward and consists of a light source, a wavelength

dispersive element sample, and detector.

Fig.1 Instrumentation of UV- Visible Spectrophotometer

Sagar Institute of Pharmaceutical Sciences, Sagar - 470 228 (M.P.) 9

3.2 Different types of spectrophotometers:

Single Beam Spectrophotometer:

Single beam spectrophotometer is an analytical instrument in which all the light waves

coming from the light source passes through the sample. Therefore, the measurements are

taken as the intensity of light before and after the light pass through the sample. These single

beam spectrophotometers are more compact and optically simpler than double beam

spectrophotometers. And also these instruments are less expensive. The sensitivity of

detection of the light beam after it passes through the sample is high since it uses a non-split

light beam (therefore, high energy exists throughout). Single beam spectrophotometers are

available in analysis at visible and ultraviolet wavelength ranges. single beam

spectrophotometer measures the concentration of an analyze in a sample by measuring the

amount of light absorbed by that analyst. Here, the Beer Lambert Law comes into operation.

This law states that the concentrationof an analyst is directly proportional to the absorbance.

Double Beam Spectrophotometer:

Double beam spectrophotometer is an analytical instrument in which the light beam coming

from the light source splits into two fractions. One fraction acts as the reference (the

reference beam) while the other fraction passes through the sample (sample beam). As a

result, the reference beam does not pass through the sample.

The Pathway of Light Beam in a Double Beam Spectrophotometer

The sample beam can measure the absorbance of the sample. The reference beam can

measure the absorption (the sample beam can be compared with the reference beam).

Therefore, the absorption is the ratio between the sample beam (after passing through

the sample) and a reference beam. A spectrophotometer has a monochromator that isolates

the desired wavelengths from a light beam.

Sagar Institute of Pharmaceutical Sciences, Sagar - 470 228 (M.P.) 10

The reference beam and sample beam recombine before moving to the monochromator.

Consequently, this avoids or compensate the electronic and mechanical effects on both

sample and reference beams, equally.

3.3 Applications:

1. Detection of Impurities:

 It is one of the best methods for determination of impurities in organic molecules.

 Additional peaks can be observed due to impurities in the sample and it can be compared

with that of standard raw material.

 By also measuring the absorbance at specific wavelength, the impurities can be detected.

2. Structure elucidation of organic compounds

 It is useful in the structure elucidation of organic molecules, such as in detecting the

presence or absence of unsaturation, the presence of hetero atoms.

3. UV absorption spectroscopy can be used for the quantitative determination of compounds

that absorb UV radiation.

4. UV absorption spectroscopy can characterize those types of compounds which absorbs

UV radiation thus used in qualitative determination of compounds. Identification is done by

comparing the absorption spectrum with the spectra of known compounds.

5. This technique is used to detect the presence or absence of functional group in

the compound. Absence of a band at particular wavelength regarded as an evidence for

absence of particular group.

6. Kinetics of reaction can also be studied using UV spectroscopy. The UV radiation is

passed through the reaction cell and the absorbance changes can be observed.

7. Many drugs are either in the form of raw material or in the form of formulation. They can

Sagar Institute of Pharmaceutical Sciences, Sagar - 470 228 (M.P.) 11

be assayed by making a suitable solution of the drug in a solvent and measuring the

absorbance at specific wavelength.

8. Molecular weights of compounds can be measured spectrophotometrically by

preparingthe suitable derivatives of these compounds.

3.4 Advantages of UV-Visible spectrophotometry:

Analytical absorption spectroscopy in ultraviolet and visible region of electromagnetic

spectrum is widely used for quantitative analysis in the field of pharmaceutical and

biomedical analysis. Although , there is steady increase in the use of other advance physical

method of analysis of analysis such as GLC,HPLC, HPTLC, and AAS is active, highest

specificity, precision and accuracy and one may be tempted to suggest the use of simple

colorimetric methods based on color reaction has diminished in importance and application.

However , it is relevant to note that even the latest edition to various compendia

(IP/BP/USP/EP) contain identification tests for different drug substances based on the color

reaction of one or more specific functional groups present in drug molecule. It is an accepted

fact that real sample analysis is often too complex for direct analysis, no matter what new

technology/instrumentation are available. Selective chemical reactions provide the method to

manipulate a sample to reveal the information desired. They also provide the means to

increase the response of an analyte to particular functional group or increase the selectivity

of analysis by targeting certain components of the sample to response to the selected

reagents.

As a matter of fact, most of the time , analytical chemist attempts to optimize compendia

identification tests based on color reaction for quantitative analysis. Further speed simplicity

and easy maintenance of such instruments (colorimeter, UV-visible spectrophotometric ) are

added advantages, so, UV-Visible spectrophotometric methods are widely used for

quantitative determination in dosage form[26].

Sagar Institute of Pharmaceutical Sciences, Sagar - 470 228 (M.P.) 12

The biggest advantage for chemists and astronomers who use UV-VIS spectrometers is the

accuracy of the device.

SmallUV-VIS spectrometers can give extremely accurate readings, which is crucial when

you are preparing chemical solutions or recording the movement of celestial bodies.

UV-VIS spectrometers are easy to use. Most UV-VIS spectrometers used in astronomy

attachto telescopes.

Most of the ones used in chemistry are comparable in size to electron microscopes and

require the same basic skills to use.

They are simple to operate, there is little chance of a UV-VIS spectrometer being used

improperly.

3.5 Disadvantages:

The main disadvantage of using a UV-VIS spectrometer is the time it takes to prepare to

useone. With UV-VIS spectrometers, setup is key.

there must clear the area of any outside light, electronic noise, or other outside

contaminantsthat could interfere with the spectrometer's reading.

If the space has been properly prepared ahead of time, UV-VIS spectrometers are

simple touse and give accurate results. if the space has not been properly prepared, even a

small bit of outside light or vibration froma small electronic device could interfere with the

results you are hoping to achieve in using a UV-VIS spectrometer.

