Phd 050 2013 Maulik Amin

STABILITY INDICATING SIMULTANEOUS

DETERMINATION OF NEW

MUSCLE RELAXANT DRUGS BY

CHROMATOGRAPHIC METHODS

A thesis submitted in partial fulfillment of therequirements

for award of the degree of

Doctor of Philosophy

Research Guide: Research Scholar:

Dr. P. U. PATEL AMIN MAULIKKUMAR R.M. Pharm., Ph.D. M. Pharm

Reg. No. : PH/007/057/08

S. K. Patel College of Pharmaceutical Education and Research,

Ganpat University, Ganpat Vidhyanagar-384012, Gujarat,

NOVEMBER-2012

CertificateThis is to certify that, the thesis entitled “Stability indicatingsimultaneous determination of new muscle relaxant drugs bychromatographic methods” Submitted by Amin MaulikkumarRameshchandra of Kalol Institute of Pharmacy, Kalol is the bonafidework completed under my supervision and guidance for the award ofDegree of Doctor of Philosophy in the Faculty of Pharmacy, GanpatVidhyanagar. The experimental work included in the thesis was carriedout at Department of Pharmaceutical chemistry, Kalol Institute ofPharmacy, Kalol and Shree S. K. Patel College of PharmaceuticalEducation and Research, under my supervision and the work is up tomy satisfaction.

Research Guide:

Dr. P. U. PATELM. Pharm., Ph.D.

Professor & Head, Department of Pharmaceutical Quality Assurance,Shree S. K. Patel College of Pharmaceutical Education and Research,Ganpat University, Ganpat Vidhyanagar-384012

Forwarded through:

Dr. R. K. PATELM. Pharm., Ph.D.

I/C Principal,Shree S. K. Patel College of Pharmaceutical Education and Research,Ganpat University, Ganpat Vidhyanagar-384012

Date:

Place: Ganpat Vidhyanagar

THESIS APPROVAL SHEETThe Ph. D. Thesis entitled “Stability indicating simultaneous

determination of new muscle relaxant drugs by chromatographic

methods” by Amin Maulikkumar Rameshchandra has been

approved for the award of the Degree of Doctor of Philosophy

under the Faculty of Pharmacy, Ganpat University, Ganpat

Vidhyanagar, Dist: Mehsana (Gujarat).

External Examiner: Research Guide:


Date:


Certificate

This is to certify that the suggestion given by doctoral

committee during pre submission seminar held on 5th May

2012, vide letter of Ganpat University no.

89/GNU/Ph.D./600/2012 dated 28th May, 2012 are duly

incorporate in the thesis.

Research Guide:


Professor & Head,Department of Pharmaceutical Quality Assurance,Shree S. K. Patel College of Pharmaceutical Education and Research,Ganpat University, Ganpat Vidhyangar-384012

Date:


DECLARATION

I hereby declare that the topic entitled “Stability indicating

simultaneous determination of new muscle relaxant drugs by

chromatographic methods” which is submitted herewith to Ganpat

University for the award of Doctor of Philosophy in the Faculty of

Pharmacy is the result of work carried out by me at Kalol Institute of

Pharmacy, Kalol and Shree S. K. Patel College of Pharmaceutical

Education and Research, under guidance of Dr.P.U.Patel, Professor &

Head, Department of Pharmaceutical Quality Assurance, Shree S. K.

Patel College of Pharmaceutical Education and Research, Ganpat

University. I further declare that results of this work have not been

previously submitted for any degree or fellowship.

Date:

Place: Ganpat Vidhyangar Amin Maulikkumar R.

M.Pharm

GANPAT UNIVERSITY

DECLARATION BY THE AUTHOR OF THE THESIS

I, Mr. Maulikkumar Rameshchandra Amin, Reg. No. PH/007/057/08 registered as a

research scholar of Ph.D. programe in the Department of Pharmacy, Ganpat University do

hereby submit my thesis, entitled: “Stability indicating simultaneous determination of

new muscle relaxant drugs by chromatographic methods” (herein referred to as ‘my

thesis’) in printed as well as in electronic forms for holding in the library of records of the

University.

I hereby declare that:

1. The electronic version of my thesis submitted herewith on CDROM is in PDF format.

2. My thesis is my original work of which the copyright vests in me and my thesis do

not infringe or violate the rights of anyone else.

3. The contents of the electronic version of my thesis submitted herewith are the same as

those submitted as final hard copy of my thesis after my viva voce and adjudication of

my thesis.

4. I agree to abide by the terms and conditions of the Ganpat University Policy on

Intellectual Property (hereinafter Policy) currently in effect, as approved by the

competent authority of the university.

5. I agree to allow the university to make available the abstract of my thesis to any user

in both hard copies (printed) and electronic forms.

6. For the University’s own, non-commercial, academic use I grant to the University the

non-exclusive license to make limited copies of my thesis in whole or in part and to

loan such copies at the University’s discretion to academic persons and bodies

approved from time to time by the University for non-commercial academic use. All

usage under this clause will be governed by the relevant fair use provisions in the

Policy and by the Indian Copyright Act in force at the time of submission of the

thesis.

7. I agree to allow the University to place such copies of the electronic version of my

thesis on the private intranet maintained by the University for its own academic

community.

8. I agree to allow the University to publish such copies of the electronic version of my

thesis on a public access website of the internet.

9. If in the opinion of the University my thesis contains patentable or copyrightable

material and if the University decides to proceed with the process of securing

copyrights and/or patents, I authorize the University to do so. I also undertake not to

disclose any of the patentable intellectual properties before being permitted by the

University to do so, or for a period of one year from the date of final thesis

examination, whichever is earlier.

10. In accordance with the Intellectual Property Policy of the University, I accept that

any commercializable intellectual property contained in my thesis is the joint property

of me, my co-workers, my supervisors and the Institute. I authorize the University to

proceed with protection of the intellectual property rights in accordance with

prevailing laws. I agree to abide by the provisions of the University Intellectual

Property Right Policy to facilitate protection of the intellectual property contained in

my thesis.

11. If I intend to file a patent based on my thesis when the University does not wish so, I

shall notify my intention to the University. In such case, my thesis should be marked

as patentable intellectual property and access to my thesis is restricted. No part of my

thesis should be disclosed by the University to any person(s) without my written

authorization for one year after my information to the University to protect the IP on

my own, within 2 years after the date of submission of the thesis or the period

necessary for sealing the patent, whichever is earliest.

Name of Research student Name of Guide

AMIN MAULIKKUMAR R. Dr. P. U. PATELM.Pharm. M.Pharm., Ph.D.

Signature of Research student Signature of Guide

Date:Place: Ganpat Vidhyanagar

ACKNOWLEDGEMENTThesis is first step of research methodology, for every student during the course of

Ph.D. Today, at the acme of my thesis, with heartiness, I gratefully remember my research

guide, parents and my colleagues and all those hands that have contributed directly or

indirectly; as one flower make no garland. This presentation would not have taken shape

without their wholehearted encouragement and live involvement.

Teacher is a guide, philosopher and friend which I could experience in my respected

guide Dr. Paresh U. Patel, M. Pharm. Ph.D., Professor, Department of Pharmaceutical Quality

Assurance of Shree S. K. Patel College of Pharmaceutical Education and Research, Ganpat

Vidhyanagar. I wish to express my sincere gratitude for his constant guidance, supervision,

unceasing encouragement which paved the way for the successful completion of this

research work. His attitude towards work, his optimistic thinking, and ideas of work and

experimental has instilled me more confidence than before. It is a pleasure and privilege for

me to acknowledge gratefully the interest and attention so generously lavished by him.

It is with great pleasure and profound gratitude of reverence that I express my

esteemed Dr. Madhabhai M. Patel, Former Principal, Kalol Institute of Pharmacy, Kalol for

his valuable guidance during the course of my research work.

I warmly extend my acknowledgement to Dr. Atulbhai Patel, Managing director of

Umiya mata sanchalit samaj seva education trust for providing facilities during this course of

investigation.

I express my special thanks to Dr. R. K. Patel, I/C Principal of Shree S. K. Patel College

of Pharmaceutical Education and Research, Ganpat University, Ganpat Vidhyanagar for the

appreciable suggestions.

I would like to express my sincere thanks to honorable Shri Anilbhai T. Patel,

President, Ganpat University and respected Prof. L. N. Patel, Vice Chancellor, Ganpat

University for permitting me to pursue this work for Ph.D. degree.

Words are an inadequate medium to express my deep sense of gratitude to

Dr. B. N. Suhagia, Dean, Faculty of Pharmacy, Dharmsinh Desai University, Nadiad for the

helpful suggestions whenever required.

It gives immense pleasure to thank Dr. Deepa Patel, Mr. Ravi Shah, Mrs. Advaita

Patel and Mr. Kandarp Patel for their valuable support, co-operation and proper guidance

during this work.

I acknowledge the support of my colleagues Dr. Bhupendra Chauhan, Dr. Rajnikant

Patel, Dr. Samir Patel, Mr. Nileshbhai Patel, Ms. Jagruti Patel, Ms. Aditi Bariya, Mr. Rajesh

Keralia, Mr Chirag Patel and Mr. Kaushik Kanada for their kind help and encouragement.

I would like to sincerely thank Dr. L. J. Patel, Professor & Head, Department of

Pharmaceutical Chemistry of Shree S. K. Patel College of Pharmaceutical Education and

Research, Ganpat Vidhyanagar for his valuable suggestion.

I express my heartfelt gratitude to Ms. Mittal Patel, a librarian, Kalol Institute of

Pharmacy, Kalol for providing excellent library facility. I am thankful to Mr. Bhavesh Patel

(Store Keeper, Kalol Institute of Pharmacy, Kalol) who provided chemicals, during my work

in laboratory when I required. I owe a special thanks to Mr. Vijaybhai Raval for helped me in

maximum utilization of computer center.

I am thankful to my college Clerk Mr. Prayag Thakore and Mr. Surubha Vaghela as

well as non-teaching staff Sanjay, Jagdish, Chirag, Tushar, Alpesh, Raju, Suresh for providing

me moral support.

Research never possible without materials so I am heartly grateful to Zydus Cadila

Ltd, Ahmedabad for providing me the gift samples. My special thanks to Mr. Jitendra

Verma, Deputy Manager, Analytical Department, Zydus Research Centre, Ahmedabad.

I would fail in duty if I do not express my overriding debt to my Father Shri

Rameshchndra Amin, Mother Smt. Hemlattaben Amin, Brother Dr. Paragkumar Amin,

Bhabhi Mrs. Alaknanda Amin and nephew Preet who contributed to this research project in

countless ways. All I know is that without their care and faith in me it was impossible for me

to reach at this stage of my life.

Words can never express love of my wife Mrs. Jinali Amin for her constant

emotional support during hardships of this project.

November-2012 Mr.Maulikkumar R. Amin

INDEX

PARTICULARS OF CHAPTERChapter Content Page

No.1 Introduction 1-46

1.1 Muscle relaxants 1-4

1.2 Drug profiles 4-9

1.3 Chromatography 9-25

1.4 Development of new analytical method 25-34

1.6 Stability indicating analytical method 34-41

1.7 References 42-46

2 Review of literature 47-75

3 Aim of the present work 76

4 Chemicals, glasswares and instruments 77-80

4.1 Chemicals 77

4.2 Active Pharmaceutical Ingredients 77

4.3 Glasswares 78

4.4 Instruments 78

4.5 Other requirements 79

4.6 References 80

5 Stability indicating method development and validation for

simultaneous estimation of Diclofenac potassium,

Chlorzoxazone and Paracetamol

81-128

5.1 Stability indicating HPLC method development and validation

for simultaneous estimation of Diclofenac potassium,

Chlorzoxazone and Paracetamol

81-103

5.2 Stability indicating HPTLC method for simultaneous estimation

of diclofenac potassium, Chlorzoxazone and Paracetamol

104-126

5.3 Statistical comparison between official and proposed methods 127

5.4 References 128

6 Stability indicating method development and validation for

simultaneous estimation of Diclofenac potassium,

Paracetamol and Methocarbamol

129-173

6.1 Stability indicating HPLC method development for simultaneous

estimation of Diclofenac potassium, Paracetamol and

Methocarbamol

129-150

6.2 Stability indicating HPLC method for simultaneous estimation

of Diclofenac potassium, Paracetamol and Methocarbamol

151-171

6.3 Statistical comparison between official and proposed methods 172

6.4 References 173

7 Summary 174-175

8 Publications 176

List of TablesTableNo.

Caption PageNo.

1.3.1 Controlling sample retention by changing solvent strength 22

1.6.1 Various degradation conditions described for the stress testing 37

2.1.1 Official methods for paracetamol 47

2.1.2 Official methods for paracetamol in combination 50

2.1.3 Official methods for diclofenac potassium in combination 50

2.1.4 Official methods for chlorzoxazone in combination 51

2.1.5 Official methods for methocarbamol 52

2.2.1 Reported methods for paracetamol 53

2.2.2 Reported methods for diclofenac potassium 60

2.2.3 Reported methods for chlorzoxazone 64

2.2.4 Reported methods for methocarbamol 66

4.2.1 List of active pharmaceutical ingredients 78

5.1.1a Results of optimization of mobile phase 89

5.1.1b Linearity and range data for DIC, CHL and PCM by HPLC 91

5.1.2 Recovery study of DIC, CHL and PCM by HPLC 92

5.1.3 Intra-day & Inter-day precision of DIC, CHL and PCM by HPLC 92

5.1.4 Robustness data for DIC, CHL and PCM by HPLC 93

5.1.5 System suitability parameters for DIC, CHL and PCM by HPLC 93

5.1.6 Forced degradation study of marketed product by HPLC 102

5.2.1 Linearity and range data for DIC, CHL and PCM by HPTLC 114

5.2.2 Recovery study of DIC, CHL and PCM by HPTLC 115

5.2.3 Results of Intra-day & Inter-day precision DIC, CHL and PCM by

HPTLC

115

5.2.4 Robustness data for DIC, CHL and PCM by HPTLC 116

5.2.5 Result of Forced degradation study of drug products 125

5.3.1 Statistical comparison for DIC, CHL and PCM between proposed

methods

127

6.1.1a Results of optimization of mobile phase 136

6.1.1b Linearity and range data for DIC, PCM and MET by HPLC 139

6.1.2 Recovery study for DIC, PCM and MET by HPLC 139

6.1.3 Intra-day & Inter-day precision for DIC, PCM and MET by HPLC 140

6.1.4 Robustness data for DIC, PCM and MET by HPLC 140

6.1.5 System suitability parameters for DIC, PCM and MET by HPLC 141

6.1.6 Forced degradation study of DIC, PCM and MET by HPLC 149

6.2.1 Linearity and range data for DIC, PCM and MET by HPTLC 160

6.2.2 Recovery study for DIC, PCM and MET by HPTLC 160

6.2.3 Intra-day & Inter-day precision for DIC, PCM and MET by HPTLC 161

6.2.4 Robustness data for DIC, PCM and MET by HPTLC 161

6.2.5 Forced degradation study of marketed product by HPTLC 170

6.3.1 Statistical comparison for DIC, PCM and MET between proposed

methods

172

List of Figures

Figure No. Caption PageNo.

1.1.1 Site of action for muscle relaxants 3

1.3.3.1 Calculation of resolution for two adjacent bands 1 and 2 20

1.3.3.2 Measuring the relative valley height for two overlapping bands 21

1.3.3.3 Solvent selectivity triangle for composition of mobile phase 23

1.6.1 Flow chart for performing stress studies for photolytic degradation 38

1.6.2 Flow chart for performing stress studies for hydrolytic degradation

under acid and alkali conditions

39

1.6.3 Flow chart for performing stress studies for hydrolytic degradation

under neutral condition (in water)

40

1.6.4 Flow chart for performing stress studies for degradation underoxidativeConditions

41

5.1.1 Overlay UV spectra of DIC, CHL and PCM 83

5.1.2a Blank Chromatogram of DIC, CHL and PCM 89

5.1.2b Chromatogram of DIC, CHL and PCM by HPLC 90

5.1.3 Calibration curve of DIC by HPLC 90

5.1.4 Calibration curve of CHL by HPLC 91

5.1.5 Calibration curve of PCM by HPLC 91

5.1.6a Chromatogram of acidic degradation of DIC by HPLC 94

5.1.6b Chromatogram of acidic degradation of CHL by HPLC 95

5.1.6c Chromatogram of acidic degradation of PCM by HPLC 95

5.1.7a Chromatogram of alkaline degradation of DIC by HPLC 95

5.1.7b Chromatogram of alkaline degradation of CHL by HPLC 96

5.1.7c Chromatogram of alkaline degradation of PCM by HPLC 96

5.1.8a Chromatogram of oxidative degradation of DIC by HPLC 96

5.1.8b Chromatogram of oxidative degradation of CHL by HPLC 97

5.1.8c Chromatogram of oxidative degradation of PCM by HPLC 97

5.1.9a Chromatogram of thermal degradation of DIC by HPLC 97

5.1.9b Chromatogram of thermal degradation of CHL by HPLC 98

5.1.9c Chromatogram of thermal degradation of PCM by HPLC 98

5.1.10a Chromatogram of neutral degradation of DIC by HPLC 98

5.1.10b Chromatogram of neutral degradation of CHL by HPLC 99

5.1.10c Chromatogram of neutral degradation of PCM by HPLC 99

5.1.11 Chromatogram of acidic degradation of marketed product by

HPLC

99

5.1.12 Chromatogram of alkaline degradation of marketed product by

HPLC

100

5.1.13 Chromatogram of oxidative degradation of marketed product by

HPLC

100

5.1.14 Chromatogram of thermal degradation of marketed product by

HPLC

100

5.1.15 Chromatogram of neutral degradation of marketed product by

HPLC

101

5.2.1 Overlay UV spectra of DIC, CHL and PCM 105

5.2.2 Chromatogram of CHL, PCM and DIC 112

5.2.3 Calibration curve for DIC by HPTLC 113

5.2.4 Calibration curve for CHL by HPTLC 113

5.2.5 Calibration curve for PCM by HPTLC 114

5.2.6a Chromatogram of acidic degradation of DIC by HPTLC 117

5.2.6b Chromatogram of acidic degradation of CHL by HPTLC 117

5.2.6c Chromatogram of acidic degradation of PCM by HPTLC 118

5.2.7a Chromatogram of alkaline degradation of DIC by HPTLC 118

5.2.7b Chromatogram of alkaline degradation of CHL by HPTLC 118

5.2.7c Chromatogram of alkaline degradation of PCM by HPTLC 119

5.2.8a Chromatogram of oxidative degradation of DIC by HPTLC 119

5.2.8b Chromatogram of oxidative degradation of CHL by HPTLC 119

5.2.8c Chromatogram of oxidative degradation of PCM by HPTLC 120

5.2.9a Chromatogram of thermal degradation of DIC by HPTLC 120

5.2.9b Chromatogram of thermal degradation of CHL by HPTLC 120

5.2.9c Chromatogram of thermal degradation of PCM by HPTLC 121

5.2.10a Chromatogram of neutral degradation of DIC by HPTLC 121

5.2.10b Chromatogram of neutral degradation of CHL by HPTLC 121

5.2.10c Chromatogram of neutral degradation of PCM by HPTLC 122


HPTLC

122


HPTLC

123


HPTLC

123


HPTLC

123


HPTLC

124

6.1.1 Overlay UV spectra of DIC, PCM and MET 131

6.1.2a Blank Chromatogram by HPLC 137

6.1.2b Chromatogram of DIC, PCM and MET by HPLC 137

6.1.3 Calibration curve of DIC by HPLC 138

6.1.4 Calibration curve of PCM by HPLC 138

6.1.5 Calibration curve of MET by HPLC 139

6.1.6a Chromatogram of acidic degradation of DIC by HPLC 142

6.1.6b Chromatogram of acidic degradation of PCM by HPLC 142

6.1.6c Chromatogram of acidic degradation of MET by HPLC 142

6.1.7a Chromatogram of alkaline degradation of DIC by HPLC 143

6.1.7b Chromatogram of alkaline degradation of PCM by HPLC 143

6.1.7c Chromatogram of alkaline degradation of MET by HPLC 143

6.1.8a Chromatogram of oxidative degradation of DIC by HPLC 144

6.1.8b Chromatogram of oxidative degradation of PCM by HPLC 144

6.1.8c Chromatogram of oxidative degradation of MET by HPLC 144

6.1.9a Chromatogram of thermal degradation of DIC by HPLC 145

6.1.9b Chromatogram of thermal degradation of PCM by HPLC 145

6.1.9c Chromatogram of thermal degradation of MET by HPLC 145

6.1.10a Chromatogram of neutral degradation of DIC by HPLC 146

6.1.10b Chromatogram of neutral degradation of PCM by HPLC 146

6.1.10c Chromatogram of neutral degradation of MET by HPLC 146

6.1.11 Chromatogram of acidic degradation of marketed product byHPLC

147

6.1.12 Chromatogram of alkaline degradation of marketed product byHPLC

147

6.1.13 Chromatogram of oxidative degradation of marketed product byHPLC

147

6.1.14 Chromatogram of thermal degradation of marketed product byHPLC

148

6.1.15 Chromatogram of neutral degradation of marketed product byHPLC

148

6.2.1 Overlay UV spectra of DIC, PCM and MET 152

6.2.2 Chromatogram of DIC, PCM and MET by HPTLC 158

6.2.3 Calibration curve for DIC by HPTLC 159

6.2.4 Calibration curve of PCM by HPTLC 159

6.2.5 Calibration curve of MET by HPTLC 160

6.2.6a Chromatogram of acidic degradation of DIC by HPTLC 162

6.2.6b Chromatogram of acidic degradation of PCM by HPTLC 162

6.2.6c Chromatogram of acidic degradation of MET by HPTLC 163

6.2.7a Chromatogram of alkaline degradation of DIC by HPTLC 163

6.2.7b Chromatogram of alkaline degradation of PCM by HPTLC 164

6.2.7c Chromatogram of alkaline degradation of MET by HPTLC 164

6.2.8a Chromatogram of oxidative degradation of DIC by HPTLC 164

6.2.8b Chromatogram of oxidative degradation of PCM by HPTLC 165

6.2.8c Chromatogram of oxidative degradation of MET by HPTLC 165

6.2.9a Chromatogram of thermal degradation of DIC by HPTLC 165

6.2.9b Chromatogram of thermal degradation of PCM by HPTLC 166

6.2.9c Chromatogram of thermal degradation of MET by HPTLC 166

6.2.10a Chromatogram of neutral degradation of DIC by HPTLC 166

6.2.10b Chromatogram of neutral degradation of PCM by HPTLC 167

6.2.10c Chromatogram of neutral degradation of MET by HPTLC 167


HPTLC

167


HPTLC

168


HPTLC

168


HPTLC

169


HPTLC

169

ABBREVIATION

GENERAL ABBREVIATION IP- Indian Pharmacopeia

BP- British Pharmacopoeia

USP- United State Pharmacopeia

FDA- Food and Drug Administration

DIC- Diclofenac Potassium

CHL- Chlorzoxazone

MET- Methocarbamol

PCM- Paracetamol

SD- Standard Deviation

RSD- Relative Standard Deviation

μl- Micro Liter

ml- Mili Liter

Ach- Acetylcholine

NaOH- Sodium hydroxide

HCl- Hydrochloric acid

H2O2- Hydrogen peroxide

Max- Maxima

HPLC- High Performance Liquid Chromatography

HPTLC- High Performance Thin Layer Liquid Chromatography

LC- Liquid Chromatography

Rf- Retention factor

tR- Retention time

AR- Analytical reagent

Min- Minute

hr- Hour

WHO- World Health Organization

Vs- Versus

H2O- Water

ODS- Octa Decyl Silane

ppm- Parts per million

AUC- Area Under Curve

LOD- Limit of Detection

LOQ- Limit of Quantification

UV- Ultra Violet

COA- Certificate of Analysis

Conc- Concentration

i.d.- Internal Diameter

Edn.- Edition

ICH- International Conference of Harmonization

KH2PO4- Mono potassium phosphate

GC- Gas Chromatography

ACN- Acetonitrile

PDA- Photo Diode Array

JOURNAL ABBREVIATION Asian J. Research Chemistry- Asian Journal of Research Chemistry