Sagar Institute of Pharmaceutical Sciences, Sagar - 470 228 (M.P.) 13

4. INSTRUMENTION:
4.1 Radiation source
The best source of light is the one which is more stable, more intense and which gives range

of spectrum from 180-360nm (up to 400 nm ). The different sources available are-:

 Tungsten lamp

 Hydrogen discharge lamp

 Deuterium lamp

 Xenon discharge lamp

 Mercury arc

 Monochromators

The monochromator is used to disperse the radiation according to the wavelength. The

essential elements of a monochromator are an entrance slip, a dispersing element and an

exit slit. The entrance slit sharply defines the incoming beam of heterochromatic radiation.

Quartz and fused silica prism which are transparent throughout the entire UV range are

widely used in UV spectrophotometers [27].

4.2 Sample cells:

The cells that are to contain samples for analysis should fulfill three main conditions:
(i) They must be uniform in construction, the thickness must be constant.

(ii) The material of construction should be inert to solvents.

(iii) They must transmit light of the wavelength used.

The most commonly used cells are made of quartz or fused silica. The path length of the

cellare 10nm to 1cm.

Sagar Institute of Pharmaceutical Sciences, Sagar - 470 228 (M.P.) 14

4.3 Solvent:

Solvent plays an important role in UV spectra, since compound peak could be obscured by

solvent peak. Hence the solvent for a sample is selected in such a way that the solvent neither

absorbs in the region of measurement nor affects the absorption of the sample.

4.4 Detectors:

Although any one of the detectors used in calorimetric can be used, photomultiplier tubes are

mainly used mainly used, since the cost of such UV spectrophotometers are high and more

accurate measurements are to be made.

Example:-

1. Barrier layer cell photo voltaic cell.

2. Photo tubes or photo emissive cell.

3. Photomultiplier tubes (PMT).

4.5 Sample cells:

The signal from the photomultiplier tubes is finally received by the recording system. The

recording is done by recorder pen. The type of arrangement is only done in recording UV

spectrophotometers[28].

The power supply serves a triple function.

(i). It decreases the line voltage is to the instruments operating level with a transformer.

(ii). It convert A.C. to D.C with a rectifier it direct current is required by the instrument.
(iii). It smoothes out any ripple which may occur in the line voltage in order to deliver a
constant voltage to the source lamp and instruments.

Sagar Institute of Pharmaceutical Sciences, Sagar - 470 228 (M.P.) 15

4.6 Application:

 Detection of functional groups.

 Determination of dissociation constant of acids and bases.

 Chemical kinetic.

 Identification of an unknown compounds.

 Extent of conjugation.

 Detection in conjugation and no-conjugation compounds.

 Detection of polynuclear hydrocarbon.

 It directly used to measure light intensity at different wavelengths.

 It is use to determine the unknown concentration of solution.

 Spectrometers can be used to determine the equilibrium constant of reaction.

5. VALIDATION:

Validation of an analytical procedure is the process by which it is established, by laboratory

studies, that the performance characteristics of the procedure meet the requirements for the

intended analytical applications.[29] Method validation provides an assurance of reliability

during normal use, and is sometime referred to as “the process for providing documented

evidence that the method does what it is intended to do.” The main objective of the

validation is to demonstrate that the analytical method is suitable for its intended purpose, is

accurate, specific and precise over the specified range that an analytic will be analyzed.

Analytical Method Validation is to be performed for new analysis methods or for

current methods when any changes are made to the procedure, composition of the drug

product and synthesis of the drugs substances.

Common types of analytical procedure that can be validated [30]

Sagar Institute of Pharmaceutical Sciences, Sagar - 470 228 (M.P.) 16

A. Identification tests;

B. Quantitative tests for impurities content;

C. Limit tests for the control of impurities;

D. Quantitative tests of the active moiety in samples of drug substance or drug product

orother selected component(s) in the drug product.

Typical validation characteristics which should be considered are listed below:[31]

 Accuracy

 Precision

 Specificity

 Detection Limit

 Quantitation Limit

 Linearity
 Range

 Robustness

The validation characteristics are to be evaluated on the basis of the type of analytical
procedures.

5.1 Methods and Terminology:

5.1.1 Accuracy

The accuracy of an analytical method is the closeness of the test results obtained by that

method to the true value.[32]This is sometimes termed trueness. It is recommended that

accuracy should be determined using a minimum of nine determinations over a minimum of

the three concentration levels, covering the specified range (3 concentrations/3 replicates

each of total analytical procedures).[33]

Sagar Institute of Pharmaceutical Sciences, Sagar - 470 228 (M.P.) 17

It is measured as the percent of analyst recovered by assay. The recovery can be determined

by the equation:

Recovery= Analytical Result x100%

True Value

The recovery should be in the range of Control limit. The following method can be applied

for calculating the Upper Control Limit (UCL) and Lower Control Limit (LCL). The method

involves the moving range, which is defined as the absolute difference between two

consecutive measurements (|xi-xi-1|).

5.1.2 Precision

The precision of an analytical method is the degree of agreement among individual test

results when the method is repeated to multiple samplings of a homogeneous sample[34].

The precision of an analytical procedure is usually expressed as the standard deviation or

relative standard deviation (coefficient of variation) of a series of measurements.

a.Repeatability

Repeatability refers to the use of the analytical procedure within a laboratory over a short

period of time using the same analyst with the same equipment.[35]Repeatability should be

assessed using a minimum of nine determinations covering the specified range for the

procedure (i.e., three concentrations and three replicates of each concentration or using

a minimum of sixdeterminations at 100% of the test concentration).[36]

b.Reproducibility

Reproducibility expresses the precision between laboratories (collaborative studies, usually

applied to standardization of methodology). Reproducibility is usually demonstrated by

means ofan inter-laboratory trial. [37]

Sagar Institute of Pharmaceutical Sciences, Sagar - 470 228 (M.P.) 18

c. Intermediate

Intermediate precision is the results from within lab variations due to random events such as

different days, different analysts, different equipment, etc.[38] The standard deviation,

relative standard deviation (coefficient of variation) and confidence interval should be

reported for each type of precision investigated.

5.1.3 Limit of quantitation:

The limit of quantitation is the minimum injected amount that produces quantitative

measurements in the target matrix with acceptable precision in chromatography, typically

requiring peak heights 10 to 20 times higher than the baseline noise. If the required precision

of the method at the limit of quantitation has been specified, the [39] approach can be used.

A number of samples with decreasing amounts of the analyte are injected six times.

Thecalculated RSD percent of the precision is plotted against the analyte amount. The

amount that corresponds to the previously defined required precision is equal to the limit of

quantitation. It is important to use not only pure standards for this test but also spiked

matrices that closely represent the unknown samples. For the limit of quantitation, the ICH

recommends, in addition to the procedures as described above, the visual inspection and the

standard deviation of the response and the slope of the calibration curve. Any results of

limits of detection and quantitation measurements must be verified by experimental tests

with samples containing the analytes at levels across the two regions. It is equally important

to assess other method validation parameters, such as precision, reproducibility and

accuracy, close to the limits of detection and quantitation.

Sagar Institute of Pharmaceutical Sciences, Sagar - 470 228 (M.P.) 19

5.1.4 Specificity:

Consider the example of a medical test for diagnosing a disease. Specificity relates to the

test's ability to correctly reject healthy patients without a condition. Specificity of a test is the

proportion of those who truly do not have the condition who test negative for the condition.

Mathematically, this can also be written as:

A positive result in a test with high specificity is useful for ruling in disease. The test rarely

gives positive results in healthy patients. A positive result signifies a high probability of the

presence of disease. A test with 100% specificity will recognize all patients without the

disease by testing negative, so a positive test result would definitely rule in the presence of

the disease. However, a negative result from a test with high specificity is not necessarily

useful for ruling out disease. For example, a test that always returns a negative test result will

have a specificity of 100% because specificity does not consider false negatives. A test like

that would return negative for patients with the disease, making it useless for ruling out the

disease.

Specificity is defined as the ability of an analytical method to measure the analyte clearly in

the presence of other components. This definition has following implications:

 Identification

 Purity tests

 Assay

5.1.5 Range: The range of the method is the interval between upper level and lower
level of analyst that have been determined with acceptable accuracy, precision and linearity.

It is determined on either a linear or nonlinear response curve and expressed in the same unit

as the test results are expressed. The range is normally expressed in the same units as test

results (e.g., percent) obtained by the analytical procedure.

Sagar Institute of Pharmaceutical Sciences, Sagar - 470 228 (M.P.) 20

 A. For Assay of a Drug Substance (or a drug product) the range should be from 80%
to 120% of the test concentration.

 B. For Determination of an Impurity: from 50% to 120% of the acceptance criterion.

 C. For Content Uniformity: a minimum of 70% to 130% of the test concentration, unless a
wider or more appropriate range based on the nature of the dosage form (e.g., metered-dose

inhalers) is justified.

 D. For Dissolution Testing: ±20% over the specified range

 E. (e.g., if the acceptance criteria for a controlled-release product cover a region from
20%, after 1 hour, and up to 90%, after 24 hours, the validated range would be 0% to 110%
of the label claim).

5.1.6 Robustness:

Ruggedness is normally evaluated during method development, typically by the originating

laboratory, before collaborating with other laboratories and is a measure how well a method

stands up to less than perfect implementation. In any method there will be certain stages,

which, if not carried out sufficiently carefully, will have a severe effect on method

performance, and may even result in the method not working at all. These stages should be

identified, usually as part of method development, and if possible, their influence on method

performance evaluated using ‘ruggedness tests’, sometimes also called ‘robustness tests.

5.1.7 Linearity
It may be demonstrated directly on the drug substance (by dilution of a standard stock

solution) and/or separate weighing of synthetic mixtures of the drug product components,

using the proposed procedure. The latter aspect can be studied during investigation of the

range. Linearity should be evaluated by visual inspection of a plot of signals as a function of

analyte concentration or content. The correlation coefficient, y-intercept, slope of the

regression line, and residual sum of squares should be submitted.

Sagar Institute of Pharmaceutical Sciences, Sagar - 470 228 (M.P.) 21

 A plot of the data should be included. According to the Beers Lambert Law, Absorbance is

the ratio of logarithm of Intensity of incident light and Intensity of transmitted light, or A =

εCT. Knowledge of the sensitivity of the color is important and the following terms are

commonly employed for expressing sensitivity. The absorbance (A) is proportional to the

concentration (C) of the absorbing species, if absorptivity (ε) and thickness of the medium (t)

are constant. When concentration is in moles per liter, the constant is called molar

absorptivity.

5.2 TYPE OF VALIDATION:

 Prospective Validation

 Concurrent Validation

 Retrospective Validation

 Revalidation

5.2.1 Prospective validation-

Prospective validation is carried out during the development stage.It includes the division of

the production process into separate steps, and the analysis of potentially critical points in the

manufacturing process e.g. mixing times, or temperature.This particular type of process

validation is normally carried out with the introduction of new products and manufacturing

processes.

Before this validation can take place, the following requirements need to be satisfied:

 The facilities & equipment must be qualified

 The operators running the validation batches must have an understanding of the process

 The design and optimization must be completed

 The pilot laboratory batches must be completed

Sagar Institute of Pharmaceutical Sciences, Sagar - 470 228 (M.P.) 22

 Product stability information is available

 At least one pilot batch has been completed which shows no significant deviations

fromthe expected performance of the process.

5.2.2 Concurrent Validation-

Concurrent validation is carried out during normal production. It requires a full

understanding of the process based on prospective work. It involves very close and

intensified monitoring of all the manufacturing steps and critical points in at least the first

three production-scale batches

Examples of in-process testing include:

 pH Value

 Tablet Hardness

 Weight Variation

 Dissolution Time

 Content Uniformity

 Viscosity or Density

 Colour or Clarity

 Particle Size Distribution

 Average Unit Potency

Sagar Institute of Pharmaceutical Sciences, Sagar - 470 228 (M.P.) 23

5.2.3 Retrospective Validation-

Retrospective validation is the analysis of accumulated results from past production batches

manufactured under identical conditions to assess the consistency of a process. It includes

trend analysis on test results and a close examination of all recorded process deviations and

their relevant investigation reports.

This type of validation is applied to established products who are considered stable where

prospective validation programs cannot be justified.

Using either data-based computer systems or manual methods the following method can be

used to perform

Retrospective validation:

 Gather the numerical data from completed batch records

 Organize this data in sequence i.e. Batch Manufacturing Date

 Include the data for at least 20-40 batches, if the number is less than 20 include all of

thedata available

 Lean the data by eliminating all of the non-critical numerical information

 Subject this data to statistical evaluation

 Analysis the state of control of the manufacturing process

 Generate a report of all findings.