Anal. Chem- Analytical Chemistry

Ann. Intern. Med.- Annals of Internal Meidicine

Arch Intern Med.- Archives of Internal Medicine

Curr Med Res Opin- Journal of Current Medical Research and Opinion

Drug Dev. Ind. Phrm.- Drug Development and Industrial Pharmacy

E Journal of Chemistry- European Journal of Chemistry

Eur J Pharmacol- European Journal of Pharmacology

Journal of AOAC Int.- Journal of AOAC International

J. of Chromatogr. B: Biomed. Sci. Appl.- Journal of Chromatography B:

Biomedical Sciences and Applications

J Biomed Sci and Res.- Journal of Biomedical Science and Research

Journal of Anal Bioanal Techniques- Journal of Analytical and Bioanalytical

Techniques

J. Chromatogr. B- Journal of Chromatography B

J Clin Pharmacol- Journal of Clinical Pharmacology

J. Pharm. Biomed. Anal.- Journal of Pharmaceutical and Biomedical Analysis

Clin Pharmacokinet.- Journal of Clinical Pharmacokinetics

J. Pharm. Biomed. Anal.- Journal of Biomedical Analysis

N Engl J Med.- The New England Journal of Medicine

Turk J Chemistry- Turkish Journal of Chemistry

SYMBOL ABBREVIATION μ- Micro

mg- Milligram

λ- Lambda

β- Beta

α- Alpha

&- And

%- Percentage

ng- Nano gram

M- Molar

R2- Coefficient of variance

g/mol- Gram per mole

Rs- Resolution

μL- Microlitre

°C -Degree celsius

mL- Mili liter

Tf- Tailing factor

Tp- Theoretical plate

v/v/v- volume/volume/volume

w/w- weight by weight

mM- Mili molar

σ - Standard deviation

DL- Detection limit

QL- Quantification limit

Chapter 1 Introduction

Ganpat University 1

1. INTRODUCTION

1.1 Muscle relaxants

Skeletal muscle relaxants are a heterogeneous group of medications that are

commonly used to treat two different types of underlying conditions: spasticity from

upper motor neuron syndromes and muscular pain or spasms from peripheral

musculoskeletal conditions. Skeletal muscle relaxants are drugs that act peripherally

at neuromuscular junction/muscle fibre itself or centrally in the cerebrospinal axis to

reduce muscle tone and paralysis. The neuromuscular blocking agents are used

primarily in conjugation with general anesthetics to provide muscle relaxation for

surgery, while centrally acting muscle relaxants are used mainly for painful muscle

spasms and neurological conditions. Although these drugs have been classified into

one class, the Food and Drug Administration (FDA) has approved only baclofen,

dantrolene, and tizanidine in this class for the treatment of spasticity; tizanidine and

the remainder of the skeletal muscle relaxant class are approved for treatment of

musculoskeletal conditions. Spasticity is a clinical condition that is “a motor disorder

characterized by increase in tonic stretch reflexes (muscle tone) with exaggerated

tendon jerks, resulting from hyper excitability of the stretch reflex, as one component

of the upper motor neuron syndrome.”Spasticity from the upper motor neuron

syndrome can result from a variety of conditions that affect the brain or the spinal

cord such as: multiple sclerosis, spinal cord injury, traumatic brain injury, cerebral

palsy, and post-stroke syndrome. In many patients with these chronic conditions,

spasticity can be disabling and painful with a marked effect on their functional ability

and quality of life.

A muscle relaxant is a drug which affects skeletal muscle function and decreases the

muscle tone. It may be used to alleviate symptoms such as muscle spasm and pain,

and hyperreflexia. The term "muscle relaxant" is used to refer to two major

therapeutic groups: neuromuscular blockers and spasmolytics. Neuromuscular

blockers act by interfering with transmission at the neuromuscular end plate and have

no CNS activity. They are often used during surgical procedures and in intensive care

and emergency medicine to cause paralysis[1-2].

Spasmolytics, also known as "centrally-acting" muscle relaxants, are used to alleviate

musculoskeletal pain and spasms and to reduce spasticity in a variety of neurological


Ganpat University 2

conditions. While both neuromuscular blockers and spasmolytics are often grouped

together as muscle relaxants the term is commonly used to refer to spasmolytics only.

Muscles can be divided into two classes, the voluntary or skeletal muscles and the

involuntary or smooth muscles. The heart muscle, the myocardium, is a unique type

of muscle that does not fit into either category. Skeletal muscles are those that are

involved in movement of arms, legs and under voluntary control. Smooth muscles are

those that are not under conscious control. The muscles in the digestive organs are

smooth muscles.

Usually it is the skeletal or striated muscles that will require therapy for painful spasm

or will need to be relaxed to allow the surgeon to gain access to the abdomen easily.

Muscle spasm may be associated with a trauma or may be brought on by multiple

sclerosis, cerebral palsy, stroke, or an injury to the spinal cord. Severe cold, an

interruption of blood supply to a muscle, or overexertion of the muscle also can lead

to spasms. A muscle spasm actually is an increase in muscle tone brought on by an

abnormality in motor control by the spinal nerves.

Skeletal muscles are controlled by large nerves in the spinal cord. The nerve cell or

neuron is part of the spinal cord, but its projections, the axon and the many dendrites

course outward to connect to muscle cells. The nerve axon is a sensory device that

senses the muscle cells current condition. The dendrites are motor fibers that deliver

the instructions to change its state to the muscle fiber. The area at which the muscle

and nerve connect is called the neuromuscular junction. It is here that the end releases

a chemical called a neurotransmitter that crosses the microscopic space between the

nerve and muscle and causes the desired response. Five such neurotransmitters have

been described: acetylcholine, serotonin, norepinephrine, glycine, and gamma-

amminobutyric acid or GABA.

Neuromuscular blocking agent (tubocurarine, pancuronium, vancuronium) cause

inhibition of muscle twitches but there was no effect if the blood supply of the hind

limb was occluded. The only possible site of action could be the neuromuscular

junction. The competitive blockers have affinity for the nicotinic cholinergic receptors

at muscle end-plate[3].

Common musculoskeletal conditions causing tenderness and muscle spasms include

fibromyalgia, tension headaches, myofascial pain syndrome, and mechanical low back

or neck pain. In these conditions, muscle spasm is related to local factors involving

the affected muscle groups. There is no increased tone or reflex. These conditions


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are usually acute and occur more commonly than spasticity in clinical practice. They

can cause significant disability and pain in some patients. Skeletal muscle relaxants

are one of several classes of medications such as anti-inflammatory drugs and pain

relievers that are used to treat these conditions.

Figure 1.1.1 site of action for muscle relaxants

Chlorzoxazone is a centrally acting muscle relaxant used to treat muscle spasm and

the resulting pain or discomfort. It acts on the spinal cord by depressing reflexes.

Aceclofenac is a phenylacetic acid derivative with analgesic and anti-iflammatory

activity and an improved gastrointestinal tolerance compared with the other NSAIDs,

such as diclofenac.

Paracetamol is a widely-used analgesic and antipyretic, unlike aspirin, it is not a very

effective anti-inflammatory agent. It is well tolerated, lacks many of the side-effects

of aspirin, and is available over-the-counter, so it is commonly used for the relief of

fever, headaches, and other minor aches and pains. Paracetamol is also useful in the

management of more severe pain, where it allows lower dosages of additional non-

steroidal anti-inflammatory drugs (NSAIDs) to be used, thereby minimizing overall

side-effects. It is also used in combination with opioid analgesics[4].


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such as diclofenac.









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such as diclofenac.









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Classification of muscle relaxant drugs:

1. Peripherally acting muscle relaxants

Mechanism of action:

Competitive block: - The site of action of both competitive and depolarizing blockers

is the end plate of skeletal muscle fibres. The competitive blockers have affinity for

nicotinic cholinergic receptors at muscle end plate, but have no intrinsic activity.

Competitive blockers also block prejunction nicotinic receptors located on motor

nerve endings. Since activation of these receptors by Ach normally facilitates

mobilization of additional quanta of Ach from the axon to the motor nerve endings,

their blockage contributes to depression of neuromuscular transmission.

Skeletal muscles:-

2. Centrally acting muscle relaxant drugs:

These drugs reduce skeletal muscle tone by selective action in the cerebrospinal axis,

without altering consciousness. They selectively depress spinal and supraspinal

polysynaptic reflexes involved in the regulation of muscle tone without significantly

affecting monosynaptically mediated stretch reflex. Polysynaptic pathways in the

ascending reticular formation which are involved in maintenance of wakefulness are

also depressed.

1.2 Drug Profiles1.2.1 Chlorzoxazone[5-7]

Systematic (IUPAC) name: 5-Chlorobenzoxazol-2-ol

Synonyms: Chlorobenzoxazolinone

Structure:

N

O

Cl

OH

Molecular Formula: C7H4 ClNO2

Mol. mass: 169.565 g/mole

Description: Colourless or white crystalline powder

Melting point: 190° to 194°C

Solubility:- Sparingly soluble in water, soluble in acetone, methanol, and

isopropanol; freely soluble in aqueous solutions of alkali hydroxides and ammonia.

Dissociation constant (pKa): 8.0


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Partition Coefficient: 1.6

Half life: 1 hr

UV detection: 280nm

Category: neuromuscular blocker

Mechanism of action[8]:

Chlorzoxazone a centrally acting acting agent for painful musculoskeletal condition it

primarily act at the level of the spinal cord and subcortical area of brain. It inhibits

degranulation of mast cells, subsequently preventing the release of histamine and

slow-reacting substance of anaphylaxis, mediators of type I allergic reactions. It

reduces release of inflammatory leukotrienes. It acts by inhibiting calcium and

potassium influx which would lead to neuronal inhibition and muscle relaxation. It

acts primarily at the level of the spinal cord and subcortical areas of brain where it

inhibits multisynaptic reflex arcs involved in producing and maintaining skeletal

muscle spasm.

Pharmacokinetics[9]:

It is absorbed orally and undergoes first pass metabolism and is excreted by kidney.

t1/2 is 2-3 hrs. It is indicated in spasticity due to neurological disorders and in painful

muscle spasms of spinal origin.

Indications:

For acute and chronic treatment of signs and symptoms of osteoarthritis and

rheumatoid arthritis.

Contraindications: hypersensitivity, liver disease, porphyria, lactation

Adverse effects: headache, gastric irritation, nausea

Special precautions: reduce dose is necessary, avoid alcohol as it has an additive

effect and increase the depression.

1.2.2 Paracetamol[10-12]

Systematic (IUPAC) name: N-(4-Hydroxyphenyl)acetamide

Synonyms: Acetaminophen; N-Acetyl–p–aminophenol

Structure:HO

NH

CH3

O


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Molecular Formula: C8H9NO2

Mol. mass: 151.2 gm/mole

Description: White crystals or crystalline powder

Melting point: 169° to 170° C

Solubility:- Very slightly soluble in cold water, considerably more soluble in hot

water; soluble in ethanol, methanol, dimethylformamide, ethylene dichloride, acetone,

and ethyl acetate; very slightly soluble in chloroform; slightly soluble in ether;

practically insoluble in petroleum ether, pentane, and benzene



Log P: 4.21

Half life: 1-3 hrs.

UV detection: 245nm

Category: analgesics

Mechanism of action:

Paracetamol has analgesic and antipyretic action with weak anti inflammatory

activity. These effects are related to inhibition of prostaglandin synthesis.

Pharmacokinetics[13]:

Paracetamol is rapidly absorbed on oral administration. Peak plasma levels are

achieved within ½ to 1 hour. It is metabolized in the liver and metabolites are

excreted in urine as conjugation products of glucuronic and sulfuric acid. The ability

of infants liver for glucuronidation of paracetamol is poor and this may result in

enhanced toxicity of the drug in neonates.

Indications:

Dosage: Paracetamol is used as analgesic and antipyretic. The total daily dose not

exceed than 2.5gm in adults. It can be used in a liquid dosage form in children.

Contraindications:

Adverse effects[14]: Paracetamol may cause fever, neutropenia, thrombocytopenia,

and skin reactions. Larger doses produce extensive damage to the liver and may cause

death due to liver failure. The drug may produce anemia as a result of haemolysis.


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1.2.3 Diclofenac potassium[15-18]:-

Systematic (IUPAC) name: 2-[(2,6-dichlorophenyl)amino]benzeneacetic acid

Synonyms: Diclophenac

Structure:

O

O

NH

Cl

Cl

K+

Molecular Formula: C14H10Cl2KNO2

Mol. mass: 296.2 gm/mole

Description: Yellowish to white crystals or crystalline powder

Melting point: 156° to 158° C

Solubility: Soluble in water and methanol



Log P: 4.21

Half life: 1-2 hrs.

UV detection: 254nm

Category: Antipyretic-analgesics agent


Diclofenac potassium tablets are a non-steroidal anti-inflammatory drug (NSAID) that

exhibits anti-inflammatory, analgesic, and antipyretic activities in animal models. It

inhibits both leukocyte migration and the enzyme cylooxygenase (COX-1 and COX-

2) which leads to peripheral inhibition of prostaglandin synthesis. As prostaglandins

sensitize pain receptors, inhibition of their synthesis is responsible for the analgesic

effects of diclofenac. Antipyretic effects may be due to action on the hypothalamus,

resulting in peripheral dilation, increased cutaneous blood flow, and subsequent heat

dissipation.

Pharmacokinetics:

It is well absorbed orally and metabolized and excreted both in urine and bile. The

plasma t1/2 is 5 hrs. Though, it has good tissue penetrability and concentration is

synovial fluid is maintained for 3 times longer period than in plasma, excreting

extended therapeutic action in joints.


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Indications[20]: It is used mainly in rheumatoid arthritis, severe osteoarthritis and in

ankylosing spondylitis.

Contraindications:

It is contraindicated in patients with peptic ulcer, hypersensitivity of diclofenac or

other NSAIDs. Also, patients having impaired cardiac, renal, hepatic function, and

asthma.

Adverse effects:

It produces mild epigastric pain, nausea, dizziness, and rashes. Gastric ulcer and

bleeding are less common.

Reversible elevation of serum aminotransferases has been reprted more commonly;

kidney damage is rare.

1.2.4 Methocarbamol[21-23]

Systematic (IUPAC) name: 3-(2-methoxyphenoxy)-l,2-propanediol-1-carbamate

Synonyms: Guaifenesin carbamate

Structure:

OH

O

O

H2N

O

O

Molecular Formula: C11H15NO5

Mol. mass: 241.2gm/mole

Description: Yellowish to white crystalline powder

Melting point: 94° to 96 ° C

Solubility: Soluble in water and methanol

Dissociation constant (pKa):

Partition Coefficient: Log P: 0.63

Half life: 1-2 hrs.

UV detection: 275nm

Category: Muscle relaxant


The mechanism of action of methocarbamol in humans has not been established, but

may be due to central nervous system depression. It has no direct action on the

contractile mechanism of striated muscle, the motor end plate or the nerve fiber11-13


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Pharmacokinetics:

Indications:

For use as an adjunct to rest, physical therapy, and other measures for the relief of

discomforts associated with acute, painful musculoskeletal conditions. It is also given

along with surgical procedure to reduce pain.

Contraindications:

Coma or pre-coma states, brain damage, myasthenia gravis, renal impairment,

epilepsy, pregnancy and lactation

Adverse effects:

It causes drowsiness, confusion, gastric irritation, and sedation. It also produces

muscular weakness, sedation, malaise, light headache, and sometimes diarrhoea.

1.3. ChromatographyIt is a special separation process for complex mixture and very similar component

with great precision. Chromatography can purify basically any soluble or volatile

substance if the right adsorbent material, carrier fluid and operating conditions are

employed. A mixture of various components enters a chromatography process and

different components are flushed through the system at different rates. These

differential rates of migration as the mixture moves over adsorptive materials provide

separation. Repeated sorption/desorption acts that take place during the movement of

sample over the stationary phase. The smaller the affinity a molecule has for

stationary phase, the shorter time spent in a column.

HPLC is one of the classes of liquid chromatography according to the nature of the

stationary and mobile phase. It has gained importance in analytical chemistry due to

its high resolution capacity, sensitivity and specificity. The separation in HPLC is

done by partitioning between a mobile phase and stationary column material and

depends on the solubilities of solute. Mobile phase is pumped at a high pressure

through a packed column with fine particles of silica or chemically modified silica,

etc. The sample is injected and the solute after separation, enter the detector, the data

from which can be quantified. Special technique used for separation in HPLC is

isocratic and gradient elution, where elution strength of elute is increased during a run

by changing polarity, pH or ionic strength. HPLC can be used to resolve and

determine the active drug and small amount of impurity in active drug.


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Pharmaceutical compounds and preparation often cause chemical or physical

instabilities. The instabilities may produce toxic reaction products, reduced activity of

compound or produce unusable product. Stability testing is therefore carried out to

ensure that deterioration does not exceed an acceptable level in order to ensure safety

of patient and maintain activity of product. Developing stability indicating method is

necessary to carry out any stability study. Stress testing of drugs was performed

according to the International Conference on Harmonization (ICH) guideline in order

to validate method. Stress testing showed that drug underwent acid, alkaline and

oxidative degradation, on other hand, it showed stability towards photo & thermal

degradation.

1.3.1 History of Chromatography

Prior to the 1970’s, few reliable chromatographic methods were commercially

available to the laboratory scientist. During 1970's, most chemical separations were

carried out using a variety of techniques including open-column chromatography,

paper chromatography, and thin-layer chromatography. However, these

chromatographic techniques were inadequate for quantification of compounds and

resolution between similar compounds. During this time, pressure liquid

chromatography began to be used to decrease flow through time, thus reducing

purification times of compounds being isolated by column chromatography. However,

flow rates were inconsistent, and the question of whether it was better to have

constant flow rate or constant pressure was debated. High-pressure liquid

chromatography was developed in the mid-1970 and quickly improved with the

development of column packing materials and the additional convenience of on-line

detectors. In the late 1970's, new methods including reverse phase liquid

chromatography allowed for improved separation between very similar compounds.

By the 1980's HPLC was commonly used for the separation of chemical compounds.

New techniques improved separation, identification, purification and quantification

far above the previous techniques. Computers and automation added to the

convenience of HPLC. Improvements in this type of columns and thus reproducibility

were made as such terms as micro-column, affinity columns, and Fast HPLC began to

immerge. The past decade has seen a vast undertaking in the development of the

micro-columns, and other specialized columns. The dimensions of the typical HPLC

column are varying in length with an internal diameter between 3-5 mm. The usual

diameter of micro-columns, or capillary columns, ranges from 3 µm to 200 µm. Fast



HPLC utilizes a column that is shorter than the typical column, with a length of about

3 mm long, and they are packed with smaller particles.

Currently, one has the option of considering over different types of columns for the

separation of compounds, as well as a variety of detectors to interface with the HPLC

in order to get optimal analysis of the compound. We hope this review will provide a

reference, which all levels of HPLC users will be able to find quick answers to their

HPLC problems. Although HPLC is widely considered to be a technique mainly for

biotechnological, biomedical and biochemical research as well as for the

pharmaceutical industry, these fields currently comprise only about 50% of HPLC

users. Currently HPLC is used by a variety of fields including cosmetics, energy, food

and environmental industries[25].

Chromatography encompasses a diverse and important group of methods that permits

the scientist to separate closely related components of complex mixtures, many of

these separations are impossible by other means. In all chromatographic separations,

the sample is dissolved in a mobile phase, which may be a gas, liquid or a

supercritical fluid. This mobile phase is the forced through an immiscible stationary

phase, which is fixed in a column or on a solid surface. The two phase or chosen so

that the components of the sample distribute themselves between mobile and

stationary phase to varying degrees. In contrast, components that are weakly held by

stationary phase travel rapidly. As a consequence of these differences in mobility,

sample components separated into discrete bands that can be analyzed qualitatively

and or quantitatively. Each component has a characteristic time of passage through

the system, called a "retention time." Chromatographic separation is achieved when

the retention time of the analyte differs from that of other components in the sample.

A chromatograph takes a chemical mixture carried by liquid or gas and separates it

into its component parts as a result of differential distributions of the solutes as they

flow around or over a stationary liquid or solid phase. Various techniques for the

separation of complex mixtures rely on the differential affinities of substances for a

gas or liquid mobile medium and for a stationary absorbing medium through which

they pass; such as paper, gelatin, alumina or silica. A chromatogram is the visual

output of the chromatograph. Different peaks or patterns on the chromatograph

correspond to different components of the separated mixture. Analytical

chromatography is used to determine the identity and concentration of molecules in a

mixture. Preparative chromatography is used to purify larger quantities of a molecular



species. Most of the following refers to analytical chromatography. This is a method

used to divide or separate mixtures[26-27].

1.3.2 Types of chromatography

It is a technique by which the components in a sample, carried by the liquid or

gaseous phase, are resolved by sorption-desorption steps on the stationary phase. A

more fundamental classification of chromatographic methods is one based upon the

type of mobile and stationary phases and the kinds of equilibrium involved in the

transfer of the solutes between phases. Classification of column chromatography

techniques is as follows.

1.3.2.1 High Performance Liquid Chromatography (HPLC)

HPLC chromatographic separation is based on interaction and differential partition of

the sample between the mobile liquid phase and the stationary phase. The commonly

used chromatographic methods can be roughly divided into the following groups, not

necessarily in order of importance:

1. Chiral

2. Ion--exchange

3. Ion--pair/affinity

4. Normal phase

5. Reversed phase

6. Size exclusion

1) Chiral Chromatography

Separation of the enantiomers can be achieved on chiral stationary phases by

formation of diastereomers via derivatizing agents or mobile phase additives on

achiral stationary phases. When used as an impurity test method, the sensitivity is

enhanced if the enantiomeric impurity elutes before the enantiomeric drug.

2) Ion-exchange Chromatography

Separation is based on the charge-bearing functional groups, anion exchange for

sample negative ion (X), or cation exchange - for sample positive ion (X), gradient

elution by pH is common.

3) Ion-pair/Affinity Chromatography

Separation is based on a chemical interaction specific to the target species. The more

popular reversed phase mode uses a buffer and an added counter-ion of opposite

charge to the sample with separation being influenced by pH, ionic strength,

temperature, concentration of and type of organic co-solvent(s). Affinity



chromatography, common for macromolecules, employs a ligand (biologically active

molecule bonded covalently to the solid matrix), which interacts with its homologous

antigen (analyte) as a reversible complex that can be eluted by changing buffer

conditions.

4) Normal Phase Chromatography

Normal phase chromatography is a chromatographic technique that uses organic

solvents for the mobile phase and a polar stationary phase. Here, the less polar

components elute faster than the more polar components.

5) Reversed Phase Chromatography

The test method most commonly used in reversed phase HPLC method. UV detection

is the most common detection technique. Reversed phase chromatography, a bonded

phase chromatographic technique, uses water as the base solvent. Separation based on

solvent strength and selectivity also may be affected by column temperature and pH.

In general, the more polar components elute faster than the less polar components.