5.2.4 Revalidation-

Periodic revalidation offers the opportunity to check that the systems are still operating as

originally validated and that no unintended changes have affected the process, system or

piece ofequipment and the end result.

Sagar Institute of Pharmaceutical Sciences, Sagar - 470 228 (M.P.) 24

Conditions requiring revalidation can be summarized into 5 main categories:

 A change to a critical raw material involved with the drug make-up

 A change or replacement of a critical piece of equipment

 A change to the facility

 A significant increase or decrease in batch size

 Sequential products that that fail to meet product and process specifications

5.3 SCOPE OF VALIDATION:

 Analytical

 Instrumental calibration

 Raw material

 Packaging material

 Equipment

 Facilities

 Manufacturing operations

 Product design

 Cleaning

5.4 IMPORTANCE OF VALIDATION:

 Reduction of quality costs

 Process optimization

 Assurance of quality

 Safety

 Increased output

 More rapid automation

Sagar Institute of Pharmaceutical Sciences, Sagar - 470 228 (M.P.) 25

5.5 NEED FOR VALIDATION:

 Basic requirement for the product quality system.

 Assures that every lot of each product that is released to the market will
consistentlymeet all the quality requirements.

 Capable of achieving the intended results.

5.6 Validation Protocol

A written plan stating how validation will be conducted and defining acceptance criteria. For

example, the protocol for a manufacturing process indentifies processing equipment, critics

process parameters/operating range, product characteristics, sampling, test data to collected,

number of validation runs, and acceptable test results.

Objectives – The main Objectives are

 To fulfill the government regulatory requirements.

 To assure the quality of the process & the procedure.

 To decrease the overall cost of the processes.

5.7 Advantages of validation

 During the process the knowledge of process increases
 Assures the repeatability of the process
 Assures the fluency of production
 Assures that the product is continuously according to the marketingauthorization
 Decreases the risk of the manufacturing problems
 Decreases the expenses caused by the failures in production
 Decreases the risks of failing in GMP
 Decreases the expenses of the every day production even though thevalidation itself
will create expense.

Sagar Institute of Pharmaceutical Sciences, Sagar - 470 228 (M.P.) 26

 CHAPTER -2
LITERATURE OF
REVIEW
 2. LITERATURE OF REVIEW:

 Abulhadi Z. Rasha, Mohammed H. Dwood.(2022). Review the new spectrophotometric

method for determination of mebendazole by oxidative coupling reaction. The result

revealed that the determination of mebendazole based on the oxidation of albendazole with

N-bromoboxinamide and then conjugation of the oxidized mebendazole with rhodamine

reagent the method was sensitive, with acceptable accuracy and applicable to pharmaceutical

preparation.

 Abdulhadi Z Rasha and Mohammed Habboo Dawood(2022). Review the indirect

spectrophotometric determination of mebendazole using methyl orange dyein the presence

of the oxidizing agent NBS. The result revealed that the method exceedingly selective and

toachy and it characterized by simplicity as it did not need to be organized the temperature

and extraction steps and the method to be organized the temperature and extraction steps and

the method has been successfully carried out to pharmaceutical preparations with desirable

accuracy and precision.

 Shaker A-L M.S, Yusra et al(2022). Spectrophotometric method for the

determination of mebendazole in different formulation. The result revealed that the determine

aqueous mebendazole solution. The method was based on the coupling of mebendazole with

catechol reagent in the presence of potassium chromate to present a colored dye which

provided maximum absorption at 402nm. The molar absorptivity is 1556.0794 l.mol-1. The

proposed method was applied for the determination of benzocaine in two synthetic

pharmaceuticals.

 BardeLokesh dr.(2022) et al. design and evaluate the mebendazole taste mask chewable

tablets using ion exchange resin kyron T-114. The result revealed that the mebendazole can

be taste mask by ion exchange resin such as kyron-T114 with good stability.

 Choukhandevanita MS et al (2022). Review the uv spectrophotometric method

development and validation for estimation of progesterone in bulk and tablet dosage

Sagar Institute of Pharmaceutical Sciences, Sagar - 470 228 (M.P.) 27

 form. The result revealed that the uv spectrophotometric method was development and

validated for quantitative determination progesterone in tablet formulation.

 Shrivastavasaurabh and kaur deep chanchal (2021). Review the development and

validation of novel UV spectrophotometric method for the determination of mebendazole in

pharmaceutical formulation. The result revealed that the analysis of validation parameters

proved that the method is reproducible and efficient for the determination of mebendazole

pure drug and in tablet formulation without any interferences from excipients. Hence, the

method can be used for routine analysis of bulk drug and formulations of can also be used

for bioequivalence studies.

 Perez Fernando et al (2021). Review the acceptability of mebendazole chewable

tablet in children aged 2 to 4 year in Peru. The result revealed that the study demonstrated

that it is an appropriate alternative to the conventional mebendazole hard tablet in preventive

chemotherapy for STH in children aged 2 to 4 years.

 Rinaranale et al (2021). Review the analytical method development and validation. The

result revealed that the development of analytical methods helps in understrongly the critical

process parameters and to reduce their effects on precision and accuracy, validation is a

necessary techniques in the pharma sector and that used to ensure that quality work is done

in the process which supports the development of medicine and products.

 Roxana Sandulovici et al (2020). Review the modeling of release kinetics of

mebendazole from solid dispersion based formulation in simulated gastric fluid. The result

revealed that the conclusion has a highly relevant methodological significance in conditions

of quasi total acceptance in literature of higuchi model as most appropriate in case of release

from supramolecular systems.

 Palmeirim S. marta et al (2020). Review the efficacy safety and acceptability of a new

chewable formulation versus the solid tablet of mebendazole against hookworm infection in

children an open label randomized controlled trial. The result revealed that the future

Sagar Institute of Pharmaceutical Sciences, Sagar - 470 228 (M.P.) 28

 studies should further explore the efficacy, safety and acceptability of the new chewable

tablet in a larger number of preschoolers.

 Murage K Johnson et al (2020). Review the development and application of a

spectrophotometric method in quality evaluation of benzimidazole anthelminthics in

nairobicity county. The result revealed that the found to work as reliably as it did for the bulk

raw material the method was found to be comparable to other validated compendia methods.

It could there for find applicability in the analysis of bulk raw material and finished product

in manufacturing establishment.

 Dhandar G. Ajinkya et al (2018). Review the development and validation of UV

spectrophotometric method for simultaneous estimation of quinfamide and mebendazole in

in-house pharmaceutical formulation. The result revealed that the development methods

were found to be simple rapid, reproducible and precise and can be used for quality control

analysis of quinfamide and mebendazole in bulk and may be conveniently extended towards

determination of drug in pharmaceutical formulations.

 Savalekishoresagar (2018). Review the uv spectrophotometric method development and

validation for quantitative estimation of mebendazole. The result revealed that the found very

simple, rapid and economical. The method is validated in compliance with ICH guidelines is

suitable for estimation of mebendazole with excellent recovery, precision and linearity.

 Ahmad Rahman naïf et al (2018). Review the A new method for estimation mebendazole

in its pharmaceutical preparation and in camel urine. The result revealed that the study a

simple, rapid, precise and accurate spectrophotometric method was developed and validated

for the determination of mebendazole in pharmaceutical preparation and spiked camel urine

sample.

 Ravishankerpanchumarthy et al (2017). Review the development and validation of uv

spectrophotometric method for the determination of bamifylline hydrochloride in

pharmaceutical dosage form. The result revealed that the rapid, simple, accurate,

Sagar Institute of Pharmaceutical Sciences, Sagar - 470 228 (M.P.) 29

 sensitive and precise. Hence the proposed method can be utilized for the routine analysis of

bamifylline hydrochloride in pharmaceutical dosage form.

 M. Derayeasayed et al (2017). Review the application of silver nanoparticles for the

spectrophotometric determination of three benzimidazole anthelmintic drugs in their

pharmaceutical preparation. The result revealed that the proposed analytical methodology is

considered a green procedure which could be applied for monitoring and optical detection of

investigated benzimidazole drugs in pure and pharmaceutical dosage form. Hence the

methods can be used in routine analysis of these drugs in quality control laboratories.

 Parka Rameshlal durgesh (2016). Review the RP-HPLC method development and

validation for quantification of mebendazole in API and pharmaceutical formulation. The

result revealed that the found to be simple, sensitive, accurate precise, and reproducible. The

value of % recovery was close bility and accuracy of the proposed method shows that the

method could find practical application hence utilized as routine quality control analysis.

 Salam abdel Khalid et al (2015). Review the spectrophotometric method for

determination of mebendazole in presence of its alkaline induced degradation product in pure

from and pharmaceutical preparation. The result revealed that the proposed method are

simple, rapid, accurate and precise and can be used for the determination of mebendazole in

pure form and in pharmaceutical dosage form as well as in presence of its degradation

product.

 Sharma kiran (2015). Review the development and validation of uv

spectrophotometric method for the estimation of curcumin in bulk drug and pharmaceutical

dosage form. The result revealed that the proposed method was found to be simple, sensitive,

accurate, precise, economical and rapid for the routine estimation of curcumin in bulk drug

and pharmaceutical dosage forms.

Sagar Institute of Pharmaceutical Sciences, Sagar - 470 228 (M.P.) 30

  Parakhrameshlaldurgesh et al (2015). Review the development and validation of

spectrophotometric method for estimation of mebendazole in bulk and pharmaceutical

formulation. The result revealed that the value of recovery was close to 100% indicating

reproducibility and accuracy of the proposed method shows that the method could find

practical application hence, utilized as routine quality control analysis.

 R.Ambadassaudagar B. Ravindrananth (2014). Review the estimation of metformin

hydrochloride by uv spectrophotometric method in pharmaceutical formulation. The result

revealed that the successfully applied for routine quantitative estimation of metformin

hydrochloride in bulk and pharmaceutical formulation it should be used for routine analysis

in industry.

 Audumbar mall et al (2014). Review the development and validation of uv

spectrophotometric method for the estimation of quinapril hydrochloride in bulk and its

formulation. The result revealed that the proposed method is simple, sensitive, accurate and

precise and can be successfully employed for the routine analysis of the quinapril

hydrochloride in bulk drug. The method was found to provide high degree of precise and

reproducibility.

 Sudhakararao G.V. et al (2014). Review the development and validation of uv

spectrophotometric method for the estimation of ondansetron in bulk pharmaceutical

formulation. The result revealed that the proposed method are simple, sensitive and cost

effective. The result are reproducible and can be used successfully for the estimation of

ondansetron in bulk and its pharmaceutical formulation.

Sagar Institute of Pharmaceutical Sciences, Sagar - 470 228 (M.P.) 31

 CHAPTER - 3
RESEARCH ENVASIAGED
& PLAN OF WORK
 3. RESEARCH ENVASIAGED

The quantitive analysis of the active constituents it an essential part

for developing a pharmaceutical dosage form. Any changes in the

quality or purity of the drug adversely affect the therapeutic value.

Therefore, there is need to develop better and reliable methods for the

estimation of drug in pharmaceutical dosage form. In pharmaceutical

industry, the quality of the manufactured drug in various dosages

form must be carefully controlled. Slight changes in the composition

or in the purity of the drug itself can affect the therapeutic value.

Therefore, to detect these, there is a greater need for development of

newer and better method of pharmaceutical analysis. Many method

are available for the quantitative analysis of pharmaceutical dosage

form like UV Spectrophotometric method.

Mebendazole is aanthelminitics category. Literature survey revealed

that, till date no uv method is available for estimation of mebendazole

in marketed formulation. In proposed research we tried to develop
simple, accurate, rapid & validated UV method for the, estimation of

mebendazole in pharmaceutical formulation as per ICH guidelines.

Sagar Institute of Pharmaceutical Sciences, Sagar - 470 228 (M.P.) 32

 PLAN OF WORK:

The purpose of project work will be performed as per below mention

plan-:

1. Literature survey
2. Procurement of drug and chemicals
3.Analytical method development by UV spectrophotometric
method
4. Validation of methodology
(a) Linearity
(b) Range
(c) Accuracy
(d) Precision
(e) Specificity
(f) Robustness
5. Statistical analysis of the results.
6. Compilation of data & thesis submission.