UV detection can be used with all chromatographic techniques. The concern for this

type of detector is the loss of sensitivity with lamp aging and varying sensitivity at the

low level depending on design and/or manufacturer. A point to note is that

observations on the HPLC chromatograms, by UV detection in combination with

reversed-phase HPLC, may not be a true indication of the facts for the following

reasons:

Compounds much more polar than the compound of interest may be masked

(elute together) in the solvent front or void volume.

Compounds very less polar than the analyte may elute either late during the

chromatographic run or retained in the column.

Compounds with lower UV extinction coefficients or different wavelength

maxima may not be detectable at the low level relative to the visibility of the

analyte since only one wavelength is normally monitored.

6) Size Exclusion Chromatography

It is also known as gel permeation or filtration, separation is based on the molecular

size or hydrodynamic volume of the components. Molecules that are too large for the

pores of the porous packing material on the column elute first, small molecules that

enter the pores elute last and the elution rates of the rest depend on their relative

sizes[28-31].



1.3.2.2 Gas Chromatography (GC)

Gas chromatography is based on the volatilized sample transported by the carrier gas

as the moving phase through the stationary phase of the column where separation

takes place by the sorption/desorption process. Samples for gas chromatographic

analysis are normally low molecular weight compounds that are volatile and stable at

high temperature. In this respect, residual solvents in drug substances and drug

products are suitable for gas chromatographic analysis. Chemical derivatives can also

be formed to achieve volatility and thermal stability.

Common detectors are flame ionization (FID) for carbon-containing compounds,

electron capture (ECD) for halogenated compounds, flame photometric (FPD) for

compounds containing sulphur or phosphorous and nitrogen-phosphorous (NPD) for

compounds containing nitrogen or phosphorous. Chiral separation also can be

achieved by gas chromatography. The capillary column that provides improved

separation by resolution and analysis speed is rapidly replacing the packed column.

The location of the analyte on the gas chromatogram is described by retention time

(R), which is similar to HPLC.

1.3.2.3 Thin-Layer Chromatography (TLC)

Thin-layer chromatography is the simplest of the more common chromatographic

techniques. Separation is based on migration of the sample spotted on a coated

(stationary phase) plate with one edge dipped in a mixture of solvents (mobile phase).

The whole system is contained in an enclosed tank Detection techniques include

fluorescence, UV and sprays (universal and specific) for compounds that are not

naturally colored. The location of the analyte on the TLC plate is described by the R

value which is the ratio of the migration distance of the compound of interest to the

mobile phase front Of the three techniques, gas, liquid and thin-layer, TLC is the most

universal test method as all components are present on the plate and with appropriate

detection techniques, all components can be observed. However, it normally is not as

accurate or sensitive as HPLC. TLC has a higher analytical variation than HPLC.

Planar Chromatography as opposed to column chromatography (e.g. GC, HPLC)

utilizes a flat (planar) stationary phase for separation. In Thin-Layer Chromatography

(TLC) this stationary phase is supported by a glass plate or a foil (plastic or

aluminum). Again unlike column separations, the TLC plate constitutes an open

system, which passes through the individual steps of the TLC analysis in an off-line

mode.The relative independence of sample application, chromatogram development,



detection, etc. in time and location makes possible the parallel analysis of many

samples on the same plate. The most advanced form of instrumental TLC is

commonly called high performance thin-layer chromatography (HPTLC), but the term

does not simply imply instrumental TLC on special high performance layers. HPTLC

is an entire concept that includes a widely standardized methodology based on

scientific facts as well as the use of validated methods for qualitative and quantitative

analysis. Sophisticated instruments, controlled by an integrated software platform

ensure to the highest possible degree the usefulness, reliability, and reproducibility of

generated data. HPTLC is therefore the term for a method that meets all quality

requirements of today’s analytical labs even in a fully regulated environment.

Initial costs for an HPTLC system as well as maintenance, and cost per sample still

remain comparatively low and all advantages derived from the planar separation

principle are certainly maintained. The possibility of visual evaluation of separated

samples on the plate is one of the most valuable aspects of TLC. It reaches a

completely new dimension in HPTLC through the use of modern techniques for

generating and evaluating digital images.

Features of HPTLC:

The advantages of this off-line arrangement as compared with an on-line process,

such as column high-performance liquid chromatography (HPLC), have been outlined

and include the following:

Technically, it is simple to learn and operate.

Several analysts work simultaneously on the system.

Lower analysis time and less cost per analysis.

Low maintenance cost.

Visual detection possible-as it is an open system.

Availability of a great range of stationary phases with unique selectivity for

mixture components. Chromatographic layer (sorbent) requires no

regeneration as TLC/HPTLC plates are disposable.

Ability to choose solvents for the mobile phase is not restricted by low UV

transparency or the need for ultra-high purity. Corrosive and UV-absorbing

mobile phases can be employed.

No prior treatment for solvents like filtration and degassing.



There is no possibility of interference from previous analysis as fresh

stationary and mobile phases are used for each analysis. No carry over, hence

no contamination.

Repetition of densitometric evaluation of the same sample can be achieved

under different conditions without repeating the chromatography to optimize

quantification, since all sample fractions are stored on the TLC/HPTLC plate.

Samples rarely require cleanup.

High sample throughput since several samples can be chromatographed

simultaneously.

Lower expenditure of solvent purchase and disposal since the required amount

of mobile phase per sample is small. In addition, it minimizes exposure risks

of toxic organic effluents and reduces possibilities of environment pollution.

Accuracy and precision of quantification is high because samples and

standards are chromatographed and measured under the identical experimental

conditions on a single TLC/HPTLC plate.

Sensitivity limits of analysis are typically at nanogram (ng) to pictogram (pg)

levels.

Use of different universal and selective detection methods

HPTLC is a modern adaptation of TLC with better and advanced separation efficiency

and detection limits.

HPTLC methodology:

Set the analytical objective first that may be quantification or qualitative identification

or separation of two components/multicomponent mixtures or optimization of

analysis time before starting HPTLC. Method for analyzing drugs in multicomponent

dosage forms by HPTLC demands primary knowledge about the nature of the sample,

namely, structure, polarity, volatility, stability, and the solubility parameter. Method

development involves considerable trial and error procedures. The most difficult

problem usually is where to start, with what kind of mobile phase.

Selection of stationary phase is quite easy, that is, to start with silica gel which is

reasonable and nearly suits all kind of drugs. Mobile phase optimization is carried out

by using three level techniques. First level involves use of neat solvents and then by

finding some such solvents which can have average separation power for the desired

drugs. Second level involves decreasing or increasing solvent strength using hexane

or water for respective purposes. Third level involves trying of mixtures instead of



neat solvents from the selected solvents of first and second level which can further be

optimized by the use of modifier like acids or bases. Analytes are detected using

fluorescence mode or absorbance mode. But, if the analytes are not detected perfectly

than it needs change of stationary phase or mobile phase or need the help of pre or

post chromatographic derivatization. Optimization can be started only after a

reasonable chromatogram which can be done by slight change in mobile-phase

composition. This leads to a reasonable chromatogram which has all the desired peaks

in symmetry and well separated. Procedure for HPTLC method development is

outlined as follow.

Stationary phase:

HPTLC can be regarded as the most advanced form of modern TLC. It uses HPTLC

plates featuring small particles with a narrow size distribution. As a result,

homogenous layers with a smooth surface can be obtained. HPTLC uses smaller

plates (10x10 or 10x20 cm) with significantly decreased development distance

(typically 6 cm) and analysis time (7-20 min). HPTLC plates provide improved

resolution, higher detection sensitivity, and improved in situ quantification and are

used for industrial pharmaceutical densitometric quantitative analysis.

Mobile phase:

The selection of mobile phase is based on adsorbent material used as stationary phase

and physical and chemical properties of analyte.

General mobile-phase systems that are used based on their diverse selectivity

properties are diethyl ether, methylene chloride, and chloroform combined

individually or together with hexane as the strength-adjusting solvent for normal-

phase TLC and methanol, acetonitrile, and tetrahydrofuran mixed with water for

strength adjustment in reversed-phase TLC.

Accurate volumetric measurements of the components of the mobile phase must be

performed separately and precisely in adequate volumetric glassware and shaken to

ensure proper mixing of the content.

Volumes smaller than 1 ml are measured with a suitable micropipette. Volumes up to

20 ml are measured with a graduated volumetric pipette of suitable size. Volumes

larger than 20 ml are measured with a graduated cylinder of appropriate size. To

minimize volume errors, developing solvents are prepared in a volume that is

sufficient for one working day.



Layer prewashing:

Plates are generally handled only at the upper edge to avoid contamination. Usually

plates are used without pretreatment unless chromatography produces impurity fronts

due to contamination of the plate. For reproducibility studies and quantitative

analysis, layers are often prewashed using 20 ml methanol (generally, methanol is

used as a prewashing solvent; however, a mixture of methanol and ethyl acetate or

even mobile phase of the method may also be used) per trough in a 20x10 cm twin-

trough chamber (TTC). Up to two 20x10 cm or four 10x10 cm plates can be

developed back-to-back in each trough of the TTC.

Preparation of plate:

Precoated layers: TLC plates can be made in any lab with suitable apparatus.

However such layers do not adhere well to the glass support. Precoated plates that use

small quantities of very high molecular weight polymer as binder overcomes most

limitations of a home-made layer. Precoated layers are reasonably abrasion resistant,

very uniform in layer thickness, reproducible, preactivated, and ready to use. They are

available with glass or aluminum or polyester support. Aluminum foil plates are less

expensive to buy, cheaper, can be cut, and therefore easy to carry around or transport

or mail. Glass plates are the best for highest quality of results. Most often, layers

containing a fluorescent indicator F 254 are used. This enables the visualization of

samples in a UV cabinet very simply, instantly, and in a nondestructive

manner.Commonly used size of plates in TLC is 20x20 cm and in HPTLC 20x10 cm

or 10x10 cm is widespread.

Sample application:

In Thin-Layer Chromatography manual sample application with capillaries is usually

performed for simple analyses. Sample volumes of 0.5 to 5 μL can be applied as spots

onto conventional layers without intermediate drying. HPTLC layers take up to 1 μL

per spot. More demanding qualitative, quantitative, and preparative analyses or

separations are made possible only by instruments for band wise application of

samples using the spray-on technique. Particularly HPTLC takes full advantage of the

gain in separation power and reproducibility available by precise positioning and

volume dosage.

Spray-on technique:

With LINOMAT, an ideal instrument for sample application for instrumental and

preparative Thin-Layer Chromatography samples are sprayed onto the



chromatographic layers in the form of narrow bands. This technique allows larger

volumes to be applied than by contact transfer (spotting). During the spraying the

solvent of the sample evaporates almost entirely concentrating the sample into narrow

band of selectable length. Starting zones sprayed on as narrow bands ensure the

highest resolution attainable with any given thin-layer chromatographic system. For

qualitative and quantitative HPTLC as well as for demanding preparative separation

spray-on application as bands is a necessity.

Key features:

Operation in standalone mode or under WINCATS.

Sample application as narrow bands using the spray-on technique.

Application of solutions onto any planar medium.

Semi-automatic operation, only changing of sample (cleaning, filling and

replacing the syringe) is performed manually.

Automatic sample application is a key factor for productivity of the HPTLC

laboratory. The requirements for an instrument serving this purpose, i.e. precision,

robustness during routine use and convenient handling are fully met by the Automatic

TLC Sampler 4. The LINOMAT offers fully automatic sample application for

qualitative and quantitative analyses as well as for preparative separations. it is suited

for routine use and high sample throughput in mass analysis. Samples are either

applied as spots through contact transfer (0.1-5 μL) or as bands or rectangles (0.5 to

>50μL) using the spray-on technique. Starting zones sprayed on as narrow bands offer

the best separation attainable with a given chromatographic system. Application in the

form of rectangles allows precise application of large volumes without damaging the

layer. Prior to chromatography, these rectangles are focused into narrow bands with a

solvent of high elution strength.

Development of chromatogram:

Thin-layer chromatography differs from all other chromatographic techniques in the

fact that in addition to stationary and mobile phases, a gas phase is present. This gas

phase can significantly influence the result of the separation.

Processes in the Developing Chamber

The classical way of developing a chromatogram is to place the plate in a chamber,

which contains a sufficient amount of developing solvent.

The lower end of the plate should be immersed several millimeters. Driven by

capillary action the developing solvent moves up the layer until the desired running



distance is reached and chromatography is stopped. The following considerations

primarily concern silica gel as stationary phase and developments, which can be

described as adsorption chromatography[32-33].

1.3.3 Basics of separation

Most chromatographer’s have some idea of how a change in experimental conditions

will affect an HPLC chromatogram. In reversed – phase separations an increase

organic phase in the mobile phase will shorten run time but usually lead to increased

band overlap. Decrease in flow rate increase run time but usually improves separation.

Sometimes changing the column will improve separation. This awareness of how

conditions affect the chromatogram is a combination of training and experience.

1.3.3.1 Resolution: General consideration

Chromatographers measure the quality of separations by resolution (Rs) of adjacent

bands. More is the Rs better is separation.

Figure 1.3.3.1: Calculation of resolution for two adjacent bands 1 and 2

Rs=2(t2-t1)w1+w2

Here t1 and t2 are the retention times of the first and second bands and W1 and W2 are

their baseline bandwidth. The resolution of two adjacent band with Rs=1 illustrated in

(Fig.1.3.3.1) Resolution Rs is equal to the distance between the peak centers divided

by the average bandwidth. To increase resolution, either the two bands must be

moved further apart, or bandwidth must be reduced.

1.3.3.1.1 Measurement of resolution

Resolution can be estimated or measured in three different ways:

1.3.3.1.1.1 Calculation based on equation

Equation 1.3.1 can be used for the measurement of resolution whenever the bands are

well separated, so that retention times and bandwidths can be determined reliably. The

manual determination of baseline bandwidth W involves the construction of tangents

















Rs=2(t2-t1)w1+w2




























Rs=2(t2-t1)w1+w2














to each side of the band, and the measurement of the distance between the

intersections of these tangents with the baseline. Calculation of Rs using this method

may not be reliable when Rs is less than one.

1.3.3.1.1.2. Comparison with standard resolution curves

A comparison of two adjacent bands with standard resolution curves can also be used

to determine value of Rs. This approach does not require any calculations, is quite

convenient, and is applicable to overlapping band. “Ideal” representation of two

overlapping bands can be calculated as a function of relative band size (height or area)

and resolution. Actual overlapping bands can be compared with the ideal curves to

match “real” and “ideal” as closely as possible. It does not matter whether the larger

band elutes first or last; just mentally transpose the peaks. Once a match has been

achieved, the Rs value for closest match is then the resolution of the real band pair.

1.3.3.1.1.3 Calculation based on the valley between the two bands

Third way of estimating Rs, based on the height of the valley between two adjacent

bands, can be used for 0.8<Rs <1.5. The procedure provides more precise value of Rs

but require slightly more effort than the standard resolution curve approach. This

valley height hv is expressed as a percentage of the height of the smaller of the two

bands. Assumes band area calculated from perpendicular drop through the valley

divides the area of the two bands for integration. (Fig. 1.3.3.2)

Figure 1.3.3.2: Measuring the relative valley height for two overlapping bands

1.3.3.2 Minimum resolution

A common objective in HPLC separation is to separate all bands of interest with some

minimum resolution for accurate Quantitation of sample components. Baseline

resolution occurs when the detector trace for the first band returns to the baseline

before the next band begins to leave the column. With baseline resolution of all bands,

the HPLC data system is able to draw an accurate baseline under each band, thereby

increasing the accuracy of band area or peak-height measurements.





























































It is convenient to define the critical band pair in each chromatogram obtained during

method development. The critical pair is that band pair with the smallest value of Rs.

In method development the separation conditions are changed systematically to

improve separation of the critical band pair. This process continues until acceptable

resolution of the entire sample is obtained.

1.3.3.3 Effect of solvent strength

Sample retention can be controlled by varying the solvent strength of the mobile

phase. A strong solvent decreases retention and weak solvent increases retention.

Table 2.1 summarizes the primary mean for varying solvent strength with different

HPLC method[34,35].

Table 1.3.1 Controlling Sample Retention by Changing Solvent StrengthHPLC method How solvent strength is usually varied

Reversed Phase Water (A) plus organic solvent (B) (eg. Water-ACN); increase in

%B decreases K

Normal phase Nonpolar organic solvent (A) plus polar organic solvent (B) (e.g.

hexane-propanol); increases in % B decreases K.

Ion Pair Same as reverse phase.

Ion exchange Buffered aqueous solution plus added salt (e.g. 5mM sodium

acetate plus 50 mM NaC1); increases in (C) ionic strength

(NaCl concentration) decreases K

1.3.3.4 Effect of selectivity

The next step in method development (after adjusting % B for 0.5<K<20) is achange

of conditions that will vary band spacing or selectivity (), changes in can be

created by a change in the mobile phase, a change in the type of column packing, or a

change in temperature. Usually, it is best to start with changes in the mobile phases.

1.3.3.4.1 Change in the mobile phase1.3.3.4.1.1 Solvent -type selectivityA change in organic solvent is a powerful way to change band spacing for both

reversed and normal phase HPLC. Usually, it is the stronger solvent component (B

solvent) that will be changed for this purpose. There are many solvents to choose

from, which complicates the selection of preferred solvents for this purpose. The

solvent selectivity triangle in figure-1.3.3.3 is a useful guide for choosing among

different solvents for the purpose of a large change in band spacing solvents are

attracted to sample molecules in the mobile phase by a combination of dipole and



hydrogen bonding interactions. As a result, solvent is expected to depend on the

dipole moment, acidity, and basicity of the solvent molecule.

Figure 1.3.3.3: Solvent selectivity triangle for composition of mobile phase

1.3.3.4.1.2 Optimizing solvent-Type Selectivity

A change of the strong solvent (B-solvent) often result in large changes in band

spacing, such that bands that were formerly overlapped are now resolved and bands

that were formerly resolved are now overlapped. As a result, a mixture of the two

strong solvents often provides intermediate band spacing and acceptable resolution. In

first experiment designed to adjust solvent strength and the range of K value. It is

advisable to start with a relatively strong mobile phase. The sample is weakly retained

and leaves the column quickly with poor resolution of the sample.

1.3.2.4.1.3 Selectivity for ionic compound

For ionic samples that contain ionized or ionizable components further changes in

mobile phase are possible as a means of varying selectivity: change of pH, use of ion

pairing reagents or amine additives, change of buffer or buffer concentration.

1.3.2.4.1.4 Selective Complexation

In rare cases it may be possible to add a complexing agent to the mobile phase that

interacts selectively with one or more sample components. If complexing agent is

used, the equilibrium between the sample compound and complexing agent must be

rapidly reversible; otherwise, broad bands and poor chromatography are likely to

result.

1.3.2.4.2 Changes in the column

The nature of the column packing can have a major effect on band spacing. In most

cases it is not practical to combine different packing into a single column, although

columns of different type have been connected in series. Therefore, a change in the



column necessarily involves an abrupt change in selectivity, as opposed to the

continuous change in selectivity that is possible by changing mobile phase

composition. This limits the ability of the column to fine-tune band spacing for

samples that contain a relatively large number of components. For this reason a

change in the column usually should be combined with changes in the mobile phase to

optimize band spacing.

Most HPLC column packing is made by bonding an organic layer on to the internal

surface of porous silica particles.

The resulting column packing can exhibit differences in selectivity as results of a

number of factors are as follows:-

I. Chemical nature or functionality of the bonded phase (e.g. C18, Phenyl or

cyano)

II. Amount of bonded phase permit surface of the silica particle.

III. Way in which the bonded phase is attached to the silica surface (e.g.

monofunctional Vs polyfunctional)

IV. Nature of the silica surface, which caries among different silica sources.

1.3.2.4.3 Changes in temperature

An increase in column temperature by 1oC will usually decrease retention (K) by 1 to

2%. A change in K can also result in change in , so temperature is a potentially

useful parameter for changing band spacing and improving resolution. One advantage

of using temperature for optimizing selectivity is convenience. No change in the

column is required, nor is it necessary to make up a new mobile phase; however, for a

large increase in temperature it may be necessary to reduce % B to maintain

0.5 < K < 20.

Temperature has not been widely used for controlling band spacing, because of

certain consideration:

1. HPLC equipment is often not equipped with a column thermostat.

2. HPLC columns are not stable at higher temperature particularly for a

mobile phase pH below 3 or above 6.

3. Solvent viscosity and vapour pressure depend strongly on temperature,

which restricts the practical range in which temperature can be varied.

4. it has been assumed that a change in temperature is usually less effective

for changing value of .



These considerations have been undergoing a reexamination and it is expected that in

the future temperature will be used increasingly for the purpose of controlling bond

spacing and facilitating HPLC method development[36-41].

1.4 Development of new analytical methodA ‘regulatory analytical procedure’ is used to evaluate a defined characteristic of the

drug substance or drug product. An ‘alternative analytical procedure’ is proposed by

the applicant for use other than regulatory analytical procedure. A ‘stability-indicating

assay’ is a validated quantitative analytical procedure that can detect the changes with

time in the pertinent properties of the drug substance and drug product. A stability

indicating assay accurately measures the active ingredients, without interference from

degradation products, process impurities, excipients, or other potential impurities[42].

Stability testing forms an important part of the process of drug product development.

The purpose of stability testing is to provide evidence on how the quality of a drug

substance or drug product varies with time under the influence of a variety of

environmental factors such as temperature, humidity and light, and enables

recommendation of storage conditions, retest periods and shelf lives to be established.

The two main aspects of drug product that play an important role in shelf life

determination are assay of active drug, and degradants generated, during the stability

study. The assay of drug product in stability test sample needs to be determined using

stability indicating method, as recommended by the International Conference on

Harmonization(ICH) guidelines[43] and USP 26[44].

The modern methods of choice for quantitative analysis are HPLC, GLC, and

HPTLC, which are highly sophisticated. Chromatographic methods are commonly

used in regulatory laboratories for the qualitative and quantitative analysis of drug

substance, drug products, raw materials and biological samples throughout all phases

of drug development, from research to quality control. High performance liquid

chromatography (HPLC) is the fastest growing analytical technique for the analysis of

drugs. Its simplicity, high specificity and wide range of sensitivity make it ideal for

the analysis of many drugs in both dosage forms and biological fluids. The rapid

growth of HPLC has been facilitated by the development of reliable, moderately

priced instrumentation and efficient columns[45]. High performance thin layer

chromatography (HPTLC) is a classical separative technique that has employed wide



spread popularity in the analysis of complex mixtures of natural origin. Now a days

HPTLC is becoming a routine analytical technique due to its advantages of low

operating cost, high sample put, and need for minimum sample clean-up. The major

advantage of HPTLC is that several samples can be run simultaneously using small

quantity of mobile phase unlike HPLC, thus lowering analysis time and cost per

analysis[46].

1.5 Validation of analytical methods[47-53]

As defined by the USP, method validation provides an assurance of reliability during

normal use, and is sometime referred to as “the proces of providing documented

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

an analytical method is to demonstrate that the procedure, when correctly applied,

produces results that are fit for purpose. To be fit for the intended purpose, the method

must meet certain validation characteristics. Typical validation characteristics, which

should be considered, are: selectivity, specificity, linearity, range, accuracy, precision,

limit of detection, limit of quantification, ruggedness, robustness and system

suitability testing.