Sagar Institute of Pharmaceutical Sciences, Sagar - 470 228 (M.P.) 33

 CHAPTER -4
DRUG PROFILE
 4. DRUG PROFILE:

MEBENDAZOLE. PHYSIOCOCHEMICAL PROFILE:

IUPAC NAME: Methyl ( 5-benzoyl-1H benzimidazol-2-yl)carbamate. Chemical formula:

C16H13N3O3

Molecular weight: 295.29

Melting point: 288.50 C (551.30F). Category: Anthelmintics.

Solubility: Practically insoluble in ethanol ether, chloroform.

Water solubility: Insoluble in water.

Description: Mebendazole is an anthelmintic agent used commonly for

roundworm (pinworm and hookworm) infections trichinosis capillaries and

toxocariasis and other parasitic worm infections. Mebendazole when given

prolonged periods in high doses has been associated with elevation in serum

enzyme levels, and rare instance of acute, clinically apparent liver injury have

been linked to its use.

Storage: 150 - 250C

Sagar Institute of Pharmaceutical Sciences, Sagar - 470 228 (M.P.) 34

 Appearance: White to slightly yellow. Water solubility: Insoluble in water.

PHARMACOKINETIC OF DRUG:

BCS CLASS: II

Bioavailability: 17%.

Metabolism: Largely metabolized primarily by the liver. Half life: 3 to 6 hours

after oral administration.

Elimination: Mostly in bile or urine. Renal clearness: 92-95%.

Side effect: Black, tarry stools.

. Cough or hoarseness.

. Fever with or without chills.

. Light colored stools.

Mechanism of action: Mebendazole acts by inhibiting the production of

microtubules via binding to colchicines binding site of B- tubulin and thereby

blocking polymerization of tubulin dimers in the intestinal cells of parasites.

Sagar Institute of Pharmaceutical Sciences, Sagar - 470 228 (M.P.) 35

 CHAPTER - 5
EXPERIMENTAL WORK
 5. EXPERIMENTAL WORK:

MATERIAL AND METHOD:

Mebendazole (API Form) was received as a gift sample form Anant pharmaceutical Pvt. Ltd.

Ambernath Maharashtra India. Marketed formulation of mebendazole is mebex and vermox

in 100mg tablet for children. And adult dose of drug 500mg. these registred trademark of

cipla, mankind etc.

MARKETED FORMULATION OF MEBENDAZOLE:

IDENTIFICATION:

The gift sample of mebendazole drug was obtained from the anant pharmaceutical company

was evaluated for the spectral analysis to check their purity and authenticity by various types

of validation parameters.

FTIR SPECTRUM OF MEBENDAZOLE:

The drug mebendazole was evaluated by the FT-IR spectroscopy . IR spectrum of

mebendazole was show in fig. and data of IR interpretation was show in table no.

PHYSIOCHEMICAL CHARACTERISTICS OF MEBENDAZOLE:

The following parameters was selected for characteristics of mebendazole.

MELTING POINT:

The melting point of a substances is the temperature at which it change state from solid to

liquid. At the melting point solid and liquid phase exist in equilibrium. The melting point of

a substances is depended on pressure and usually specified at a standard pressure. Melting

point of mebendazole was analyzed by digital melting point apparatus. Melting point of

mebendazole was found to be283-2910 C. that defined the purity of drug sample and was

shown in table no.2.

Sagar Institute of Pharmaceutical Sciences, Sagar - 470 228 (M.P.) 36

 SOLUBILITY:

The solubility of mebendazole was carried out by various solvent system, and data of

solubility will help in the designing and selection of solvent for the UV analysis. Different

solvent were used for the solubility analysis of drug and was shown in table no.3.

MOBILE PHASE:

UV SPECTROMETRIC ANALYSIS:

UV spectrum analysis is used to analyses the chemical properties of material. It can be used

to determine concentration identify unknown compound and provide information about the

physicaland electronic structure of organic and inorganic compound . The drug mebendazole

was scanned in UV ranges for the determination of maximum absorbance. Uv spectra of pure

mebendazole scanned in UV range i.e 200-400nm and λ max was found to be 247nm.

PREPRATION OF STANDARD SOLUTION AND CALIBRATION CURVE:

Accurately weighed 100 mg of pure Mebendazole drug and transferred into a 100 ml
volumetric flask. A small quantity of Phosphate buffer 6.8 was added to ensure complete
solubilization of the drug, and finally, volume was made up to the mark with the same buffer
solution. The solution was sonicated for 5 min to dissolve and remove air completely.
CALIBRATION OF CURVE:

Preparation of Calibration Curve: From the working solution, 0.5, 1.0, 1.5, 2.0, 2.5, and

3.0 ml solution was transferred into a series of calibrated 10 ml volumetric flasks, and the

volume was made up using Phosphate buffer 6.8. The solutions were scanned in the range of

200 to 400 nm against a blank (Phosphate buffer 6.8).The calibration curve was plotted over

a concentration range 1-10µg/ml for mebendazole and mean absorbance of the drug was

calculated and the calibration curve was plotted against the absorbance and concentration

of drug was representedin.

Sagar Institute of Pharmaceutical Sciences, Sagar - 470 228 (M.P.) 37

 WORKING STANDARD SOLUTION:

Working standard solution of mebendazole was made from the stock solution. The drug

mebendazole 25mg of IP transfer to 50ml volumetric flask add 10ml of formic acid and

dissolve, dilute with methanol to volume and mix. The solvent system to give concentration

rate of 2,4,6,8,10 and 12µg/ml. The mebendazole absorbance was recorded at 247nm against

a reagent and calibration curve.

Sagar Institute of Pharmaceutical Sciences, Sagar - 470 228 (M.P.) 38

 CHAPTER – 6
RESULT AND DISCUSSION
 6. RESULT AND DISCUSSION:

Characterization of drug Structure of mebendazole

FTIR Spectra of mebendazole

The mebendazole was characterized by IR spectroscopy for identification and IR spectrum of

same was shown in figure.1. and data of IR interpretation was shown in table no. 1.

FIGURE 2 .FTIR Spectra of mebendazole in A,B,C polymorphs.

Sagar Institute of Pharmaceutical Sciences, Sagar - 470 228 (M.P.) 39

 Table no. 1. Interpretation of FTIR spectra of mebendazole.

S. Functional Peak wave number (cm-1)

NO group
.
Theoretical Experimental

1. N-H str. 3369 3057

2. N-O str. 3345 3071

3. O-H str. 3404 3024

Melting point of mebendazole

The drug was identified by the melting point determination for the purity and was shown in

table no. 2.

Table no. 2: Melting point of mebendazole Solubility study of drug

Melting point (0C)

Drug

288.5 0C
MEBENDAZOLE

Sagar Institute of Pharmaceutical Sciences, Sagar - 470 228 (M.P.) 40

 Different solvent were used for the solubility analysis of drug and was
shown in table no.3.
Table no. 3. Solubility of drug

Solvents Mebendazole

Insoluble in water
Distilled water

Very soluble
Ethanol

Sparingly soluble
DMSO

Soluble
Acetonitrile

Slightly soluble
Isopropanol

Soluble
N-propanol

Methanol Freely soluble

PBS Buffer Sparingly soluble

Sagar Institute of Pharmaceutical Sciences, Sagar - 470 228 (M.P.) 41

 Simultaneous estimation of mebendazole by UV spectrophotometric method: Determination
ofλ max mebendazole The spectrophotometric method in uv reagion has been developed and
used for determination of mebendazole in pharmaceutical formulations, Uv spectra of pure
mebendazole scanned in UV range i.e 200-400nm and λ max was found to be 247nm.

FIGURE NO. 3 UV SPECTRUM OF MEBENDAZOLE Calibration curve of

mebendazole at 247nm

Various concentration range tried to find out linearity in calibration of mebendazole

Correlation
S. No. Conc. Range
betw
(µg/ml)
eenAbs. and
Conc.
1. 3-5 Linear

2. 12-22 Non-linear

3. 27-42 Non-linear

Table no.4. CONCENTRATION RANGE OF MEBENDAZOLE

Sagar Institute of Pharmaceutical Sciences, Sagar - 470 228 (M.P.) 42

 Individual aliquots mebendazole and mebendazole degradation product each equivalent to

(10– 90 µgml-1), were transferred from their standard working solutions (100 µg ml-1) into

two separate series of 10 ml volumetric flasks and volume was made with methanol. The

spectra of prepared standard solutions were scanned from 200 - 400 nm against methanol as

blank. First derivative (1D) spectra of mebendazole and its degradation product were

recorded. The amplitude at 248.2 nm was measured for each drug concentration. The

calibration curve was constructed relating the amplitudes of the first derivative values at

248.2 nm to the corresponding concentrations in µg ml-1 of mebendazole, the regression

equation was derived.A regression analysis of Beer's law plot at 247 nm revealed a good

correlation (r=0.999, n=7) The graph of the absorbance versus the concentration of

mebendazole showed a low intercept (- 0.017) and slope (0.028) described by a regression

equation Y = ax + b (where x is the concentration of mebendazole in µg /ml, Y is the

absorbance, a is the slope and b is the intercept. The apparent molar absorptivity was

1.346×104 l. mol -1 .cm-1.

FIGURE NO.4.Concentration of mebendazole

Sagar Institute of Pharmaceutical Sciences, Sagar - 470 228 (M.P.) 43

 Table no.5.Optical characteristics and statistical for regression equation

Parameter Value

λ Max nm 247

Beer’s law limits (µg.ml-1) 20-280

Molar absorpitivity (1.mol-

1 3.32X104
.cm-1)

Determination coefficient
0.9987
(r2)

Regression equation
(y=ax+b)

Slope (a) 0.0785

Intercept (b) 0.0077

METHOD OF VALIDATION:

The simple and sensitive method of UV spectrophotometric has been developed

for the determination of various types of drugs in a bulk and pharmaceuticals

formulations. The mebendazole was validated as per ICH guidelines for its

linearity, accuracy, precision, robustness, the limit of detection, the limit of

quantitation, and ruggedness.

Sagar Institute of Pharmaceutical Sciences, Sagar - 470 228 (M.P.) 44

 Linearity: The UV spectrum and the calibration curve of Mebendazole in

phosphate buffer 6.8 at 247 nm are shown in figure, respectively. The linearity

of Mebendazole was found to be in the range of 5-30 μg/ml with a correlation

coefficient of 0.999.

Table no.6.Linearity of mebendazole

Concentration (µg/ml) Absorbance value

5
0.176

10
0.336

15
0.488

20
0.674

25
0.84

0.987
30

Sagar Institute of Pharmaceutical Sciences, Sagar - 470 228 (M.P.) 45

 Accuracy: The accuracy of the proposed method was estimated by a recovery study at the

three-level of percentage addition. The percentage recovery of Mebendazole was found to be

in the range of 97.22 to 97.54. The results of the recovery studies undoubtedly demonstrate

the accuracy of the proposed method.

Table no. 7. Accuracy of mebendazole

Concentr Level of Amount % Mean
ation addtion of drug recovery
(µg/ml) (%) found + % RSD
(µg/ml)
Mean +
SD

10 80 07.92+0.03 97.22+0.13

100 09.95+0.02
120 11.89+0.01

15 80 11.86+0.03 97.34+0.08

100 14.97+0.02
120 17.88+0.01

20 80

100 15.88+0.04 97.54+0.10

120 19.95+0.02
23.91+0.01

Sagar Institute of Pharmaceutical Sciences, Sagar - 470 228 (M.P.) 46

 Ruggedness:In the ruggedness study, the influence of small, deliberate variations of the
analytical parameters on absorbance of drug was examined. The factor selected was change

in analyst. Results of ruggedness study indicate that the selected factor remained unaffected

by small variations with % RSD of 0.3223-0.2243, which confirms the ruggedness of

method.

Table no.8. Ruggedness data of mebendazole

Absorb
S. NO. Con. ance %RSD SE
(µg/ml) (Mean
+SD)

1 4.8 0.406+0. 0.3223 0.002

0017

2 6 0.55277 0.3189 0.0013

+0.0021

3 7.2 0.8863+ 0.2243 0.00083

0.0015

Sagar Institute of Pharmaceutical Sciences, Sagar - 470 228 (M.P.) 47

 Robustness: In the robustness study, the influence of small, deliberate variations of the

analytical parameters on absorbance of drug was examined. The factor selected was change

in wavelength. Results of robustness study indicate that the selected factor remained

unaffected by small variations with % RSD of 0.155-0.0896, which confirms the robustness

of method.