1.5.1 Specificity

Specificity is the ability to assess unequevalently the analyte in the presence of

components, which may be expected to be present. Typically it might be include

impurities, degradants, etc. Specificity investigation should be conducted during the

validation of identification tests, the determination of impurities and the assay. The

procedures used to demonstrate Specificity will depend on the intended objective of

the analytical procedure. It is not always possible to demonstrate that an analytical

procedure as specific for a particular analyte. In this case a combination of two or

more analytical procedures is recommended to achieve the necessary level of

discrimination.

Suitable identification test should be able to discriminate between compounds of

closely related structures which are likely to be present. The discrimination of a

procedure may be confirmed by obtaining positive results (perhaps by comparison

with a known reference material) from samples containing the analyte, coupled with

negative results from sample which does not contain the analyte. The choice of such

potentially interfering materials should be based on sound scientific Judgment with a

consideration of the interferences that could occur.



Assay and impurity test(s): For chromatographic procedure, representative

chromatograms should be used to demonstrate specificity and individual components

should be labeled. Critical separation in chromatography should be investigated at an

appropriately level for critical separation Specificity can be demonstrated by the

resolution of t he two components which elute closest to each other. In case where a

non-specific assay is used, other supporting analytical procedure should be used to

demonstrate overall Specificity. For example, where a titration is adopted to assay the

drug substance for release, the combination of the assay and a suitable test for

impurities can be used. The approach is similar for both assay and impurity tests:

Impurities are available: For the assay, this should involve demonstration of the

discrimination of the analyte in the presence of impurities and/or excipients;

practically, this can be done by spiking pure substance (drug substance or drug

product) with appropriate levels of impurities and /or excipients and demonstrating

that the assay result is unaffected by the presence of these materials (by comparison

with the assay result obtained on unspiked samples). For the impurity test the

discrimination may be established by spiking drug substance or drug product with

appropriate levels of impurities and demonstrating the separation of these impurities

individually and/or from other components in the sample matrix. For the impurity test

the discrimination may be established by spiking drug substance or drug product with

appropriate levels of impurities and demonstrating the separation of these impurities

individually and/or from other components in the sample matrix. Impurities are not

available: If impurity or degradation product, standards are unavailable, specificity

may be demonstrated by comparing the test. Results of samples containing impurities

or degradation products to a second well-characterized procedure e.g. pharmacopoeial

appropriate, this should include samples stored under relevant stress conditions: light,

heat, humidity, acid/base hydrolysis and oxidation.

For the assay, the two results should be compared. For the impurity tests the impurity

profiles should be compared. Peak purity tests may be useful to show that the analyte

chromatographic peak is not attributable to more than one component (e.g. diode

array, mass spectrometry).



1.5.2 Accuracy

The accuracy of an analytical procedure expresses the closeness of agreement

between the value which is accepted either as a conventional true value or an accepted

reference value and the value found. This is sometimes termed trueness. Accuracy

should be established across the specified range of the analytical procedure. Drug

substance: Several methods of determining accuracy are available: a) Application of

an analytical procedure to an analyte of known purity (e.g. reference material) b)

Comparison of the results of the proposed analytical procedure with those of a second

well-characterized procedure, the accuracy of which state and/or defined; c) Accuracy

may be inferred once precision, linearity and specificity have been established.

Drug product: Several methods for determining accuracy are available: a) Application

of the analytical procedure to synthetic mixture of the drug products components to

which known quantities of the drug substance to be analyzed have been added. b) In

cases where it is impossible to obtain samples of all drug product components, it may

be acceptable either to add known quantities of the drug product or to compare the

result obtained from a second, well characterized procedure, the accuracy of which is

stated and/or defined. c) Accuracy may be inferred once precision, linearity and

specificity have been established. Impurities (Quantitation): Accuracy should be

assessed on samples (drug substance/drug product) spiked with known amounts of

impurities. In cases where it is impossible to obtain samples of certain impurities

and/or degradation products, it is considered acceptable to compare results obtained

by an independent procedure. The response factor of the drug substance can be used.

It should be clear how the individual or total impurities are to be determined e.g.

weight/weight or area percent, in all cases with respect to the major analyte.

Recommended data: Accuracy should be assessed using a minimum of 9

determinations over a minimum of 3 concentration levels covering the specified range

(e.g.3 concentrations/3 replicates each of the total analytical procedure). Accuracy

should be reported as percent recovery by the assay of known added amount of

analyte in the sample or as the difference between the mean and the accepted true

value together with the confidence intervals.

1.5.3 Precision

The precision of an analytical procedure expresses the closeness of agreement (degree

of scatter) between a series of measurements obtained from multiple sampling of the

same homogeneous sample under the prescribed conditions. Precision may be



considered at three levels: repeatability, intermediate precision and reproducibility.

Precision should be investigated using homogeneous, authentic samples. However, if

it is not possible to obtain a homogeneous sample it may be investigated using

artificially prepared samples or a sample solution. The precision of an analytical

procedure is usually expressed as the variance, standard deviation or coefficient of

variation of a series of measurements. Repeatability: Repeatability expresses the

precision under the same operating conditions over a short interval of time.

Repeatability is also termed intra-assay precision. Repeatability should be assessed

using: a) A minimum of 9 determinations covering the specified range for the

procedure (e.g.3concentrations/3replicates each) or b) A minimum of 6

determinations at 100% of the test concentration.

Intermediate precision: Intermediate precision expresses within-laboratories

variations: different, days, different analyst, different equipment, etc. the extent to

which intermediate precision should be established depends on the circumstances

under which the procedure is intended to be used. The applicant should establish the

effects of random events on the precision of the analytical procedure. Typical

variations to be studied include days, analysts, equipment, etc. it is not considered

necessary to study these effects individually. The use of an experimental design

(matrix) is encouraged.

Reproducibility: is assessed by means of an inter laboratory trial. Reproducibility

should be considered in case of the standardization of an analytical procedure, for

instance, for inclusion of procedures in pharmacopoeias, these data are not part of the

marketing authorization dossier. Reproducibility expresses the precision between

laboratories (collaborative studies, usually applied to standardization of

methodology). Validation of tests for assay and for quantitative determination of

impurities includes an investigation of precision. Recommended data: The standard

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

should be reported for each type of precision investigated.

1.5.4 Detection limit

The detection limit of an individual analytical procedure is the lowest amount of

analyte in a sample which can be detected but not necessarily quantitated as an exact

value. Several approaches for determining the detection limit are possible depending

on whether the procedure is a non-instrumental or instrumental, approaches other than

those listed below may e acceptable.



Based on visual evaluation: Visual evaluation may be used for non instrumental

methods but may also be used with instrumental methods. The detection limit is

determined by the analysis of samples with known concentrations of analyte and by

establishing the minimum level at which analyte can be reliably detected.

Based on signal-to-noise: This approach can only be applied to analytical procedures

which exhibit baseline noise. Determination of the signal-to-noise ratio is performed

by comparing measured signals from samples with known low concentrations of

analyte with those of blank samples and establishing the minimum concentration at

which the analyte can be reliable detected. A signal-to-noise ratio between 3 or 2:1 or

generally considered acceptable for estimating the detection limit.

Based on the standard deviation of the response and the slope: The detection limit

(DL) may be expressed as: DL =3.3 δ/ S

Where δ = the standard deviation of the response

S = the slope of the calibration curve The slope S may be estimated from the

calibration curve of the analyte. The estimate of δ may be carried out in a variety of

ways, for example; (A) Based on the standard deviation of the blank: Measurement of

the magnitude of analytical background response is performed by analyzing an

appropriate number of blank samples and calculating the standard deviation of these

responses. (B) Based on the calibration curve: A specific calibration curve should be

studied using samples containing an analyte in the range of DL. The residual standard

deviation of a regression line or the standard deviation of y-intercepts of regression

lines may be used as the standard deviation.

Recommended data: The detection limit and the method used for determining the

detection limit should be presented. If DL is determined based on visual evaluation or

based on signal to noise ratio, the presentation of the relevant chromatograms i s

considered acceptable for justification. In cases where an estimated value for the

detection limit is obtained by calculation or extrapolation, this estimate may

subsequently be the independent analysis of a suitable number of samples known to

be near or prepared at the detection limit.

1.5.5 Quantitation limit

The quantitation limit of an individual analytical procedure is the lowest amount of

analyte in a sample which can be quantitatively determined with suitable precision

and accuracy. The quantitation limit is a parameter of quantitative assays for low

levels of compound in sample matrices, and is used particularly for the determination



of impurities and/or degradation products. Several approaches for determining the

quantitation limit are possible, depending on whether the procedure is a non-

instrumental or instrumental. Approaches other than those listed below may be

acceptable.

Based on visual evaluation: Visual evaluation may be used for non-instrumental

methods but may also be used with instrumental methods. The quantitation limit is

generally determined by the analysis of samples with known concentrations of analyte

and by establishing the minimum level at which the analyte can be quantified with

acceptable accuracy and precision.

Based on signal-to-noise approach: This approach can only be applied to analytical

procedures that exhibit baseline noise. Determination of the signal-to-noise ratio is

performed by comparing measured signals from samples with known low

concentrations of analyte with th ose of blank samples and by establishing the

minimum concentration at which the analyte can be reliably quantified. A typical

signal-to-noise ratio is 10:1

Based on the standard deviation of the response and the slope: The quantitation

limit (QL) may be expressed as: DL =10 δ/S


S = slope of the calibration curve

The slope S may be estimated from the calibration curve of the analyte. The estimate

of may be carried out in a variety of ways for example:

Based on Standard Deviation of the Blank: Measurement of the magnitude of

analytical background response is performed by analyzing an appropriate number of

blank samples and calculating the standard deviation of these responses.

Based on the calibration curve: A specific calibration curve should be studied using

samples, containing an analyte in the range of QL. The residual standard deviation of

a regression line or the standard deviation of y-intercepts of regression lines may be

used as the standard deviation.

Recommended data: The quantitation limit and the method used for determining the

quantitation limit should be presented. The limit should be subsequently validated by

the analysis of a suitable number of samples known to be near of prepared at the

quantitation limit.



1.5.6 Linearity

The linearity of an analytical procedure is its ability (within a given range) to obtain

test results which are directly proportional to concentration (amount) of analyte in the

sample. A linear relationship should be evaluated across the range of the analytical

procedure. 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. If there is a linear

relationship, test results should be evaluated by appropriate statistical methods, for

example, by calculation of a regression lime by the method of least squares. In some

cases to obtain linearity between assays and sample concentration, the data may need

to be subjected to a mathematic al transformation prior to the regression analysis.

Data from the regression lie itself may be helpful to provide mathematical estimates

of the degree of linearity. The correlation coefficient, y-intercept, slope of the

regression line and residual sum of square should be submitted. A plot of the data

should be included. In addition, an analysis of the deviation of the actual data points

from the regression line may also be helpful for evaluating linearity. Some analytical

procedures, such as immunoassays, do not demonstrate linearity after any

transformation. In this case, the analytical response should be described by an

appropriate function of the concentration (amount) of an analyte in a sample. For the

establishment of linearity, a minimum of 5 concentrations is recommended.

1.5.7 Range

The range of an analytical procedure is the interval between the upper and lower

concentration (amounts) of analyte in the sample (including these concentrations) for

which it has been demonstrated that the analytical procedure has a suitable level of

precision, accuracy and linearity. The specified range is normally derived from

linearity studies and depends on the intended application of the procedure. It is

established by confirming that the analytical procedure provides an acceptable degree

of linearity, accuracy and precision when applied to samples containing amounts of

analyte within or at the extremes of the specified range of the analytical procedure.

The following minimum specified range should be considered. a) For the assay of a

drug substance or a finished (drug) product; normally from 80 to 120 percent of the

test concentration. b) For content uniformity, covering minimum of 70 to 130 percent



of the test concentration unless a wider more appropriate range. Based on the nature

of the dosage form (e.g. metered dose inhalers), is justified. C) For dissolution

testing:+20% over the specified range.

For the determination of an impurity: from the reporting level of an impurity to 120%

of the specification. If assay and purity are performed together as one test and only a

100% standard is used, linearity should cover the range from the reporting level of the

impurities to 120% of the assay specification.

1.5.8 Robustness

The robustness of an analytical procedure is a measure of its capacity to remain

unaffected by small, but deliberate variation in method parameters and provides an

indication of its reliability during normal usage. The evaluation of robustness should

be considered during the development phase and depends on the type of procedure

under study. It should show the reliability of an analysis with respect to deliberate

variation in method parameters. If measurements are susceptible to variations in

analytical condition the analytical conditions should be suitably controlled or a

precautionary statement should be included in the procedure. One consequence of the

evaluation of robustness should be that a series of system suitability parameters (e.g.

resolution test) is established to ensure that the validity of the analytical procedure is

maintained whenever used. Examples of typical variations are:

a) Stability of analytical solutions. b)Extraction time. In the case of liquid

chromatography, examples of typical variation are: a) Influence of variations of pH in

a mobile phase. b) Influence of variations in mobile phase composition. c) Different

columns (different lots and/or suppliers). d) Temperature. e) Flow rate.

1.5.9 Ruggedness

Method ruggedness is defined as the reproducibility of results when the method is

performed under actual condition. This includes different analysis laboratories,

instruments source of reagents, chemicals, solvents and so on. The strategies for

determining method ruggedness will very delivery on the type and complexity of the

method and the time available for validation. Often, the real ruggedness of a method

can only be determined over time by experience in different laboratories.

1.5.10 System suitability testing

System suitability testing is an integral part of many analytical procedures. The tests

are based on the concept that the equipment, electronics, analytical operations and

samples to be analyzed constitute an integral system that can be evaluated as such.



System suitability test parameters to be established for a particular procedure depend

on the type of procedure being validated. The relationship between validation and

other phase of the method evaluation/ utilization process is often unclear. The

hierarchical concept of method utilization wherein the stage of method development

validation and utilization are distinct and sequential represents a situation which is

practically undesirable and potentially inefficient. After the method has been

conceived and operationally developed its performance is validated with respect to

several different performance parameters. Successful completion of the validation

results in a method which can reliable be used to characterize “real” samples. The

objective of the analytical procedure should be clearly understood since this will

govern the validation characteristics, which need to be evaluated. International

conference on harmonization recently published its own list of important validation

characterization for various procedures.

1.6 Stability indicating method1.6.1 Introduction

The stability indicating assay is a method that is employed for the analysis of stability

samples in pharmaceutical industry. With the advent of International Conference on

Harmonisation (ICH) guidelines, the requirement of establishment of stability

indicating assay method (SIAM) has become more clearly mandated. The guidelines

explicitly require conduct of forced decomposition studies under a variety of

conditions, like pH, light, oxidation, dry heat, etc. and separation of drug form

degradation products. The method is expected to allow analysis of individual

degradation products[54]. For the development of a sound scientific protocol for the

stability studies, an understanding of the conditions under which a drug degrades as

well as the mechanism of the breakdown is needed. This is established through a

series of stress studies designed to elucidate the intrinsic stability of the new molecule

by establishing its degradation pathway[55]. A stress stability study is often referred to

as a ‘pre-formulation study’ or ‘characterization study’. The result of these studies are

the basis for developing appropriate dosage forms, formulations and manufacturing

processes, selecting appropriate packaging and storage conditions and the analytical

methods to be used in stability studies. The information derived from stress testing

can be used to establish the methodology employed, the parameters followed and

specifications for long-term testing under accelerated and normal storage condition



conditions[56]. Stress studies are different from accelerated studies because the former

is carried under more sever conditions. Stress testing is testing under extreme

conditions, used to characterize only the drug substance. Accelerated testing applies

both to the drug substance and drug product and involves testing under conditions

more severe than normal, which serve to generate data useful in predicting what might

happen during storage under normal conditions[57].

1.6.2 Practical conduct of stress testing

The ICH guideline Q1A suggests the following conditions to be employed: (i) 10°C

increments above the accelerated temperatures (e.g. 50°C, 60°C, etc.), (ii) humidity

where appropriate (e.g.75% or greater), (iii) hydrolysis across a wide range of pH

values, (iv) oxidation and (v) photolysis. However, the guideline provides no details

on how hydrolytic, photolytic and oxidative studies have to be actually performed. In

other words, the practical aspects concerning the conduct of stress testing are

addressed neither by the regulatory guidelines nor by any other document, leaving the

performance of these studies to the prudence of the applicant. On the other hand, the

information is available in literature but in staggered way, with suggested approaches

differing a lot from one another. Few approaches towards the practical conduct of

stress studies are reported in literature[58]. A comprehensive document providing

guidance on the practical conduct and issues related to stress testing under variety of

ICH prescribed conditions has been published. This report from the authors proposes

a classification scheme and offers decision trees to help in the selection of the right

type of stress condition in a minimum number of attempts. This guidance document

on the conduct of stress tests to determine inherent stability of drugs will be followed

in the current study From the guidance on the conduct of stress tests to determine

inherent stability of drugs, the following observations were made clearly: the

condition used to study decomposition in acid revealed that 0.1N hydrochloric acid

was most commonly used. A few reports indicate the use of 1N HCl and even higher

strength and the use of sulfuric acid in varying strengths. Large variations were also

seen in the reaction (temperature) conditions and periods of study. The temperatures

varied between 40°C and 110°C. The reaction time varied, for example, drugs being

kept at 100°C or at boiling conditions, for periods ranging from a few minutes to as

long as 2 months. The extent of decompositions also varied. For example, a 35% loss

of retinoic acid was observed on refluxing in 0.1N HCl for just 5min, whereas no drug

decomposition was reported after refluxing nabilone in 0.1N acid for a week.It is



observed that NaOH is most often used for the hydrolysis of drugs in alkaline

conditions, at strengths of 0.1N and 1N, with the occasional use of potassium

hydroxide. As with acidic degradation, great variation is observed in the time and

temperature of alkali exposure. Depending on their inherent stability, some drugs (for

example, nabilone) show no degradation even after refluxing in 0.1N NaOH for a

week, whereas, others (such as trifluoperazine) undergo complete degradation in 0.1N

alkali for 24 h at 30°C[59]. At neutral pH, no significant degradation was obtained

when the temperature was 37°C for celiprolol, and refluxing conditions were used for

sertraline. The testing is generally done in water. The slow rate of decomposition in

neutral conditions is understandable because reactions at neutral pH are non-catalytic

and hence long periods at exaggerated temperatures may be required to obtain

sufficient quantities of degradation products. The most commonly used oxidizing

agent, hydrogen peroxide, is used in varying strengths between 1% and 30%. Some

drugs (ranitidine HCl and cimetidine HCl) degrade when exposed to 3 %H2O2 for

short periods at room temperature (RT)[59]. In other cases, exposure to high

concentration of H2O2, even at extreme conditions, does not cause any significant

degradation (for example sertraline HCl). This could happen since non-oxidizable

drugs are not expected to show any change-even in the presence of high concentration

of oxidizing agents. Photolytic studies are done on drugs in either solid form or

solution, in water or in acidic and alkaline solutions and also on drugs dissolved in

either methanol or acetonitrile. Mostly drugs are exposed to short /long wavelength

UV or fluorescent light of varying illumination (approximately 4300-17000 lux). The

Various degradation conditions described for the stressed testing are shown in Table

1.6.1.



Table1.6.1 Various degradation conditions described for the stress testing

Decompositi

on condition

Recently used

most

frequently

Temperatu

re

Reaction time Extent of

degradation

Acidic HCl(0.1N/1N/5N)

H2SO4

40°C-110°C Few min-2months

Negligible-complete

Alkaline NaOH(0.1N/1N5N)

80°C/Reflux 5 min-21days Insignificant-Extensive

Neutral Water 85°C/Reflux Very longperiods

No significantdegradation

Oxidation H2O2(1%-3%) RT-Refluxing

Few hours-week

Insignificant-Extensive

Photolytic UV/Fluorescentlight (4300 to

17000 lux)

RT Few hours-months

Nil-Complete

RT= room temperature

Flow charts or decision trees (Figures 1.6.1-1.6.4) for investigating the different types

of stress conditions for new drug substances are shown which assume that the new

drug is labile in nature to the stress conditions. Depending on the results, the strength

of the reaction condition may be increased or decreased. The change, if required, is

done stepwise and stress conditions are accepted when sufficient decomposition is

obtained. The term ‘sufficient decomposition’ is taken in the broadest sense, meaning

80%-100% decomposition if the objective is isolation of the degradation products, or

between 20-80% decompositions when the objective is to establish degradation

pathways.



No Degradation TotalDegradation

SufficientDegradation

No DegradationSufficient

Degradation

Figure 1.6.1 Flow chart for performing stress studies for photolytic degradation

START

1.2 x106 Lux hr

6.0 x106 Lux hr Reduce exposure

Declare drug to bepractically stable

ACCEPT



No Degradation Total Degradation

Sufficient Degradation

Sufficient Degradation Sufficient Degradation


SufficientDegradation Sufficient

Degradation



No Degradation

Figure 1.6.2: Flow chart for performing stress studies for hydrolytic degradation

under acid and alkali conditions

START

0.1N HCl/ NaOH,8 h, Reflux

1 N HCl /NaOH,12 h, Reflux

0.01HCl/ NaOH,8 h, at 40°c

ACCEPT2 N HCl/ NaOH,24 h, Reflux

1N HCl/ NaOH,2 h at, 25°C

5 N HC l/ NaOH,24 h, Reflux

Carry out studiesunder milderconditions

Declare drug tobe practicallystable





Sufficient Degradation Sufficient Degradation


Sufficient SufficientD degradation Degradation



No Degradation

Figure 1.6.3: Flow chart for performing stress studies for hydrolytic degradation

under neutral condition (in water)

START

Water/ 12 h,Reflux

Water/ 1 day,Reflux

ACCEPT

Water/ 8 hr, at40°C

Water/ 2 day,Reflux

Water/ 2 hr, at25°C

Water/ 5 dayReflux

Carry out studiesunder milder

conditions

Declare drug tobe practically

stable




Sufficient SufficientDegradation Degradation


Sufficient SufficientDegradation Degradation

No degradation Total Degradation


Figure 1.6.4: Flow chart for performing stress studies for degradation under oxidative

conditions.

START

3% H2O2/ 6 h,RT

3% H2O2/ 24 h,RT

1% H2O2/ 3 h,RT

ACCEPT10% H2O2/ 24h, RT

1% H2O2/ 30 min,RT

30% H2O2/ 24 h,RT

Carry out studiesunder milder

conditions

Declare drug tobe practically

stable



1.7 References

1. Tripathi KD, Essentials of Medical Phamacology, 6th Ed., Jaypee Brothers

Medical Publishers, 2008; 339-50.

2. Rang HP, Dale MM, Ritter JM, Flower RJ, Rang and Dale’s Pharmacology,

6th Ed., Elsevier, 2005: 385-386.

3. Bras H, Jankowska E, Noga B, Skoog B, Comparison of Effects of Various

Types of NA and 5-HT Agonists on Transmission from Group II Muscle

Afferents in the Cat, European Journal of Neuroscience, 1990; 2(12): 1029-

1039.

4. Chou R, Qaseem A, Snow V et al., Diagnosis and treatment of low back pain:

a joint clinical practice guideline from the American College of Physicians

and the American Pain Society, Ann. Intern. Med., 2007; 147 (7): 478-91.

5. Frye RF and Stiff J, Analysis of Chlorzoxazone in plasma and urine by high

performance liquid chromatography, J. of Chromatogr. B: Biomed. Sci.

Appl.,1996; 68(2): 291-296

6. Dong DL, Luan Y, Feng TM, Fan CL, Yue P, Sun ZJ, Gu RM, Yang BF,

Chlorzoxazone inhibits contraction of rat thoracic aorta., Eur J Pharmacol.,

2006; 545(2): 161-6.

7. Sweetman SC, Martindale: The Complete Drug Reference, 37th Ed. London:

Pharmaceutical Press, 2011; 1392-93.

8. Wan J, Ernstgard L, Song BJ, Shoaf SE, Chlorzoxazone metabolism is

increased in fasted Sprague-Dawley rats., J Pharm Pharmacol., 2006; 58(1):

51-61

9. Park JY, Kim KA, Park PW, Ha JM, Effect of high-dose aspirin on CYP2E1

activity in healthy subjects measured using Chlorzoxazone as a probe., J Clin

Pharmacol., 2006; 46(1): 109-114.

10. Forrest JA, Clements JA, Prescott LF, Clinical pharmacokinetics of

Paracetamol, Clin Pharmacokinet.,1982; 7: 93-107.

11. O’Neil MJ, The Merck Index: An Encyclopedia of chemicals, drugs and

biological, 14th Ed., Merck Research Laboratories, 2006; 9.

12. Sweetman SC, Martindale: The Complete Drug Reference, 37th edition Ed.

London: Pharmaceutical Press, 2011; 76-79



13. Levy G, Comparative pharmacokinetics of aspirin and acetaminophen, Arch

Intern Med. 1981; 141: 279-281.

14. Satoskar RS, Bhandarkar SD, Ainapure SS, Pharmacology and

pharmacotheraputics, 6th Ed., Popular prakashan, 1999; 162-163.

15. Solomon DH, Avorn J, Sturmer T, Glynn RJ, Mogun H, Schneeweiss S,

Cardiovascular outcomes in new users of coxibs and nonsteroidal

antiinflammatory drugs: high-risk subgroups and time course of risk, Arthritis

Rheum., 2006; 54(5): 1378-89.

16. Gan TJ, Diclofenac: an update on its mechanism of action and safety profile.,

Curr Med Res Opin., 2010; 26(7): 1715-31.

17. FitzGerald GA, Patrono C, The coxibs, selective inhibitors of cyclooxygenase-

2 N Engl J Med. 2000; 345(6): 433-42.

18. Sweetman SC, Martindale: The Complete Drug Reference, 37th edition Ed.

London: Pharmaceutical Press, 2011; 32-3.

19. Satoskar RS, Bhandarkar SD, Ainapure SS, Pharmacology and

pharmacotheraputics, 6th Ed., Popular prakashan, 1999; 164-166.

20. Goodman & Gilman’s The Pharmacological Basis of Therapeutics, 11th Ed.,

Mcgraw-hill medical publishing division, 1634-1644.

21. Forist AA, Judy RW, Comparative pharmacokinetics of chlorphenesin,

carbamate and methocarbamol in man, Journal of Pharmaceutical Sciences,

1971; 60: 1686-1688.

22. Weng N, HPLC and CE Methods for Pharmaceutical Analysis, J. Chromatogr.

B Biomed. Appl., 1994; 654: 287-292.

23. O’Neil MJ, The Merck Index: An Encyclopedia of chemicals, drugs and

biological, 14th Ed., Merck Research Laboratories, 2006; 1033.

24. Sica DA, Comstock TJ, Davis J, Manning L, Powell R, Melikian A, Wright G,

Pharmacokinetics and protein binding of methocarbamol in renal insufficiency

and normals. Eur J. Clin Pharmacol., 1990; 39(2): 193-94.

25. Roy D, Implementation of Revised Schedule ‘M’ and SSI, The Indian

Pharmacist, Dec. 2003; 8-11.

26. Brown P, Deanotonis K, Settle F, High Performance Liquid Chromatography,

Handbook of Instrumental Techniques for Analytical Chemistry Prentice Hall,

Inc., A Simon and Schuster Company, New Jersey, 1997; 147-159.

27. United States pharmacopoeia, XXII, 2007; 1740.



28. Mant CT, Hodges RS, High-Performance Liquid Chromatography of Peptides

and Proteins: Separations, Analysis, and Conformation, CRC Press: Boston,

1991; 158-163.

29. McClure WF, Process Analytical Chemistry & Spectroscopic tools, 2nd Ed.,

Wiley Publishers, 1994; 66: 43-54.

30. Brown PR, Weston A, Principle and practices of High Performance Liquid

Chromatography & Capillary Electrophoresis, 1st Ed., Academic Press, 1990;

62: 995-1008.

31. Davidson AG, Beckett AH, Stenlake JB, Chromatography In; Practical

Pharmaceutical Chemistry, 4th Ed., CBS Publishers and Distributors, New

Delhi, 1997; 2: 85-174.

32. Sethi PD, Charegaonkar D, Identification of drugs in Pharmaceutical

Formulations by Thin Layer Chromatography, 2nd Ed., CBS Publishers &

Distributers, 2008; 2-15.

33. Patel RB, Patel MR, Patel BG, Experimental Aspects and Implementation of

HPTLC, New York: Springer, 2011; 41- 54.

34. Willard HH, Merritt LL, Dean JA, Settle FA, HPLC Theory and

Instrumentation, Instrumental Methods of Analysis, 8th Ed., CBS Publishers

and Distributors, New Delhi, 2002; 581-617.

35. Miyabe K, Gniochon G, Brown, PR and Grushka E, Fundamental

Interpretation of the Peak in Linear Reversed Phase Liquid Chromatography,

Advances in Chromatography, Marcel Dekker, Inc., Newyork, 2000; 40: 1-

107.

36. Snyder LR, Kirkland JJ, Glajch LJ, Getting Started, Practical HPLC Method

Development, 2nd Ed., John Willey and Sons, Inc., Newyork, 1997; 5-1: 351-

360.

37. Willard HH, Merritt LL, Dean JA, Settle FA, HPLC Theory and

Instrumentation, Instrumental Methods of Analysis, 8th Ed., CBS Publishers

and Distributors, New Delhi, 2002; 1-12.

38. Snyder RI, Kirkland JJ, Glajesh JI, Basics of Separation: Practical HPCL

Method development, 2nd Ed., Published By John Wiley and sons Inc, New

York, 1997; 21-50.



39. Remington, Chromatography; The Science and Practice of Pharmacy Printed

by the Make Printing Company Eston, pennsylvania, 19th Ed. 1995; 1: 537-

544.

40. Sethi PD, HPLC Quantitative Analysis of Pharmaceutical Formulations, CBS

Publisher and distribors, New Delhi, 2001; 58-67.

41. Backett AH, Stenlke JB, Davidson A, Instrumental Methods in the

development and use of medicines; practical pharmaceutical chemistry, 4th

Ed., CBS Publishers and Distributors New Delhi, 2002; 11: 1-8.

42. Guidance for Industry: Analytical procedure and method validation chemistry,

manufacturing and controls documentation. Centre for drug evaluation and

research, Rockville, Maryland, August, 2004.

43. International Conference on Harmonization: Draft Revised Guidance on Q1A

Stability Testing New drug substances and products, Federal Register, 2000;

65: 246.

44. The United States Pharmacopoeia, 26th Ed., US Pharmacopoeial Convention,

Rockville, MD, 2003; 1151.

45. Munson JW, High-performance liquid chromatography: Theory,

instrumentation, and pharmaceutical applications. In; Munson, JW, Eds.

Pharmaceutical analysis modern methos part B, New York, Marcel Dekker,

Inc., 2001; 16-17.

46. Munson JW, Quantitative thin-layer chromatography. In; Munson, J.w., Eds.

Pharmaceutical analysis modern methods part B, New York, Marcel Dekker,

Inc., 2001; 155-156.

47. Albert R, Horwitz W, Validation of analytical procedure, Anal. Chem., 1997;

69: 789.

48. Singh S, Garg S, Understanding of analytical method validation and

characteristics, Pharma Times, 1999; 15-20.

49. Felinger A, Brown P R, Grushka E, Mathematical analysis of multi component

chromatograms, advance in chromatography, Marcel Dekker Inc., New York,

1998; 39: 201-248.

50. Ahuja S, Seypinski S, Handbook of modern pharmaceutical analysis,

academic press, New York, 2001; 346-356: 415-441.

51. The United State Pharmacopoeia, 24th Ed., US Pharmacopoeial Convention,

Rockville, MD, 2000; 1923.



52. ICH, Q2B Validation of Analytical Procedure: Methodology, in: Proceeding

of the International Conference on Harmonization, Geneva, March 1996.

53. ICH, Guidance on Analytical Method Validation, in: Proceedings of

International Convention on Quality for the Pharmaceutical Industry, Toronto,

Canada, September 2002.

54. Bakshi M, Singh S, Development of validated stability-indicating assay

methods-critical review, Journal of Pharmaceutical and Biomedical Analysis,

2002; 28: 1010-1040.

55. Rhodes CT, Introductory overview, Drug Stability: Principle and practices,

J.T.Carstensen, C.T.Rhodes (ed.) Marcel Dekker, New York, 2000: 1-12.

56. Matthews BR, Regulatory aspects of Stability Testing in Europe in: Drug

Stability:Principles and Practices, Carstensen JT and Rhodes C T, 3rd Ed.,

Marcel Dekker, New York, 2000; 107: 580.

57. Hong DD, Shah M, Development and validation of HPLC stability-indicating

assays.in: Drug Stability: Principles and Practices, J.T. Carstensen and C.T.

Rhodes, 3rd Ed., Marcel Dekker, New York, 2000; 107: 329-384.

58. Caviglioli G, Stability Indicating HPLC Assay for Retinoic Acid in Hard

Gelatin Capsules Containing Lactose and as Bulk Drug Substance, Drug

Dev.Ind.Phrm., 1994; 20: 2395-2408.

59. Floery K, Analytical profile of Drug Substances, Academic Press.London.UK,

1979; 8: 217-223.

Chapter 2 Review of Literature


2. REVIEW OF LITERATURE

The drugs are official in IP, BP, and USP. The official methods are based on UV and

HPLC techniques. Some of them require an internal standard, a tedious sample

preparation procedure, costly solvents and maintenance of column temperature etc.

The reported methods are mainly developed keeping in view the requirements of research

mainly in biological fluids using high analytical methods like HPLC-MS, HPTLC, ion

pair chromatography, fluorimetric detection using extraction procedures for sample

preparation. Consequently, many methods are focused on the analysis of the drug in

biological tissues and fluids.

Analytical methods for quantification of muscle relaxant drugs under study are discussed

in this chapter. Table 2.1.1and table 2.2.2 gives a summary of the official methods of

drugs taken under study. Table 2.2.1 gives a review of reported methods drugs as single

and combined formulations.

Several methods have been reported for the analysis of muscle relaxant drugs either in

single drug or mixture of drugs. Analytical methods for muscle relaxant drugs are

summarized in following table.

2.1 OFFICIAL METHODS FOR DRUG UNDER STUDY

Table 2.1.1 Official methods for Paracetamol

Drug Method Description Reference

Paracetamol Liquid

chromatography

Stationary phase:- stainless steel

column, 25cm x 4mm long packed with

octylsilane silica gel

Mobile phase:- Disodium hydrogen

phosphate: methanol (75:25 v/v)

Detection:- 243 nm

Flow rate:- 1.0 mL/min

1

Paracetamol

syrup

Liquid

chromatography


column, 20cm x 4.6mm long packed with

octadecylsilyl silica gel

Mobile phase:- 0.01 Soidumbutane

1



sulphonate: methanol (85:15 v/v)

Detection:- 243 nm


Paracetamol

tablets

Liquid

chromatography

method


column, 20cm X 4.6mm long packed

with octadecylsilane silica gel

Mobile phase:- 0.01 Sodium

butanesuphonate in 85ml water:

methanol and 0.4 volumes of formic acid

Detection:- 243 nm


1

Paracetamol Titration Titrant:- 0.1 M Cerium sulphate 2

Paracetamol

capsule

HPLC Stationary phase:- Nitrile gp bonded

with porus silica (3-10µm) 3.9mm x

30cm

Mobile phase:- water: methanol (90:10

v/v)

Detection:- 243 nm


3

Paracetamol

oral solution



30cm


v/v)

Detection:- 243 nm


3

Paracetamol UV spectroscopy Wavelength: 243nm 3

Paracetamol

effervescent

oral solution



30cm


v/v)

3



Detection:- 243 nm


Paracetamol

suppository



30cm


v/v)

Detection:- 243 nm


3

Paracetamol

oral

suspension



30cm


v/v)

Detection:- 243 nm


3

Paracetamol

tablet



30cm


v/v)

Detection:- 243 nm


3

Paracetamol

extended

release



30cm


v/v)

Detection:- 243 nm


3



Table 2.1.2 Official methods for Paracetamol in combination


Paracetamol

and Aspirin



30cm

Mobile phase:- Chloroform:

methanol: glacial acetic acid (70:20:1

v/v/v)

Detection:- 280 nm


3

Paracetamol,

Aspirin and

Caffeine



10cm



v/v/v)

Detection:- 275 nm


3

Paracetamol

and Caffeine



10cm



v/v/v)

Detection:- 275 nm


3

Table 2.1.3 Official methods for Diclofenac potassium in combination


Diclofenacpotassium

Liquidchromatography

Stationary phase:-

Mobile phase:- Methanol: phosphate

buffer (pH 2.5) (70:30 v/v)

4



Detection:- 254 nm

Flow rate:- 1.0 mL/minDiclofenacpotassiumtablets

HPLC Stationary phase:-


buffer (pH 2.5) (70:30 v/v)

Detection:- 254nm


4

Diclofenacpotassiumdelayedrelease


Stationary phase:-


buffer (pH 2.5) (70:30 v/v)

Detection:- 254 nm


4

Diclofenactablet

TLC Stationary phase:- Plate coated with

silica gel GF254

Mobile phase:- Toluene: hexane:

formic acid (90:5:5 v/v/v)

Detection:- 254 nm

5

Diclofenacsodiuminjection

TLC Stationary phase:- Plate coated with

silica gel GF254 plate


acetone: formic acid (90:5:5 v/v/v)

Detection:- 254 nm

5

Diclofenacpotassium

Potentiometric Solvent:- Anhyrous acetic acid

Titrant:- 0.1M perchloric acid

6

Table 2.1.4 Official methods for Chlorzoxazone in combination


Chlorzoxazonetablets


Stationary phase:-

Mobile phase:- Water: acetonitrile:

glacial acetic acid (70:30:1 v/v/v)

Detection:- 280 nm


7



Table 2.1.5 Official methods for Methocarbamol


Methocarbamol UV Detection:- 274 nm 8

Methocarbamol

injections

UV Detection:- 274 nm 8

Methocarbamol

tablet

Liquid

chromatography

Stationary phase:-

Mobile phase:- Phospate

buffer(pH 4.5): methanol (75:25

v/v)

Detection:- 280 nm


8



2.2 REPORTED METHODS FOR DRUG UNDER STUDY

Table 2.2.1 Reported methods for Paracetamol

Drug Method and description ReferenceParacetamol Title:-

Reverse Phase HPLC Method for Determination of

Aceclofenac and Paracetamol in Tablet Dosage Form.

Description

Stationary phase:- ODS C18

Mobile phase:- methanol: water (70:30 v/v)


Detection:- 245 nm

9

Paracetamol Title:-

Reverse phase HPLC method for determination of

aceclofenac and paracetamol in tablet dosage forms

Description

Stationary phase:- ODS C18



Detection:- 255 nm

10

Paracetamol Title:-

RP-HPLC method for determination of aceclofenac,

chlorzoxazone and paracetamol in bulk and

pharmaceutical formulation

Stationary phase:- Reverse Phase C18

Mobile phase:- acetonitrile: water (60:40 v/v)


Detection:- 230 nm

11



Paracetamol Title:-

Validation of a RP-HPLC method for simultaneous

determination of paracetamol, methocarbamol, and

diclofenac potassium in tablets

Description:-

Stationary phase:- C18 column ODS Hypersil

Mobile phase:- 25mM phosphate buffer: acetonitrile

(65:35 v/v)


Detection:- 225 nm

12

Paracetamol Title:-

Simultaneous determination of paracetamol,

pseudoephedrine, dextrophan and chlorpheniramine in

human plasma by liquid chromatography–tandem mass

spectrometry

Description:-

Stationary phase:- C18 column ODS

Mobile phase:- acetic acid: methanol (20:80 v/v)


Detection:- 275 nm

13

Paracetamol Title:-

HPLC Method for the Analysis of Paracetamol,

Caffeine and Dipyrone

Description:-

Stationary phase:- C8 column (µ Bondapack)

Mobile phase:- methanol: acetonitrile: isopropyl alcohol

(40:30:30 v/v)


Detection:- 215 nm

14



Paracetamol Title:

Spectrophotometric Methods for Simultaneous

Estimation of Dexibuprofen and Paracetamol

Description:

Solvent:- methanol

Wavelength:- 249.5 nm

15

Paracetamol Title:

Simultaneous determination of paracetamol and

lornoxicam by RP-HPLC in bulk and tablet formulation

Description:

Stationary phase:- C18 column ODS

Mobile phase:- methanol : water (85:15v/v)


Detection:- 259 nm

16

Paracetamol Title:

Determination of paracetamol and tramadol

hydrochloride in pharmaceutical mixture using HPLC

and GC-MS

Description:

Stationary phase:- C18 column

Mobile phase:- phosphate buffer: acetonitrile (90:10 v/v)

Detection:- 220 nm

17

Paracetamol Title:

Sensitive liquid chromatography–tandem mass

spectrometry method for the simultaneous determination

of paracetamol and guaifenesin in human plasma

Description:


Mobile phase:- phosphate buffer: methanol (80:20 v/v)

Detection:- 225 nm

18



ParacetamolTitle:-

Simultaneous Determination of Paracetamol and

Caffeine in Human Plasma by LC–ESI–MS

Description:

Stationary phase:- C18

Mobile phase:- formic acid: methanol (60:40 v/v)

Detection:- 254 nm

19

Paracetamol Title:

UV spectrophotometric and RP-HPLC method

development for simultaneous determination of

paracetaomol and etodolac in pharmaceutical dosage

form

Description:

Solvent:- methanol

Wavelength:- 226 nm

20

Paracetamol Title:

Stability indicating method for the determination of

paracetamol in its pharmaceutical preparations by TLC

densitometric method

Description:

Stationary phase:- silica gel 60F254

Mobile phase:- acetic acid: benzene: acetic acid

(1:1:0.05 v/v/v)

Detection:- 250 nm

Range:- 5-20 µg/spot

21



Paracetamol Title:-

The quantification of Paracetamol glucuronide and

Paracetamol sulphate in plasma and urine using a single

high-performance liquid chromatography assay

Description:

Wavelength:- 254 nm

Mobile phase:- KH2PO4: isopropanol-TGF (98:1.5:0.1

v/v/v)

22

Paracetamol Title:-

Liquid chromatographic determination of cetrizine

hydrochloride and paracetamol in human plasma and

pharmaceutical formulations

Description:




Detection:- 230 nm

23

Paracetamol Title:-

Determination of paracetamol and orphenadrine citrate in

dosage formulations and in human plasma

Description:




Detection:- 215 nm

24



Paracetamol Title:-

A validated method for the determination of

paracetamol and its glucuronide and sulphate

metabolites in the urine of HIV/AIDS patients using

wavelength-switching UV detection

Description:


Mobile phase:- 50mM sodium acetate buffer :

acetonitrile (96:4 v/v)


Wavelength:- 254 nm

25

Paracetamol Title:-

Simultaneous estimation of Paracetamol and

Tolperisone hydrochloride in tablet by UV

spectrophotometric methods

Description:

Solvent:- metanol

Wavelength:- 254 nm

26

Paracetamol Title:-

Simultaneous Determination of Paracetamol and

Piroxicam in Tablets by Thin Layer Chromatography

Combined with Densitometry

Description:

Stationary phase:- silica gel GF254

Mobile phase:- n-Dichloroethane: methanol:

triethylamine (10:2.5:1 v/v/v)

Detection:- 288 nm

Range:- 1.625-14.625 µg/spot

27



Paracetamol Title:-

RP-UPLC method for combinational assay of

lornoxicam and paracetamol in combined tablet dosage

form

Description

Stationary phase:- UPLC BEH C18

Mobile phase:- acetonitrile:30mM Ammonium acetate

(40:50, v/v)


Detection:- 260 nm

28

Paracetamol Title:-

Simultaneous determination of aceclofenac,

paracetamol, and chlorzoxazone by RP-HPLC in

pharmaceutical dosage form.

Description

Stationary phase:- Column:- C18 (Zorbax )

Mobile phase:- acetonitrile : buffer (40:60 v/v)


Detection:- 220 nm

29

Paracetamol Title:-

Simultaneous HPTLC Determination of paracetamol

and dexketoprofen trometamol in pharmaceutical dosage

form

Description

Stationary phase:- Silica gel G 60F254 plates

Mobile phase:- toluene: ethyl acetate: acetic acid (6: 4:

0.2 v/v/v)

Detection:- 256 nm

Range:- 25-150 ng/spot

30



Table 2.2.2 Reported methods of Diclofenac potassium

Diclofenac Title:-

Validated stability indicating RP-HPLC method for

simultaneous determination & in vitro dissolution studies of

thiocolchicoside and diclofenac potassium from tablet

dosage forms.