Table no.9.Robustness of mebendazole

Wavelength Absorbance
S.NO. Nm (Mean+SD) %RSD SE

1 243 0.645+0.01 0.155 0.00064

2 245 0.6507+0.0015 0.2348 0.00092

3 248 0.6443+0.0006 0.0896 0.00038

Precision: The precision of an analytical method expresses the degree of scatter between a
series of measurements obtained from multiple sampling of the same homogeneous sample

under prescribed conditions. Intra day precision refers to the use of analytical procedure

within a laboratory over a short period of time using the same operator with the same

equipment whereas Inter day precision involves estimation of variations in analysis when a

method is used within a laboratory on different days, by different analysts.

Sagar Institute of Pharmaceutical Sciences, Sagar - 470 228 (M.P.) 48

 Precision of proposed method was determined by Intra-day and Inter-day precision and it

was expressed in terms of percent relative standard deviation (% RSD). For Intra-day and

Interday precision % RSD were found in the range of 0.3372-0.3159 and 0.1883-0.3667

respectively. Whereas, standard error were found in the range of 0.00057-0.00156 for both.

Results of precision study are summarized in table no. 10 and 11.

Table no.10. Intra-day precision study

Absorbance
Conc. SD %RSD SE
(µg/ml) (Mean+SD)

4.8 0.507 0.001 0.1872 0.00057

6 0.6507 0.00153 0.2437 0.00086

7.2 0.79 0.00265 0.3159 0.00156

Table no.11.Inter-day precision study

Absorbance
Conc. SD %RSD SE
(µg/ml) (Mean+SD)

4.8 0.503 0.001 0.1883 0.00057

6 0.6477 0.0021 0.3214 0.0012

7.2 0.786 0.0026 0.3667 0.00156

Sagar Institute of Pharmaceutical Sciences, Sagar - 470 228 (M.P.) 49

 Specificity: The specificity of proposed method was ascertained by performing study at
three concentration levels i.e. 80%, 100% and 120%. The mean recovery of added excipient

at each level was found to be 97.82- 99.18%. The % RSD was found to be 0.04402-0.1533.

The percent recovery obtained indicates non-interference from the excipients used in the

formulation. The results of specificity study are given in Table no.12.

Table no.12.Specificity of mebendazole

Level Standard Aerosil 200 Mean % Recovery

of API added area+SD (RSD)+SE
addition (µg/ml) (µg/ml)

80 48 48 2892868 98.83+1467.99
+2542.6
4
10 60 60 3748055 99.18+3317.13
0 +5745.4
4
12 72 72 4521880 97.82+1149.36
0 +1990.7
5

The results of validation parameters showed that proposed method was found to be simple,

accurate, economic, sensitive, and precise and can be adopted for estimation of mebendazole

in API and formulation.

Sagar Institute of Pharmaceutical Sciences, Sagar - 470 228 (M.P.) 50

 Limit of Detection (LOD) and Limit of Quantification

(LOQ): The LOD and LOQ study was performed to check the sensitivity of the proposed
developed method. The LOD were found to be 0.896 µg/ml and 2.883 µg/ml, at 223nm

respectively and LOQ value were found to be 0.967 µg/ml and 2.954 µg/ml at 247nm for

mebendazole respectively. The obtained results, it can be easily interpreted that this UV

method is highly sensitive to analyze Mebendazole.

Table no.13.Limit of Detection of mebendazole

Mebendazole

At 223nm At 247nm

LOD 0.896µg/ml 0.967µg/ml

LOQ 2.883µg/ml 2.954µg/ml

Sagar Institute of Pharmaceutical Sciences, Sagar - 470 228 (M.P.) 51

 CHAPTER - 7
SUMMARY & CONCLUSION
 7 SUMMARY AND CONCLUSION:

SUMMARY:
The quantitive analysis of the active constituents it an essential part for
developing a pharmaceutical dosage form. Any changes in the quality or purity
of the drug adversely affect the therapeutic value. Therefore, there is need to
develop better and reliable methods for the estimation of drug in
pharmaceutical dosage form. In pharmaceutical industry, the quality of the
manufactured drug in various dosages form must be carefully controlled. Slight
changes in the composition or in the purity of the drug itself can affect the
therapeutic value. Therefore, to detect these, there is a greater need for
development of newer and better method of pharmaceutical analysis. Many
method are available for the quantitative analysis of pharmaceutical dosage
form like UV Spectrophotometric method.

Mebendazole is anthelminitics category. Literature survey revealed that, till

date no uv method is available for estimation of mebendazole in marketed

formulation. In proposed research we tried to develop simple, accurate,rapid &

validated UV method for the estimation of mebendazole in pharmaceutical

formulation as per ICH guidelines.

The process validation of mebendazole IP (500 mg). has been carried out as per

approve validation protocol and sample plan. All equipments and instruments

used for validation in process testingare calibrated and qualified. All the In

process parameters and process variables werechecked and found well within

the limit.

Sagar Institute of Pharmaceutical Sciences, Sagar - 470 228 (M.P.) 52

 CONCLUSION:

In the present research work, a successful attempt was made for “UV

spectrophotometric method development and validation for estimation of

mebendazole in tablet formulation.”This was developed by experimentation

based on literature survey and statistical parameters of validation.

In this study the estimation of mebendazole in standard laboratory by the uv

spectrophotometric method. The parameters of validation Linearity, Range,

Accuracy, Precision, Specificity, Ruggedness and Robustness are completely

fulfill the objective of research work of estimation of the drug in the marketed

formulation. The proposed methods were find to be linearity range of 0.999,

accuracy 97.54, ruggedness 0.323, robustness 0.2348, precision 0.3159 and

0.3667, specificity 99.18.The statistical parameters are proving that the methods

are reproducible and selective for the estimation of mebendazole in marketed

tablet formulations.

These methods are describe for quantification of mebendazole in marketed

formulation is successfully employed for routine analysis in quality control

laboratories. The result established that the method is simple, accurate, precise,

reproducible and sensitive. The method could be applied successfully for the

estimation of mebendazole.

Sagar Institute of Pharmaceutical Sciences, Sagar - 470 228 (M.P.) 53

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