Description

Stationary phase:- C18 column (Zorbax SB CN)

Mobile phase:- 5 mM sodium dihydrogen phosphate:

methanol (60:40 v/v)


Detection:- 258 nm

31

Diclofenac Title:-

Development of UV spectrophotometric methods for

simultaneous estimation of Famotidine and Diclofenac

potassium in combined dosage form using simultaneous

equation method

Description

Solvent:- methanol

Detection:- 286 nm

32

Diclofenac Title:-

New Stability-Indicating RP-HPLC Method for

Determination of Diclofenac Potassium and Metaxalone

from Their Combined Dosage Form

Description

Stationary phase:- C18 column (Hibar Lichrosphere-100)



Detection:- 280 nm

33



Diclofenac Title:-

A validated RP-HPLC method for simultaneous

estimation of paracetamol and diclofenac potassium in

pharmaceutical formulation

Description

Stationary phase:- C18 column (Phenomenex LUNA)

Mobile phase:- acetonitrile: sodium dihydrogen ortho

phosphate (70:30 v/v)


Detection:- 278 nm

34

Diclofenac Title:-

Simultaneous estimation of diclofenac and rabeprazole

by high performance liquid chromatographic method in

combined dosage forms

Description

Stationary phase:- C18 column (HiQ SiL)

Mobile phase:- water: methanol: acetonitrile (20:40:40

v/v)


Detection:- 284 nm

35

Diclofenac Title:-

Validated HPLC Method for Simultaneous Quantitation

of

Diclofenac Sodium and Misoprostol in Bulk Drug and

Formulation

Description

Stationary phase:- C18 (Hypersil BDS)



Detection:- 220 nm

36



Diclofenac Title:-

High Performance Thin Layer Chromatographic method

for determination of diclofenac sodium in pharmaceutical

formulations

Description

Stationary phase:- silica gel 60 F254 aluminium plate

Mobile phase:- toluene : ethyl acetate : glacial acetic acid

(60:40:1 v/v/v)

Detection:- 282 nm

Linearity:- 5-80 µg/mL

37

Diclofenac Title:-

Validated HPTLC method for simultaneous estimation of

diclofenac potassium and metaxalone in bulk drug and

formulation

Description

Stationary phase:- silica gel 60 F254 aluminum plate

Mobile phase:- chloroform: methanol (8:1 v/v)

Detection:- 282 nm

Linearity:- 100-180 ng/mL

38

Diclofenac Title:-

Development and validation of a bioanalytical method for

direct extraction of diclofenac potassium from spiked

plasma

Description

Stationary phase:- C18 column (Hypersil)

Mobile phase:- acetonitrile: triethylamine (80:20 v/v)


Detection:- 276 nm

39



Diclofenac Title:-

A RP-HPLC method for determination of diclofenac

with rabeprazole in solid dosage form

Description

Stationary phase:- C18 column (Phenomenox)

Mobile phase:- acetonitrile: 50mM ammonium acetate

buffer (60:40 v/v)


Detection:- 254 nm

40

Diclofenac Title:-

Development and validation of a HPTLC method for

simultaneous densitometric analysis of diclofenac

potassium and dicyclomine hydrochloride as the bulk

drugs and in the tablet dosage form

Description

Stationary phase:- silica gel 60 F254 plates

Mobile phase:- toluene:methanol:acetic acid (8:2:0.1

v/v/v)

Wavelengh:- 272 nm

Linearity:- 0.2-1.6 ng/spot

41



Table 2.2.3 Reported methods for Chlorzoxazone

Chlorzoxazone Title:-

Simultaneous estimation of paracetamol, chlorzoxazone and

diclofenac potassium in pharmaceutical formulation by RP-

HPLC method

Description


Mobile phase:- methanol-0.01M monobasic sodium

phosphate (70:30 v/v)


Detection:- 254 nm

42


Simultaneous determination of paracetamol, diclofenac

sodium and chlorzoxazone by HPLC from tablet

Description

Stationary phase:- C18 column (Sodex)

Mobile phase:- methanol: water: triethylamine (55:45:0.1

v/v/v)


Detection:- 275 nm

43


Determination of 6-hydroxychlorzoxazone and

chlorzoxazone in porcine microsome samples

Description

Stationary phase:- C18 column (ODS-AQ)

Mobile phase:- phosphoric acid: methanol (60:40 v/v)


Detection:- 287 nm

44




Simultaneous estimation of tramadol hydrochloride and

chlorzoxazone by absorbance correction method

Description

Solvent:- methanol

Wavelength:- 225 nm

45


Determination of Cytochrome P450 2El Activity in

Microsomes by Thin-Layer Chromatography Using [2-14C]

Chlorzoxazone

Description

Stationary phase:- silica gel 60 F254 plates

Mobile phase:- acetone: hexane (45:55 v/v)


Detection:- 287 nm

46


The Simultaneous Estimation of aceclofenac, paracetamol and

chlorzozazone in pharmaceutical dosage forms by HPTLC

Description

Stationary phase:- silica gel G60F254

Mobile phase:- toluene: ethyl acetate: glacial acetic acid

(17.5:10:0.5 v/v)

Detection:- 271 nm

47


Simultaneous determination of paracetamol and chlorzoxazone

using orthogonal functions ratio spectrophotometry

Description

Solvent:- methanol

Wavelength:- 287 nm

48



Table 2.2.4 Reported methods for Methocarbamol

Methocarbamol Title:-

Simultaneous spectrophotometric estimation of ibuprofen

and methocarbamol in tablet dosage form

Description

Solvent:- methanol

Wavelengh of ibuprofen:- 222 nm

Wavelengh of methocarbamol:- 224 nm

49


Simultaneous determination of paracetamol and

methocarbamol in tablets by ratio spectra derivative

spectrophotometry and LC

Description

Solvent:- methanol

Wavelengh of paracetamol:- 243 nm

Wavelengh of methocarbamol:- 230 nm

50


Determination of acetaminophen and methocarbamol in

Bilayered tablets using RP-HPLC

Description

Stationary phase:- C18 column (ODS-MG-5)

Mobile phase:- water: methanol: glacial acetic (600:400:15)


Detection:- 273 nm

51


Determination of methocarbamol concentration in human

plasma by high performance liquid chromatography–

tandem mass spectrometry

Description

Range:- 150-12000 ng/mL

52




A stability-indicating high-performance liquid

chromatographic method for the determination of

methocarbamol in veterinary preparations

Description


Mobile phase:- methanol: water: tetrahydrofuran (25:65:10

v/v/v)


Detection:- 274 nm

53


Stability indicating HPLC method for simultaneous

determination of methocarbamol and nimesulide from tablet

matrix

Description

Stationary phase:- supercosil LC-8



Detection:- 276 nm

54


Simultaneous HPLC Determination of methocarbamol,

paracetamol and diclofenac Sodium

Description

Stationary phase:- supelcosil LC-8

Mobile phase:- methanol: water: glacial acetic acid

(400:600:05 v/v/v)


Detection:- 275 nm

55



Methocarbamol

Title:-

Simultaneous determination of diclofenac potassium and

methocarbamol in ternary mixture with guaifenesin by

reversed phase liquid chromatography

Description

Stationary phase:- C18 column (Symmetry Waters)

Mobile phase:- phosphate buffer: acetonitrile (20:80 v/v)


Detection:- 282 nm

56



References1. Indian Pharmacopoeia Vol.II, Government of India, Ministry of health &

family welfare, published by the Indian Pharmacopoeial Commission, 2007;

1514-1516.

2. British Pharmacopoeia Vol.II, Department of health, social services & public

safety, London: stationary office, 2007: 1575-1576.

3. United States of Pharmacopoeia NF vol.II, The official compendia of

standards, Asian edition, 2007; 1388-1392.

4. Indian Pharmacopoeia Vol.II, Government of India, Ministry of health &

family welfare, published by the Indian Pharmacopoeial Commission, 2007;

1020-22.

5. United States of Pharmacopoeia NF Vol.I, The official compendia of

standards, Asian edition, 2007; 1921-1924.

6. British Pharmacopoeia Vol.I, Department of health, social services & public

safety, London: stationary office, 2007: 665.

7. United States of Pharmacopoeia NF Vol.II, The official compendia of

standards, Asian edition, 2007; 1741.

8. United States of Pharmacopoeia NF Vol.II, The official compendia of

standards, Asian edition, 2007; 2610.

9. Godse VP, Deodhar MN, Bhosale AV, Sonawane RA, Sakpal PS, Borkar DD,

Bafana YS, Reverse Phase HPLC method for determination of aceclofenac

and paracetamol in Tablet Dosage Form, Asian J. Research Chemistry, 2009;

(2)1: 37-40.

10. Momin MY, Yeole PG, Puranik MP, Wadher SJ, Reverse phase HPLC

method for determination of aceclofenac and paracetamol in tablet dosage

forms, Indian Journal of Pharmaceutical Science, 2006; 68(3): 387-389.

11. Yadav AH, Kothapalli LP, Barhate AN, Pawar I, Pawar CR, RP-HPLC

method for determination of aceclofenac, chlorzoxazone and paracetamol in

bulk and pharmaceutical formulation, International Journal of Pharma

Research and Development, 2009; 1(10): 1-7.



12. Subraminian G, Vasudevan M, Ravishakar S, Suresh B, Validation of a RP-

HPLC method for simultaneous determination of paracetamol,

methocarbamol, and diclofenac potassium in tablets, Indian Journal of

Pharmaceutical Sciences, 2005; 4(2): 260-263.

13. Hong Gang L, Hong Y, Ruan Z, Jiang B, Simultaneous determination of

Paracetamol, pseudoephedrine, dextrophan and chlorpheniramine in human

plasma by liquid chromatography-tandem mass spectrometry, Journal of

Chromatography B, 2010; 878: 682-688.

14. Levent AM, HPLC Method for the Analysis of Paracetamol, caffeine and

dipyrone, Turk J Chemistry, 2002; 26(1): 521-528.

15. Chitlange S, Soni R, Wankhede S, Kulkarni A, Spectrophotometric Methods

for Simultaneous Estimation of Dexibuprofen and Paracetamol, Asian J.

Research Chem. 2009; 2(1): 30-33.

16. Jagtap AS, Yadav SS ,Rao JS, Simultaneous determination of paracetamol

and lornoxicam by RP-HPLC in bulk and tablet formulation, Pharmacie

Globale, 2011; 9(4): 1-4.

17. Belal T, Awad T, Clark C R, Determination of paracetamol and tramadol

hydrochloride in pharmaceutical mixture using HPLC and GC-MS, Journal of

chromatographic science, 2009; 47(10): 849-854.

18. Chen X, Huang J, Kong Z, Zhong D, Sensitive liquid chromatography-tandem

mass spectrometry method for the simultaneous determination of paracetamol

and guaifenesin in human plasma, Journal of Chromatography B, 2005;

817(2): 263-269.

19. Anna Wang, Jin Sun, Haijun Feng, Shuo Gao and Zhonggui He, Simultaneous

Determination of Paracetamol and Caffeine in Human Plasma by LC–ESI–

MS, Chromatographia, 2010; 67(3): 281-285.

20. Jadi MK, Narayan UL, UV spectrophotometric and RP-HPLC method

development for simultaneous determination of paracetaomol and etodolac in

pharmaceutical dosage form, 2009; 56(1): 169-174.



21. Nadia M. Mostafa, Stability indicating method for the determinationof

paracetamol in its pharmaceutical preparations by TLC densitometric method,

Journal of Saudi Chemical Society 2010; 14: 341-344.

22. Jensen LS, Valentine J, Milne RW, Evans AM, The Quantification of

Paracetamol, Paracetamol Glucuronide and Paracetamol Sulphate in Plasma

and Urine Using a Single High-Performance Liquid Chromatography Assay,

J. Pharm. Biomed. Anal.,2004; 34: 585-593

23. Basavaraj S, Nagaralli J, Seetharamappa B, Gowda G, Mahaveer B, Liquid

Chromatographic Determination of Ceterizine and Paracetamol in Human

Plasma and Pharmaceutical Formulations, J. Chromatogr. B., 2003; 798: 49-

54.

24. Arayne MS, Determination of paracetamol and orphenadrine citrate in dosage

formulations and in human plasma, Journal of Chinese Chemical Society,

2009; 56: 169-174.

25. Girolamo O, Neill M, Wainer IW, A Validated Method for the Determination

of Paracetamol and its Glucuronide and Sulphate Metabolites in the Urine of

HIV/AIDS Patients Using Wavelength-Switching UV Detection, J. Pharm.

Biomed. Anal., 1998; 17: 1191-1197.

26. Patel MG, Parmar RR, Nayak PP, Shah SA, Simultaneous estimation of

Paracetamol and Tolperisone hydrochloride in tablet by UV

spectrophotometric methods, Journal of Pharmaceutical Science and

Bioscientific Research, 2012; 2(2) 63-67.

27. Atul A, Shirkhedkar, Afsar M. Shaikh and Sanjay J. Surana, Simultaneous

determination of paracetamol and piroxicam in tablets by TLC combined with

densitometry, Shirkhedkar A A, Shaikh A M, Surana S J, Eurasian Journal of

Analytical Chemistry, 2008; 3(2): 258-267.

28. Srinivasu T, Rao BN, Annaoura MM, Chandrashekhar TG, RP-UPLC method

for combinational assay of lornoxicam and paracetamol in combined tablet

dosage form, International Journal of Pharma. Science & Research, 2012;

3(4): 1149-1154.



29. Shaikh KA, Devkhile AB, Simultaneous determination of aceclofenac,

paracetamol, and chlorzoxazone by RP-HPLC in pharmaceutical dosage form,

Journal of Chromatographic Sciences, 2008; 46(7): 649-652.

30. Rao JR, Mulla TS, Bharekar VV, Yadav SS, Rajput MP, Simultaneous

HPTLC determination of paracetamol and dexketoprofen trometamol in

pharmaceutical dosage form, Der Pharma Chemica, 2011; 3(3): 32-38.

31. Jadhav SD, Butle SR, Patil SD, Jagdap P K, Validated stability indicating RP-

HPLC method for simultaneous determination & in vitro dissolution studies of

thiocolchicoside and diclofenac potassium from tablet dosage forms., Arabian

Journal of Chemistry, 2012, Article in Press.

32. Mehta SA, Umarkar AR, Chaple DR, Thote LT, Development of UV

Spectrophotometric Methods for Simultaneous Estimation of Famotidine and

Diclofenac Potassium in Combined Dosage Form Using Simultaneous

Equation Method, Journal of Pharmacy Research, 2011; 4(7), 2045-2046.

33. Panda SS, Patnaik d, Ravikumar B, New Stability-Indicating RP-HPLC

Method for

Determination of Diclofenac Potassium and Metaxalone from Their

Combined Dosage Form, Scientia Pharmaceutica, 2012, Article in Press.

34. Gowramma B, Rajan S, Muralidharan S, Meyyanathan S, and Suresh B, A

validated RP-HPLC method for simultaneous estimation of paracetamol and

diclofenac potassium in pharmaceutical formulation, International Journal of

Chemtech Research, 2010; 2(1), 676-680.

35. Choudhary B, Goyal A, Khokra S , Kaushik B, Simultaneous estimation of

diclofenac and rabeprazole by high performance liquid chromatographic

method in combined dosage forms, International Journal of Pharmaceutical

Science and drug research, 2009; 1(1): 43-45.

36. Dhaneshwar SR, Bhusari VK, Validated HPLC method for simultaneous

quantitation of diclofenac sodium and misoprostol in bulk drug and

formulation, Pelagia Research Library Der Chemica Sinica, 2010; 1(2): 110-

118.



37. Thongchai W, Liawruangrath B, Thongpoon C, MachanHigh T, High

Performance Thin Layer Chromatographic method for determination of

diclofenac sodium in pharmaceutical formulations, Chiang Mai J. Sci. 2006;

33(1): 123-128.

38. Rajput MP, Bharekar VV, Yadav SS, Mulla TS, Rao JR,Validated HPTLC

method for simultaneous estimation of diclofenac potassium and metaxalone

in bulk drug and formulation, Pharmacie Globale International Journal of

Comprehensive Pharmacy, 2011; 12 (04): 1-4.

39. Sarfraz A, Sarfraz M, Ahmad M, Development and validation of a

bioanalytical method for direct extraction of diclofenac potassium from spiked

plasma, Tropical Journal of Pharmaceutical Research, 2011; 10(5): 663-69.

40. Arunadevi S, Subramania M, Suresh B, A RP-HPLC method for

determination of diclofenac with rabeprazole in solid dosage form, Pharma

science monitor, 2011; 2(2): 171-178.

41. Potawale SE, Nanda RK, Bhagwat VV, Hamane SC, Deshmukh S,

Puttamsetti S, Development and validation of a HPTLC method for

simultaneous densitometric analysis of Diclofenac potassium and

Dicyclomine hydrochloride as the bulk drugs and in the tablet dosage form,

Journal of Pharmacy Research, 2011; 4(9): 3116-3118.

42. Biswas A, Basu A, Simultaneous estimation of paracetamol, chlorzoxazone

and diclofenac potassium in pharmaceutical formulation by RP-HPLC

method, International Journal of Pharma and Bio Sciences, 2010; 1(2): 1-5.

43. Shinde VM, Desai BS, Tendolkar NM, Simultaneous determination of

paracetamol, diclofenac sodium and chlorzoxazone by HPLC from tablet,

Indian Journal of Pharma. Sciences, 1995; 57(1): 35-37.

44. Sherry K, Tina H, Joe B, Determination of 6-hydroxychlorzoxazone and

Chlorzoxazone in Porcine Microsome samples, J. Chromatogr. B, 2003; 78:

111-116.

45. Thakur AD, Hajare AL, Nikhade RD, Chandewar AV, Simultaneous

estimation of tramadol hydrochloride and chlorzoxazone by absorbance

correction method, Journal of Pharmacy Research, 2011,4(6): 1683-1684.



46. Zerillia A, Lucas D, Berthou F, Determination of Cytochrome P450 Activity

in Microsomes by Thin-Layer Chromatography Using Chlorzoxazone, J.

Chromatogr. B, 1996, 677: 156-160.

47. Yuvaraj G, Pavan Kumar P, Elangovan N, The Simultaneous Estimation of

Aceclofenac, Paracetamol and Chlorzozazone in Pharmaceutical Dosage

Forms by HPTLC, International Journal of Chemical and Pharmaceutical

Sciences, 2010; 1(2): 24-27.

48. Wahbi AM, Gazy AA, Abdel-Razak O, Mahgoub H, Moneeb MS,

Simultaneous determination of paracetamol and chlorzoxazone using

orthogonal functions- ratio spectrophotometry, Saudi Pharmaceutical Journal,

2003; 11(4): 192-200.

49. Satheeshamanikandan TRS, Wali DC, Banwal J, Kadam SS, Dhaneshwar SR,

Simultaneous spectrophotometric estimation of ibuprofen and methocarbamol

in tablet dosage form, Indian Journal of Pharmaceutical Sciences, 2004; 16:

810-813.

50. Erk N, Zkan Y, Banog E, Simultaneous Determination of Paracetamol and

Methocarbamol in Tablets by Ratio Spectra Derivative Spectrophotometry

and LC, J. Pharm. Biomed. Anal.24, 2000; 14: 469-475.

51. Mallikarjuna RB, Madhavi BR, DurgaDevi NK, Praveen PS, Mrudula BS,

Neelima RT, Determination of Acetaminophen and Methocarbamol in

Bilayered tablets using RP-HPLC, International Journal of Chemical and

Analytical Science 2010; 1(7): 158-160.

52. Zha W, Zhu Z, Determination of methocarbamol concentration in human

plasma by high performance liquid chromatography–tandem mass

spectrometry, J Chromatogr B Analyt Technol Biomed Life Sci., 2010;

15(10): 831-835

53. Rosasco MA, Ceresole RS, Forastieri CC, Segall AI, A stability-indicating

high-performance liquid chromatographic method for the determination of

methocarbamol in veterinary preparations, J AOAC Int, 2009; 92(5): 1602-

1605.



54. Manmode RS, Dhamankar AK, Manwar JV, and Laddha SS, Stability

indicating HPLC method for simultaneous determination of methocarbamol

and nimesulide from tablet matrix, Pelagia Research Library Der Chemica

Sinica, 2011; 2(4): 81-85.

55. Hafsa D, Chanda S, Prabhu P, Simultaneous HPLC Determination of

Methocarbamol, Paracetamol and Diclofenac Sodium, E Journal of

Chemistry, 2011; 8(4): 1620-1625.

56. Elkady EF, Simultaneous determination of diclofenac potassium and

methocarbamol in ternary mixture with guaifenesin by reversed phase liquid

chromatography, Talanta, 2010; 82(4): 1604-1607.

Chapter 3 Aim of the present work


3. AIM OF THE PRESENT WORKThere are rapidly increasing number of drug groups (and increasing numbers of drugs

within each group) available to treat muscle constriction. A day by day number of newer

muscle relaxant formulations either in single or in combined dosage forms are marketed

as well as is under investigation. Literature reveals that numbers of methods are available

for analysis of muscle relaxant drugs but so far stability indicating methods have not been

reported.

The objective of this work was to develop and validate chromatographic methods, which

would serve as stability indicating assay method for drug product. So the present

investigation was undertaken with a view to develop and validate new chromatographic

methods which could be applied for routine analysis of muscle relaxant drugs in

formulations. It will be tried that the methods should be,

Simple

Accurate

Precise

Selective

Specific

Reproducible

Highly sensitive

Stability indicating

The proposed methods can be made useful in routine analysis of such drugs in bulk and

in pharmaceutical formulations in single as well as in multi drug combinations in a

simple, convenient, and cost effective way.

THUS AIM OF THE PRESENT WORK INCLUDES:

To develop and validate stability indicating RP-HPLC and HPTLC method for

simultaneous estimation of Diclofenac Potassium, Chlorzoxazone and

Paracetamol in bulk powder and their pharmaceutical formulations.

To develop and validate stability indicating RP-HPLC and HPTLC method for

simultaneous estimation of Diclofenac Potassium, Paracetamol and

Methocarbamol in bulk powder and their pharmaceutical formulations.

Chapter 4 Chemicals, glasswares and instruments


4. CHEMICALS, GLASSWARES AND INSTRUMENTS

Chemicals used were of analytical (AR) grade except specified and the glasswares used

in the experimental work were calibrated in the laboratory and out of all only those were

in calibration limit were selected for work[1]. The glasswares were cleaned according to

the pharmacopoeial procedure before they were used for the experiment[2]. While the

instruments were used for the work were passed through routine calibration procedure.

4.1 Chemicals

Water-triple distilled

Methanol-HPLC grade (S.D. Fine Chemicals, Mumbai)

Acetonitrile-HPLC grade (S.D. Fine Chemicals, Mumbai)

Disodium hydrogen phosphate (Merck Ltd, Mumbai )

Sodium dihydrogen phosphate (Merck Ltd, Mumbai)

Dimethyl formamide (Rankem Ltd, New Delhi )

Ammonium acetate (Rankem Ltd, New Delhi)

Sodium hydroxide (Rankem Ltd, New Delhi )

Hydrochloric acid (Merck Ltd, Mumbai )

Hydrogen peroxide (Merck Ltd, Mumbai)

Glacial acetic acid (Rankem Ltd, Mumbai)

O-Phosphoric acid (Merck Ltd, Mumbai)



4.2 Active Pharmaceutical Ingredients (API)

Table 4.2.1: List of Active Pharmaceutical Ingredient

Sr.

No.

Name Batch No A.R.No. Supplier

1 Diclofenac

potassium

DFK/19040018 DFK/09/0018 Zydus Cadila

Ltd

2 Chlorzoxazone MNB/94/2 CHA/10/0153 Zydus Cadila

Ltd

3 Paracetamol 0509/11 FPC/PRL/0509/11 Zydus Cadila

Ltd

4 Methocarbamol CN/1127 ARD3170185 Zydus Cadila

Ltd

4.3 Glasswares

Volumetric flask (10, 25, 50, 100, 250 mL)

Graduated pipette (1, 2, 5, 10 mL)

Measuring cylinder (50, 100 mL)

Thermometer (100°C)

Petri-dish

Conical flask (100, 250 mL)

Glass beaker (50, 100, 250, 500 mL)

Plastic beaker (250, 500 mL)

Funnel

Round bottom flask (250 mL)

Condenser

4.4 Instruments

1. HPLC

System: Younglin

Pump: Solvent delivery modal LC-10ATVP

Column: Varian C-18 (250 Χ 4.6 mm i.d, 5 μm particle size)

Injector: Microliter syringe (Rheodyne)



Detector: Dual wavelength UV Detector

Software: Autochro-3000

2. HPTLC

System: CAMAG

Sample applicator: Linomat-V

Injector: Hamilton syringe (100μl)

Detection: CAMAG TLC Scanner 3

Developing chamber: CAMAG TLC chamber (20x10cm, 20x20cm)

Operation software: WinCATs

3. UV-Visible spectrophotometer

System: ELICO SL 210

Spectral range: 190 to 1100nm

Bandwidth: 1.8nm

Stray light: <0.05%T at 220nm with Nal 10g/L

Light source: Deuterium Lamp (D2) & Tungsten (W) Lamp

Monochromator: Concave holographic grating with 1200lines/mm

Detector: Photo Diode

Control: Microprocessor and Microcontroller (Computer-Optional)

4.5 Other requirements

1. Ultrasonic cleaner

Model-D compact 1.5 litre (EIE Instruments Pvt. Ltd.)

2. Digital analytical balance

Electronic Balance BL-220H Shimadzu Corporation Japan (1mg sensitivity)

3. pH meter (digital)

LI127 Elico Limited

4. FIlter

Ultipore N66 Nylon membrane (Pall India Pvt. Ltd.) (0.45µm and 0.2µm)



4.6 References

1. Indian Pharmacopeia, Government of India ministry of health and family welfare,

1996, Vol-II, published by the controller of publications, Delhi, Appendix 1.5, A-

8.

2. Indian Pharmacopeia, Government of India ministry of health and family welfare,

1996, Vol-II, published by the controller of publications, Delhi, Appendix 12.5,

A-138.

Chapter 5 Stability indicating methods for muscle relaxant drugs


5. STABILITY INDICATING METHOD DEVELOPMENT AND

VALIDATION FOR SIMULTANEOUS ESTIMATION OF

DICLOFENAC POTASSIUM, CHLORZOXAZONE AND

PARACETAMOL

5.1 STABILITY INDICATING HPLC METHOD DEVELOPMENT AND

VALIDATION FOR SIMULTANEOUS ESTIMATION OF DICLOFENAC

POTASSIUM, CHLORZOXAZONE AND PARACETAMOL5.1.1 Experimental

5.1.1.1 Solubility

The Solubility of Diclofenac potassium (DIC), Chlorzoxazone (CHL) and Paracetamol

(PCM) were performed in different solvent like distilled water, 0.1N HCl, 0.1N NaOH,

methanol, dimethylformamide, acetonitrile, ethanol and chloroform. All three drugs were

found to be soluble in methanol.

5.1.1.2 Preparation of mobile phase

The mobile phase methanol:phosphate buffer pH 4.0 in the ratio of 70:30, v/v

respectively was used. The pH was adjusted with glacial acetic acid. The mobile phase

was filtered through 0.45μ filter paper to remove particulate matter and then degassed by

sonication.

5.1.1.3 Preparation of standard solutions

5.1.1.3.1 Preparation of standard stock solutions

5.1.1.3.1.1 Preparation of standard stock solution of DIC

Accurately weighed DIC (50mg) was transferred in 100ml volumetric flask. The drug

was dissolve in methanol with sonication and final volume was adjusted with methanol

upto mark to prepare a 500μg/ml stock solution.

5.1.1.3.1.2 Preparation of standard stock solution of CHL

Accurately weighed CHL (250mg) was transferred in 100ml volumetric flask. The drug





5.1.1.3.1.3 Preparation of standard stock solution of PCM

Accurately weighed PCM (32.5mg) was transferred in 10ml volumetric flask. The drug



5.1.1.3.1.2 Preparation of standard working solutions

5.1.1.3.1.2.1 Preparation of standard working solution of DIC

From the stock solution (500μg/ml), an aliquot quantity 1, 2, 3, 4, 5 and 6 ml were

transferred into separate 100ml volumetric flask and final volume was adjusted with

methanol upto mark to prepare 5-30μg/ml solutions.

5.1.1.3.2.2 Preparation of standard working solution of CHL

From stock solution (2500µg/ml), an aliquot quantity 1, 1.8, 2.6, 3.4, 4.2 and 5.0 ml were

transferred into 100 ml volumetric flask and final volume was adjusted with methanol

upto mark to prepare 25-125µg/ml solutions.

5.1.1.3.2.3 Preparation of standard working solution of PCM

From stock solution (3250µg/ml), an aliquot quantity 1, 1.9, 2.8, 3.8, 4.7 and 5.6 ml were

transferred into 100 ml volumetric flask and final volume was adjusted with methanol

upto mark to prepare 32.5-182.5 µg/ml solutions.

5.1.1.4 Preparation of sample solution

Twenty tablets were weighed accurately and finely powdered. Powder exactly equivalent

to 50 mg of DIC, 250 mg of CHL and 325 mg of PCM was transferred to a 100 ml

volumetric flask. The powder was dissolved in 60 ml of methanol with sonication for 15

minutes and volume was made up with methanol. 1ml of the solution was transferred into

10ml volumetric flask and diluted upto mark with methanol.

5.1.1.5 Determination of wavelength maxima

Standard solutions of DIC, CHL and PCM were scanned between 200 and 400nm. UV

spectra of all three drugs show maximum absorbance at 280nm. (Figure 5.1.1)



Figure 5.1.1 Overlay UV spectra of DIC, CHL and PCM

5.1.1.6 Chromatographic conditions

The chromatographic separations were performed using Varian C-18 (250 Χ 4.6 mm i.d,

5 μm particle size) column at room temperature. The optimum mobile phase consisted of

methanol:phosphate buffer pH 4.0 (pH adjusted with glacial acetic acid) in the ratio of

70:30, v/v respectively. A 20 μl sample was injected for analysis. The analysis was done

with flow rate of 0.8 ml/min at 280 nm wavelength using Dual wavelength UV Detector.

Method validation[1-2]

5.1.1.7.1 Linearity and range

The calibration curve was plotted over the concentration range of 5-30μg/ml for DIC, 25-

125μg/ml for CHL and 32.5-182.5μg/ml for PCM. All the solution were filtered through

0.2μm membrane filter and injected, chromatograms were recorded and it was repeated

for six times. A calibration graph was plotted between the mean peak area Vs respective

concentration and regression equation was derived.

5.1.1.7.2 Accuracy

The accuracy of the method was determined by calculating recoveries of DIC, CHL and

PCM by standard addition method. Known amount of standard solution of DIC, CHL and

PCM (80, 100 and 120% level) were added to pre-analysed samples.



5.1.1.7.3 Precision

Precision was evaluated in terms of intraday and interday precision. The intraday

precision was investigated using different concentrations of sample solutions in

triplicates. The intraday and interday precision of the proposed method was determined

by analyzing the corresponding concentration three times on the same day and on

different days respectively.

5.1.1.7.4 Robustness

Robustness was determined by the analysis of the samples under a variety of conditions

making small changes in the buffer pH, in the ratio of mobile phase, in the flow rate, in

the temperature conditions and changing the wavelength.

5.1.1.7.5 Limit of detection and quantification

The LOD may be expressed as: DL =3.3 δ/ S

Where δ = the standard deviation of the response, S = the slope of the calibration curve

The LOQ may be expressed as: QL =10 δ/ S


5.1.1.7.6 System suitability

The system suitability parameters like theoretical plates (N), resolution (Rs), retention

time (RT) and tailing factor (Tf) reported in European Pharmacopoeia[2] were calculated

by LC solution software. The HPLC system was equilibrated with the initial mobile

phase composition, followed by six injections of same standard.

5.1.1.8 Analysis of marketed formulation

The response of sample solution was measured under chromatographic condition as

described above in section 5.1.1.6. The amount of DIC, CHL and PCM were calculated

by regression equation.

5.1.1.9 Forced degradation study of drug substance[3]

5.1.1.9.1 Acidic degradation

5.1.1.9.1.1 Acidic degradation of DIC

Accurately weighed (10mg) of DIC was, transferred into 100ml volumetric flask and

dissolved in 50ml methanol. 50ml of 0.1N HCl was added and refluxed at 85°C for 24

hours. 0.5ml of the solution was diluted upto 10ml with methanol (5µg/ml) and analysed

by HPLC.



5.1.1.9.1.2 Acidic degradation of CHL

Accurately weighed CHL (10mg) was transferred into 100ml volumetric flask and

dissolved in 50ml methanol. 50ml of 5N HCl was added and refluxed at 85°C for 24

hours. 2.5 ml of the solution was diluted upto 10ml with methanol (25µg/ml) and

analysed by HPLC.

5.1.1.9.1.2 Acidic degradation of PCM

Accurately weighed PCM (10mg) was transferred into 100ml volumetric flask and


hours. 3.25ml of the solution was diluted upto 10ml with methanol (32.5μg/ml) was

analysed by HPLC.

5.1.1.9.2 Alkaline degradation

5.1.1.9.2.1 Alkaline degradation of DIC

Accurately weighed DIC (10mg) was transferred into 100ml volumetric flask and

dissolved in 50ml methanol. 50ml of 0.1N NaOH was added and refluxed at 80°C for 6


by HPLC.

5.1.1.9.2.2 Alkaline degradation of CHL


dissolved in 50ml methanol. 50ml of 0.1 N NaOH was added and refluxed at 80°C for 6


by HPLC.

5.1.1.9.2.3 Alkaline degradation of PCM


dissolved in 50ml methanol. 50 ml of 5N NaOH was added and refluxed at 80°C for 6

hours. 3.25ml of the solution was diluted upto 10ml with methanol. The solution

(32.5µg/ml) was analysed by HPLC.

5.1.1.9.3 Oxidative degradation

5.1.1.9.3.1 Oxidative degradation of DIC


dissolved in 50ml methanol. 50ml of 3% H2O2 was added and kept at room temperature



for 6 hours. 0.5ml of the solution was diluted upto 10ml with methanol (5µg/ml) and

analysed by HPLC.

5.1.1.9.3.2 Oxidative degradation of CHL



for 6 hours. 2.5ml of the solution was diluted upto 10ml with methanol (25µg/ml) and

analysed by HPLC.

5.1.1.9.2.3 Oxidative degradation of PCM

Accurately weighed PCM (10mg) was, transferred into 10ml volumetric flask and


for 6 hours. 3.25ml of the solution was diluted upto 10ml with methanol (32.5µg/ml) and

analysed by HPLC.

5.1.1.9.4 Thermal degradation

5.1.1.9.4.1 Thermal degradation of DIC

Accurately weighed DIC (10mg) was kept at 105°C for 8 hours and transferred in 100ml

volumetric flask. Dissolved in 50ml of methanol then dilute upto mark with methanol. 0.5

ml of the solution was diluted upto 10ml with methanol (5µg/ml) and analysed by HPLC.

5.1.1.9.4.2 Thermal degradation of CHL

Accurately weighed CHL (10mg) was kept at 105°C for 8 hours and transferred in 100ml

volumetric flask. Dissolved in 50ml of methanol then dilute upto mark with methanol. 2.5

ml of the solution was diluted upto 10ml with methanol (25µg/ml) and analysed by

HPLC.

5.1.1.9.4.3 Thermal degradation of PCM

Accurately weighed PCM (10mg) was kept at 105°C for 8 hours and transferred in 10ml

volumetric flask. Dissolved in 5ml of methanol then dilute upto mark with methanol

(32.5µg/ml) and analysed by HPLC.

5.1.1.9.5 Neutral degradation

5.1.1.9.5.1 Neutral degradation of DIC


dissolved in 50ml methanol. 50ml of water was added and refluxed at 85°C for 24 hours.



0.5ml of the solution was diluted upto 10ml with methanol (5µg/ml) and analysed by

HPLC.

5.1.1.9.5.2 Neutral degradation of CHL



2.5ml of the solution was diluted upto 10ml with methanol (25µg/ml) and analysed by

HPLC.

5.1.1.9.5.3 Neutral degradation of PCM


dissolved in 50ml methanol. 50ml of water was added and reflux at 85°C for 24 hours.

The solution (32.5µg/ml) was analysed by HPLC.

5.1.1.10 Forced degradation study of marketed product

5.1.1.10.1 Acidic degradation of marketed product


to 50mg of DIC, 250mg of CHL and 325mg of PCM were transferred to a 100ml

volumetric flask and dissolved in 50ml methanol. 50ml of 0.1N HCl was added and

refluxed at 85°C for 24 hours. 1ml of the solution was diluted upto 100ml with methanol

and analysed by HPLC.

5.1.1.10.2 Alkaline degradation of marketed product


to 50mg of DIC, 250mg of CHL and 325mg of PCM was transferred to a 100ml

volumetric flask and dissolved in 50ml methanol. 50ml of 0.1N NaOH was added and

refluxed at 80°C for 6 hours. 1ml of the solution was diluted upto 100ml with methanol

and analysed by HPLC.

5.1.1.10.3 Oxidative degradation of marketed product



volumetric flask and dissolved in 50ml methanol. 50ml of 3% H2O2 was added and kept

at room temperature for 6 hours. 1ml of the solution was diluted upto 100ml with

methanol and analysed by HPLC.



5.1.1.10.4 Thermal degradation of marketed product


to 50mg of DIC, 250mg of CHL and 325mg of PCM was kept at 105°C for 8 hours and

transferred in 100ml volumetric flask. The powder was dissolved in 50ml of methanol

and diluted upto mark with methanol. 1ml of the solution was diluted upto 100ml with

methanol and analysed by HPLC.

5.1.1.10.5 Neutral degradation of marketed product



volumetric flask and dissolved in 50ml methanol. 50ml of water was added and refluxed

at 85°C for 24 hours. 1ml of the solution was diluted upto 100ml with methanol and

analysed by HPLC.

5.1.2 Results and discussion

5.1.2.1 Method optimization

The aim of this study was to develop a stability indicating HPLC method for

simultaneous analysis of DIC, CHL and PCM. The review of literature for estimation of

other muscle relaxant drugs either alone or in combination and knowledge of molecule

suggest that reverse phase liquid chromatography (RP-LC) is suitable for simultaneous

analysis of DIC, CHL and PCM. In RP-HPLC various columns are available but C18

column was preferred over other columns. To optimize mobile phase initially methanol

and water in different ratios were tried. But CHL gave broad peak shape, so water was

replaced by phosphate buffer pH 4.0, and mixture of methanol and phosphate buffer pH

4.0 in different ratios were tried. It was found that methanol: phosphate buffer pH 4.0 in

ratio of 70:30 v/v respectively gave acceptable retention time (tR 3.25min for DIC, tR 5.60

min for CHL and tR 9.40 min for PCM) and good resolution for DIC, CHL and PCM with

flow rate of 0.8 ml/min at 280 nm (Table 5.1.1a). The method parameters were optimized

to analyse DIC, CHL and PCM in marketed product. A chromatogram has been shown in

Figure 5.1.2a and 5.1.2b.



Table 5.1.1a Results of optimization of mobile phase

Mobile phase Ratio Result

Methanol: water 50:50 Poor elution of drugs

Methanol: water 80:20 No good shape of drug and

poor resolution of peaks

Methanol: phosphate buffer pH 4.0 50:50 High retention time with bad

shape

Methanol: phosphate buffer pH 4.0 70:30 Less retention time with good

shape and better separation

Figure 5.1.2a: Blank Chromatogram



Figure 5.1.2b: Chromatogram of DIC, CHL and PCM by HPLC

5.1.2.2 Method validation

Linearity and range

The calibration curve was plotted over the concentration range of 5-30g/ml for DIC, 25-

125µg/ml for CHL and 32.5-182.5 g/ml for PCM. A calibration graph was plotted

between the mean peak area Vs respective concentration and regression equation was

derived (Figure 5.1.3, 5.1.4, 5.1.5). The results were shown in Table 5.1.1.

Figure 5.1.3: Calibration curve of DIC by HPLC

y = 5018.1x + 362R² = 0.9998

0

20000

40000

60000

80000

100000

120000

140000

160000

0 5 10 15 20 25 30 35Concentration (µg/ml)

Peak

area



Figure 5.1.4: Calibration curve of CHL by HPLC

Figure 5.1.5: Calibration curve of PCM by HPLC

Table 5.1.1b Linearity and range data for DIC, CHL and PCM by HPLC

Drug Linearity rangeY=mx+c

r2*

Slope* Intercept*DIC 5-30 µg/ml 5018.1 362 0.9998CHL 25-125 µg/ml 4987.5 2058 0.9997PCM 32.5-182.5 µg/ml 5051.4 14445 0.9997

*= Average result of six replicate samples

5.1.2.2.2 Accuracy (Recovery study)

The recovery for DIC, CHL and PCM were found between 98% and 101%. The results

were shown in Table 5.1.2.

y = 4987.5x - 20588R² = 0.9997

0

100000

200000

300000

400000

500000

600000

700000

0 20 40 60 80 100 120 140

Concentration (µg/ml)

Peak

area

y = 5051.4x - 14453R² = 0.9997

0100000200000300000400000500000600000700000800000900000

1000000

0 50 100 150 200

Peak

area




Table 5.1.2 Recovery study of DIC, CHL and PCM by HPLC

DrugConc. Of

Form.(µg/ml)

Conc. OfStd. added

(µg/ml)

Conc.Recovered

(µg/ml) ±SD*% recovery±SD*

DIC5 4 8.89±0.12 98.78%±0.18

5 5 9.97±0.30 99.70%±0.755 6 10.92±0.02 99.27%±1.89

CHL25 24 48.49±0.07 98.96%±0.0125 25 48.90±0.71 98.16%±0.1825 26 50.58±0.05 99.17%±1.09

PCM32.5 34 65.54±1.51 98.56%±0.8632.5 32.5 65.28±0.08 100.41%±0.1532.5 36 69.07±0.16 100.83%±0.11


5.1.2.2.3 Precision

The %RSD of intraday and inter-day precision study for DIC, CHL and PCM were found

to be <2. The results are shown in Table 5.1.3.

Table 5.1.3 Intra-day & Inter-day precision of DIC, CHL and PCM by HPLC

Drug Intra day Inter dayMean %assay* %RSD* Mean %assay* %RSD*

DIC 99.34%±1.16 0.36 99.15%±1.82 1.08CHL 99.83%±1.63 0.12 100.05%±0.57 1.12PCM 98.94%±0.01 1.16 98.86%±0.12 1.52



To determine the robustness of the developed method, experimental conditions were

deliberately altered and the responses of all drugs were recorded. The results of change in

ratio of mobile phase, flow rate and wavelength are shown in Table 5.1.4.



Table 5.1.4 Robustness data for DIC, CHL and PCM by HPLC

Chromatographic

factorlevel

Retention time* Tailing factor*

DIC CHL PCM DIC CHL PCM

Flow rate

0.7ml/min 3.98±0.02 6.01±0.0

19.96±0.0

10.95±0.

02 1.02±0.01 0.97±0.01

1.0ml/min 2.95±0.02 5.04±0.0

18.66±0.0

30.96±0.

01 1.03±0.01 0.96±0.01

Methanol:phosphate

buffer(v/v)

75:25 3.91±0.03 6.02±0.03

9.95±0.02

0.95±0.02 1.02±0.01 0.96±0.0

2

85:15 3.96±0.02 5.05±0.02 8.61±.02 0.96±0.

01 1.02±0.01 0.97±0.01

Detectionwave-length

275nm 3.24±0.04 5.60±0.01

9.41±0.01

0.95±0.01 1.03±0.01 0.97±0.0

3

285nm 3.26±0.03 5.60±0.01

8.64±0.02

0.95±0.02 1.02±0.01 0.97±0.0

1*= Average result of six replicate samples


The LOD for DIC, CHL & PCM were found to be 0.15μg/ml, 1.82μg/ml and 2.40μg/ml

respectively, while LOQ were 0.47μg/ml, 5.53μg/ml and 7.29μg/ml respectively.


The parameters like retention time, asymmetric factor, number of theoretical plates and

tailing factors were evaluated for DIC, CHL & PCM. The results were shown in Table

5.1.5.

Table 5.1.5 System suitability parameters for DIC, CHL and PCM by HPLC

Parameter RT* AUC*

No. of

theoretical

plates*

Tailing

factor*

DIC 3.25±0.05 25012.20±283.43 4204.4±9.52 0.95±.002CHL 5.60±0.06 105410.2±312.53 3637.4±139.80 1.02±0.01PCM 9.40±0.09 150453.12±3923.11 1263.12±7.005 0.97±0.001

*=Average result of six replicate samples



5.1.2.3 Analysis of marketed product

Market formulation containing 50mg DIC, 250mg CHL and 325mg PCM was analysed

by HPLC.

5.1.2.4 Forced degradation study

Forced degradation studies were performed for bulk drug and marketed product, to

provide an indication of the stability indicating property. The degradation was attempted

to stress conditions like acid hydrolysis, alkaline hydrolysis, oxidative hydrolysis,

thermal treatment and neutral degradation, in order to evaluate the ability of the proposed

method to separate drug from its degradation products[3]. During forced degradation

experiments, DIC showed more degradation under oxidative hydrolysis compared to

other conditions. CHL was degraded in alkaline, oxidative and thermal stress conditions

whereas PCM was degraded only under oxidative stress conditions. Table 5.1.7 indicates

the extent of degradation of marketed product under various stress conditions. Figures

5.1.6 to 5.1.15 shows the chromatograms of forced degraded samples. The degradation

products were well resolved from drug, confirming the stability indicating power of the

method.

Figure 5.1.6a: Chromatogram of acidic degradation of DIC by HPLC



Figure 5.1.6b: Chromatogram of acidic degradation of CHL by HPLC

Figure 5.1.6c: Chromatogram of acidic degradation of PCM by HPLC

Figure 5.1.7a: Chromatogram of alkaline degradation of DIC by HPLC



Figure 5.1.7b: Chromatogram of alkaline degradation of CHL by HPLC

Figure 5.1.7c: Chromatogram of alkaline degradation of PCM by HPLC

Figure 5.1.8a: Chromatogram of oxidative degradation of DIC by HPLC



Figure 5.1.8b: Chromatogram of oxidative degradation of CHL by HPLC

Figure 5.1.8c: Chromatogram of oxidative degradation of PCM by HPLC

Figure 5.1.9a: Chromatogram of thermal degradation of DIC by HPLC



Figure 5.1.9b: Chromatogram of thermal degradation of CHL by HPLC

Figure 5.1.9c: Chromatogram of thermal degradation of PCM by HPLC

Figure 5.1.10a: Chromatogram of neutral degradation of DIC by HPLC



Figure 5.1.10b: Chromatogram of neutral degradation of CHL by HPLC

Figure 5.1.10c: Chromatogram of neutral degradation of PCM by HPLC

Figure 5.1.11: Chromatogram of acidic degradation of marketed product by HPLC



Figure 5.1.12: Chromatogram of alkaline degradation of marketed product by

HPLC

Figure 5.1.13: Chromatogram of oxidative degradation of marketed product by

HPLC

Figure 5.1.14: Chromatogram of thermal degradation of marketed product by

HPLC



Figure 5.1.15: Chromatogram of neutral degradation of marketed product by

HPLC



Table 5.1.6 Forced degradation study of marketed product by HPLC


Degradation condition Drug

Conc.Of

drug(µg/ml)

RT ofobserved

peak*AUC* %drug* %degradation

*

Acidic

DIC 5 3.25 24018 98.14%±1.04 0.00%±0.00

CHL 25

5.59 101541 91.98%±1.31 0.00%±0.00

5.19(I) 8931 0.00%±0.00 8.79±1.31

PCM 32.5 9.40 149901 99.65%±1.45 0.00%±0.00

Alkaline

DIC 5

3.25 24010 94.62%±1.24 0.00%±0.00

2.92(I) 1432 0.00%±0.00 5.96%±1.21

CHL 255.62 94421 91.56%±1.57 94.10%±1.38

5.49(II) 3410 0.00%±0.00 3.61%±1.416.12(III) 1431 0.00%±0.00 1.51±1.30

PCM 32.5 9.41 149097 99.57%±1.34 0.00%±0.00

Oxidative

DIC 5

3.25 23120 91.41%±1.32 0.00%±0.00

2.72(I) 1981 0.00%±0.00 8.98%±1.54

CHL 25 5.60 102384 97.00%±1.03 0.00%±0.005.12(II) 2398 0.00%±0.00 2.64%±0.95

PCM 32.5

9.41 148320 93.84%±1.09 0.00%±0.00

10.21(III) 9908 0.00%±0.00 6.68±1.41

ThermalDIC 5 3.25 25320 99.90%±1.12 0.00%±0.00CHL 25 5.61 105908 99.88%±1.08 0.00%±0.00PCM 32.5 9.41 149959 99.84%±1.24 0.00%±0.00

Neutral

DIC 5 3.25 24906 98.45%±1.4 0.00%±0.00

CHL 25 5.60 103956 91.88%±1.23 0.00%±0.004.93(I) 9327 0.00%±0.00 8.64%±0.95

PCM 32.5 9.41 104903 99.84%±1.21 0.00%±0.00



5.1.3 Conclusion

The RP-HPLC method developed for the analysis of ternary mixtures of DIC, CHL and

PCM as bulk drug and in pharmaceutical preparation is simple, accurate, precise, and

repeatable with short run time. The developed method is stability indicating and can

separate degradants and be used to determine the assay of pharmaceutical preparation and

also stability samples.



5.2 STABILITY INDICATING HPTLC METHOD FOR

SIMULTANEOUS ESTIMATION OF DICLOFENAC POTASSIUM,

CHLORZOXAZONE AND PARACETAMOL5.2.1 Experimental

5.2.1.1 Solubility

Solubility of Diclofenac potassium(DIC), Chlorzoxazone(CHL) and Paracetamol(PCM)

were performed in different solvent like distilled water, 0.1N HCl, 0.1N NaOH,

methanol, dimethyl formamide, acetonitrile, ethanol and chloroform. All three drugs were

found to be soluble in methanol.


The mobile phase Toluene: ethylacetate: glacial acetic acid in the ratio of 1:2:0.5,v/v/v

respectively was used.







5.2.1.3.1.2 Preparation of standard stock solution of CHL

Accurately weighed CHL (10mg) was transferred in 100ml volumetric flask. The drug




Accurately weighed PCM (10mg) was transferred in 100ml volumetric flask. The drug





to 50mg of DIC, 250mg of CHL and 325mg of PCM were transferred to a 100ml

volumetric flask. The powder was dissolved in 50ml of methanol with sonication for 15

minutes and volume was made up with methanol.




Standard solutions of DIC, CHL and PCM were scanned between 200 and 400nm. UV

spectra of all three drugs show maximum absorbance at 280nm.

Figure 5.2.1 Overlay UV spectra of DIC, CHL and PCM

5.2.1.6 Chromatographic conditionsChromatography was performed on 10x10cm Precoated Silica Gel G60F254 aluminium

sheets. The samples were applied to the plates as 6mm band, 10 mm apart, by means of

Linomat-V sample applicator with help of 100μl Hamilton syringe. It was developed in

CAMAG TLC chamber (20x10cm, 20x20cm) which was already saturated for 30 min. with

mobile phase at room temperature. The optimum mobile phase consisted of Toluene: ethyl

acetate: glacial acetic acid in the ratio of 1:2:0.5, v/v/v respectively. After development the

plate was scanned at 280 nm by means of CAMAG TLC Scanner 3 controlled by WinCATs

software.

5.2.1.7 Method validation [1-2]


The calibration curve was plotted over the concentration range of 10-60ng/spot for DIC,

50-300ng/spot for CHL and 100-350ng/spot for PCM. All the solution was spotted on



precoated TLC plate, chromatograms were recorded and it was repeated for six times. A

calibration graph was plotted between the mean peak height Vs respective concentration

and regression equation was derived.

5.2.1.7.2 Accuracy

The accuracy of the method was determined by calculating recoveries of DIC, CHL and

PCM by standard addition method. Known amount of standard solution of DIC, CHL and

PCM (80, 100 and 120% level) were added to preanalysed samples.

5.2.1.7.3 Precision


precision was investigated using different concentrations of sample solutions in triplicate.

The intraday and interday precision of the proposed method was determined by analyzing

the corresponding concentration three times on the same day and on different days.



making small changes in the ratio of mobile phase, in the saturation time, changing the

wavelength.







8, 10 and 12μl of the sample solution as described in 5.2.1.4 were spotted on precoated

TLC plate. 1ml of sample solution (5.2.1.4) was diluted upto 10ml with methanol and 8,

10 and 12μl of this solution were spotted on precoated TLC plate. 1ml of above solution

diluted upto 10ml with methanol and 8, 10 and 12μl of this solution were spotted on

precoated TLC plate. The TLC plate was developed and analysed as described under

chromatographic condition. The amount of DIC, CHL and PCM were determined by

regression equation.






50mg of DIC was weighed accurately, transferred into 100ml volumetric flask and


hours. 1.0ml of the solution is diluted upto 10ml with methanol then 1.0μl of the solution

was spotted on precoated TLC plate (50ng/spot), developed and analysed as described

under chromatographic condition.

5.2.1.9.1.2 Acidic degradation of CHL

25mg of CHL was weighed accurately, transferred into 100ml volumetric flask and


hours. 1ml of the solution diluted upto 10ml with methanol then 8μl of the solution was

spotted on precoated TLC plate (250ng/spot), developed and analysed as described under

chromatographic condition.


32.5mg of PCM was weighed accurately, transferred into 100ml volumetric flask and


hours. 1ml of the solution diluted upto 10ml with methanol then 1.0μl of the solution was







hours. 1ml of the solution diluted upto 10ml with methanol then 1.0μl of the solution was



5.2.1.9.2.2 Alkaline degradation of CHL





hours. 1.0μl of the solution was spotted on precoated TLC plate (250ng/spot), developed

and analysed as described under chromatographic condition.



dissolved in 50ml methanol. 50ml of 5N NaOH was added and refluxed at 80°C for 6







for 6 hours. 1ml of the solution diluted upto 10ml with methanol. 1.0μl of the solution

was spotted on precoated TLC plate (50ng/spot), developed and analysed as described

under chromatographic condition.

5.2.1.9.3.2 Oxidative degradation of CHL



for 6 hours. 1.0μl of the solution was spotted on precoated TLC plate (250ng/spot),

developed and analysed as described under chromatographic condition.








50mg of DIC was weighed accurately, kept at 105°C for 8 hours and transferred into

100ml volumetric flask. The powder was dissolved in 50ml methanol and diluted upto

mark with methanol. 1ml of the solution diluted upto 10ml with methanol then 1.0μl of

the solution was spotted on precoated TLC plate (50ng/spot), developed and analysed as

described under chromatographic condition.



5.2.1.9.4.2 Thermal degradation of CHL

25mg of CHL was weighed accurately, kept at 105°C for 8 hours and transferred into


mark with methanol. 8μl of the solution was spotted on precoated TLC plate

(250ng/spot), developed and analysed as described under chromatographic condition.


32.5mg of PCM was weighed accurately, kept at 105°C for 8 hours and transferred into


mark with methanol. 1.0μl of the solution was spotted on precoated TLC plate






1ml of the solution diluted upto 10ml with methanol then 1.0μl of the solution was



5.2.1.9.5.2 Neutral degradation of CHL



1.0μl of the solution was spotted on precoated TLC plate (250ng/spot), developed and

analysed as described under chromatographic condition.











to 50mg of DIC, 25mg of CHL and 32.5mg of PCM was transferred to a 100ml


refluxed at 85°C for 24 hours. 1.0ml of the solution diluted upto 10ml with methanol then

1.0μl of the solution was spotted on precoated TLC plate (50ng/spot DIC), developed and

analysed as described under chromatographic condition. Moreover, 1.0μl of the solution

was spotted on precoated TLC plate (250ng/spot CHL and 325ng/spot PCM), developed







1.0μl of the solution was spotted on precoated TLC plate (50ng/spot DIC), developed and






to 50mg of DIC, 25mg of CHL and 32.5mg of PCM was transferred to a 100 ml


at room temperature for 6 hours. 1.0ml of the solution diluted upto 10ml with methanol

then 1.0μl of the solution was spotted on precoated TLC plate (50ng/spot DIC),

developed and analysed as described under chromatographic condition. Moreover, 1.0μl

of the solution was spotted on precoated TLC plate (250ng/spot CHL and 325ng/spot

PCM), developed and analysed as described under chromatographic condition.



to 50mg of DIC, 25mg of CHL and 32,5mg of PCM was kept at 105°C for 8 hours and




and diluted upto mark with methanol. 1.0ml of the solution diluted upto 10ml with

methanol then 1.0μl of the solution was spotted on precoated TLC plate (50ng/spot DIC),

developed and analysed as described under chromatographic condition. Moreover, 1.0μl

of the solution was spotted on precoated TLC plate (250ng/spot CHL and 325ng/spot

PCM), developed and analysed as described under chromatographic condition.





at 85°C for 24 hours. 1.0ml of the solution diluted upto 10ml with methanol then 1.0μl of

the solution was spotted on precoated TLC plate (50ng/spot DIC), developed and




5.2.2 Results and discussion5.2.2.1 Method optimization

For optimization, different mobile phases and composition were employed to achieve the

good separation. The method development was initiated with using a mobile phase of

toluene:ethyl acetate:ammonium sulphate in various proportions. In the above conditions

only two drugs were separated. All three drugs were separated using mobile phase

consisting of different ratio of glacial acetic acid. Finally mobile phase consisting mixture

of toluene: ethyl acetate: glacial acetic acid in ratio of 1:2:0.5,v/v/v respectively gave

reasonable resolution (Rf 0.78 for DIC, 0.31 for CHL and 0.53 for PCM) and sharp band

for all three drugs. Saturation of TLC chamber for 30 min assured better reproducibility

and better resolution. All three drugs were detected at 280 nm by means of CAMAG TLC

Scanner 3. A chromatogram is shown in Figure 5.2.2.



Figure 5.2.2 Chromatogram of CHL, PCM and DIC



The calibration curve was plotted over the concentration range of 10-60ng/spot for DIC,

50-300ng/spot for CHL and 100-350ng/spot for PCM. A calibration graph was plotted

between the mean peak height Vs respective concentration and regression equation was

derived (Figure 5.2.3, 5.2.4, 5.2.5). The results were shown in Table 5.2.1.



Figure 5.2.3 Calibration curve for DIC by HPTLC

Figure 5.2.4 Calibration curve for CHL by HPTLC

y = 99.626x + 57.267R² = 0.9994

0

1000

2000

3000

4000

5000

6000

7000

0 10 20 30 40 50 60 70Concentration (ng/spot)

y = 40.129x + 3023.7R² = 0.9999

0

2000

4000

6000

8000

10000

12000

14000

16000

0 50 100 150 200 250 300 350Concentration (ng/spot)

Peak

Area

Peak

Area



Figure 5.2.5 Calibration curve for PCM by HPTLC

Table 5.2.1 Linearity and range data for DIC, CHL and PCM by HPTLC


r2*

Slope* Intercept*DIC 10-60ng/spot 99.62 57.26 0.9994CHL 50-300ng/spot 40.129 3023.7 0.9999PCM 100-350ng/spot 41.006 2900.1 0.9997



Recovery studies were performed to validate the accuracy of developed method. To a

pre-analysed sample solution, a definite concentration of standard drug was added and

recovery was studied. The recovery for DIC, CHL and PCM were found between 99%-

101 %.( Table 5.2.2).

y = 41.006x + 2900.1R² = 0.9997

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

0 100 200 300 400

Concentration (ng/spot)

Peak

Area



Table 5.2.2 Recovery study of DIC, CHL and PCM by HPTLC

DrugConc. Of

Form.(ng/spot)

Conc. OfStd. added(ng/spot)

Conc.Recover(ng/spot)

% recovery±SD*

DIC50 40 89.96 99.96%±0.02150 60 108.95 99.04%±0.03250 50 100.02 100.02%±0.019

CHL250 240 489.15 99.82%±0.012250 250 497.11 99.42%±0.01250 260 508.32 99.68%±0.04

PCM325 315 638.40 99.75%±0.030325 325 648.83 99.82%±0.018325 335 661.47 100.22%±0.036


5.2.2.2.3 Precision

The %RSD of intraday and interday precision study for DIC, CHL and PCM were found

to be <2. The results were shown in Table 5.2.3.

Table 5.2.3 Results of Intra-day & Inter-day precision DIC, CHL and PCM by

HPTLC


DIC 99.98%±0.02 0.012 97.96%±0.048 0.038CHL 98.97%±0.41 0.014 98.98%±0.047 0.040PCM 99.10%±0.04 0.020 99.19%±0.01 0.01

*= Average result of six replicate sample







Table 5.2.4 Robustness data for DIC, CHL and PCM by HPTLC

Chromatographicfactor level Amont found*(ng/spot)

DIC CHL PCM

Mobile phase ratio 1.2:1:4v/v/v 39.92±0.051 199.82±0.032 249.98±0.086

1:1.2:4v/v/v 39.90±0.062 199.84±0.028 249.16±0.034

Saturation time32 minutes 39.94±0.060 199.97±0.04

3 249.97±0.047

28 minutes 39.98±0.062 199.93±0.042 249.99±0.037

Detection wavelength 275nm 39.95±0.038 199.95±0.043 248.95±0.058

285nm 39.96±0.022 201.00±0.02 250.10±0.024*= Average result of six replicate samples


The LOD for DIC, CHL and PCM were found to be 0.005 ng/spot, 1.41 ng/spot and 3.17

ng/spot respectively, while LOQ were 0.87 ng/spot, 4.25 ng/spot and 9.61 ng/spot

respectively.


The market formulation containing 50mg DIC, 250mg CHL and 325mg PCM was

analysed by HPTLC.


The degradation was attempted to stress conditions like acid hydrolysis, alkaline

hydrolysis, oxidative hydrolysis, thermal treatment and neutral degradation, in order to

evaluate the ability of the proposed method to separate drug from its degradation

products[3]. HPTLC studies of the samples obtained during the stress testing of DIC, CHL

and PCM under different conditions using toluene: ethyl acetate: glacial acetic acid in the

ratio of 1:2:0.5, v/v/v respectively as the mobile phase shows different degradation peaks

as shown in Figures 5.2.6 to 5.2.15. CHL degraded under acidic, alkaline, oxidative and

neutral condition. Moreover, it showed highest degradation in alkaline condition and

degradation products appear at Rf of 0.19. DIC was degraded only in alkaline and

oxidative stress condition whereas PCM remain stable in all condition except oxidative



condition. Table 5.2.5 indicates the extent of degradation of marketed product under

various stress conditions.

Figure 5.2.6a: Chromatogram of acidic degradation of DIC by HPTLC

Figure 5.2.6b: Chromatogram of acidic degradation of CHL by HPTLC



Figure 5.2.6c: Chromatogram of acidic degradation of PCM by HPTLC

Figure 5.2.7a: Chromatogram of alkaline degradation of DIC by HPTLC

Figure 5.2.7b: Chromatogram of alkaline degradation of CHL by HPTLC



Figure 5.2.7c: Chromatogram of alkaline degradation of PCM by HPTLC

Figure 5.2.8a: Chromatogram of oxidative degradation of DIC by HPTLC

Figure 5.2.8b: Chromatogram of oxidative degradation of CHL by HPTLC



Figure 5.2.8c: Chromatogram of oxidative degradation of PCM by HPTLC

Figure 5.2.9a: Chromatogram of thermal degradation of DIC by HPTLC

Figure 5.2.9b: Chromatogram of thermal degradation of CHL by HPTLC



Figure 5.2.9c: Chromatogram of thermal degradation of PCM by HPTLC

Figure 5.2.10a: Chromatogram of neutral degradation of DIC by HPTLC

Figure 5.2.10b: Chromatogram of neutral degradation of CHL by HPTLC



Figure 5.2.10c: Chromatogram of neutral degradation of PCM by HPTLC

Figure 5.2.11: Chromatogram of acidic degradation of marketed product by

HPTLC



Figure 5.2.12: Chromatogram of alkaline degradation of marketed productby HPTLC

Figure 5.2.13: Chromatogram of oxidative degradation of marketed productby HPTLC

Figure 5.2.14: Chromatogram of thermal degradation of marketed productby HPTLC













Figure 5.2.15: Chromatogram of neutral degradation of marketed productby HPTLC



Table 5.2.5 Result of Forced degradation study of drug products

Degradation condition Drug

Conc.of

drug(ng/spot)

Rf valueof

observedpeak*

Peakarea* %of drug*

%ofdegradation

*

Acidic

DIC 50 0.73 2041.2 99.91%±0.21 0.00%±0.00

CHL 250

0.29 4694.94 91.89%±1.01 0.00%±0.00

0.21(I) 464.1 0.00%±0.00 9.88±0.91

PCM 325 0.51 6392.9 99.92%±1.05 0.00%±0.00

Alkaline

DIC 50

0.72 2064.32 89.42%±0.94 0.00%±0.00

0.89(I) 210.6 0.00%±0.00 10.17%±0.91

CHL 250 0.29 4487.52 95.27%±0.77 0.00%±0.000.19(II) 208.39 0.00%±0.00 4.63%±0.29

PCM 325 0.51 6219.9 99.89%±0.34 0.00%±0.00

Oxidative

DIC 50

0.72 2138.71 95.39%±1.22 0.00%±0.00

0.84(I) 108.8 0.00%±0.00 5.05%±0.69

CHL 250 0.29 4599.6 96.00%±1.03 0.00%±0.000.38(II) 196.45 0.00%±0.00 4.26%±0.96

PCM 325 0.32 5999.34 96.95%±0.19 0.00%±0.000.24(III) 210.1 0.00%±0.00 3.50%±0.96

ThermalDIC 50 0.72 2014.56 99.93%±0.82 0.00%±0.00CHL 250 0.29 4689.87 99.87%±1.08 0.00%±0.00PCM 325 0.51 6382.98 99.92%±0.24 0.00%±0.00

Neutral

DIC 50 0.71 1996.75 98.92%±1.03 0.00%±0.00

CHL 2500.29 4499.12 99.93%±0.63 0.00%±0.00

0.21(I) 291.3 0.00%±0.00 6.46%±0.96PCM 325 0.52 6779.99 99.91%±0.21 0.00%±0.00




5.2.3 ConclusionThe developed HPTLC technique is simple, accurate, sensitive, precise, rapid and

repeatable. The method was successfully used for determination of DIC, CHL, and PCM

as bulk drug and in pharmaceutical formulation. After exposing the drugs to different

stress condition, the drug peak area was observed to decrease and also degradants peak

were observed. The developed method is stability indicating and separate degradants and

can be used to determine the assay of pharmaceutical preparation and also stability

samples.



5.3 STATISTICAL COMPARISON BETWEEN OFFICIAL

REPORTED AND PROPOSED METHODS[4-5]

The assay result for DIC, CHL and PCM in their dosage form obtained using HPLC and

HPTLC methods were compared between official reported methods by applying the

paired t-test and F-test. The calculated t-value for DIC (0.0011), CHL (0.030) and PCM

(0.0057) for HPLC as well as DIC (0.096), CHL (0.213), and PCM (0.1854) for HPTLC

is less than the tabulated t-value (2.571) at the 95% confidence interval. Moreover,

calculated F-value for DIC (0.0252), CHL (0.1711) and PCM (0.1741) for HPLC as well

as DIC (0.3567), CHL (0.011) and PCM (0.1854) for HPTLC is also less than the

tabulated F-value (4.28). Therefore, there is no significant difference in a determined

content of DIC, CHL and PCM by HPLC and HPTLC methods. Table 5.3.1 indicates

statistical comparison between proposed methods.

Table 5.3.1 Statistical comparison for DIC, CHL and PCM between proposed

methods

Drug Methods Mean SD n

Paired t-test F-test

Tabulated

value

Calculated

value

Tabulated

value

Calculated

value

DIC

Official 99.11 0.06 6

2.571

0.0011

4.28

0.0252HPLC 99.64 0.19 6

Official 99.11 0.06 6 0.096 0.3567HPTLC 99.29 0.20 6

CHL

Official 99.78 0.27 6

2.571

0.030

4.28

0.1711HPLC 99.56 0.12 6

Official 99.78 0.27 6

0.2130.011

HPTLC 99.55 0.07 6

PCM

Official 99.65 0.24 6

2.571

0.00574.28

0.1741HPLC 99.25 0.14 6

Official 99.65 0.24 6 0.1854 0.1854HPTLC 99.67 0.15 6



5.4 References1. ICH, Q2(R1) Validation of Analytical Procedure: Text and Methodology,

International Conference on Harmonization, Geneva, October 1994.

2. The United States Pharmacopeia, The National Formulary, USP 30 NF 25, Asian

Edition, Volume 1, 2007; 680-683.

3. Bakshi M, Singh S, Development of validated stability-indicating assay methods

critical review, Journal of Pharmaceutical and Biomedical Analysis, 2002;

28:1010-40.

4. Christian GD, Analytical Chemistry, 6th Ed., University of Washington, 2007; 90-

97.

5. Sanford Bolton, Charles Bon, Pharmaceutical Statistics Practical and Clinical

Application, 4th Ed., Revised and Expanded, 2004; 417-436.



6. STABILITY INDICATING METHOD DEVELOPMENT AND

VALIDATION FOR SIMULTANEOUS ESTIMATION OF

DICLOFENAC POTASSIUM, PARACETAMOL AND

METHOCARBAMOL

6.1 STABILITY INDICATING HPLC METHOD DEVELOPMENT FOR


PARACETAMOL AND METHOCARBAMOL

6.1.1 Experimental

6.1.1.1 Solubility

Solubility of Diclofenac potassium (DIC), Paracetamol (PCM) and Methocarbamol

(MET) were performed in different solvent like distilled water, 0.1N HCl, 0.1N NaOH,

methanol, dimethyl formamide, acetonitrile, ethanol and chloroform. All three drugs were

soluble in methanol.


The mobile phase methanol: water in the ratio of 80:20, v/v respectively was used. The

mobile phase was filtered through 0.45μ filter paper to remove particulate matter and

then degassed by sonication.








Accurately weighed PCM (32.5mg) was transferred in 100ml volumetric flask. The drug





6.1.1.3.1.3 Preparation of standard stock solution of MET

Accurately weighed MET (50mg) was transferred in 50ml volumetric flask. The drug



6.1.1.3.2 Preparation of standard working solutions

6.1.1.3.2.1 Preparation of standard working solution of DIC

From the stock solution (500μg/ml), an accurately measured 0.2, 0.4, 0.6, 0.8, 1.0 and

1.2ml was transferred into separate 10ml volumetric flask and final volume was adjusted

with methanol upto mark to prepare 10-60μg/ml solutions.

6.1.1.3.2.2 Preparation of standard working solution of PCM

From the stock solution (325μg/ml), an accurately measured 2, 4, 6, 8, 10 and 12ml was

transferred into separate 10ml volumetric flask and final volume was adjusted with

methanol upto mark to prepare 65-390μg/ml solutions.

6.1.1.3.2.3 Preparation of standard working solution of MET

From the stock solution (1000μg/ml), an accurately measured 1, 2, 3, 4, 5 and 6ml was

transfer into separate 10ml volumetric flask and final volume was adjusted with methanol

upto mark to prepare 100-600μg/ml solutions.



to 50mg of DIC, 32.5mg of PCM and 500mg of MET was transferred to a 100ml


minutes and volume was made up with methanol. 1ml of the solution was transferred into

10ml volumetric flask and diluted upto mark with methanol.


Solutions of DIC, PCM and MET were scanned between 200 and 400nm. UV spectra of

all three drugs show maximum absorbance at 225nm. (Figure 6.1.1)



Figure 6.1.1 Overlay UV spectra of DIC, PCM and MET


The chromatographic separations were performed using Varian C-18 (250 Χ 4.6 mm i.d,

5 μm particle size) column at room temperature. The optimum mobile phase consisted of

methanol: water in the ratio of 80:20, v/v respectively. 20 μl sample was injected for

analysis. The analysis was done with flow rate of 0.8 ml/min at 225 nm wavelength using

Dual wavelength UV Detector.

6.1.1.7 Method validation[1-2]


The calibration curve was plotted over the concentration range of 10-60μg/ml for DIC,

65-390μg/ml for PCM and 100-600μg/ml for MET. All the solution were filtered through

0.2μm membrane filter and injected, chromatograms were recorded and it was repeated

for six times. A calibration graph was plotted between the mean peak area Vs respective


6.1.1.7.2 Accuracy

The accuracy of the method was determined by calculating recoveries of DIC, PCM and

MET by standard addition method. Known amount of standard solution of DIC, PCM

and MET (80, 100 and 120% level) were added to pre-analysed samples.



6.1.1.7.3 Precision


precision was investigated using three different concentrations of sample solutions. The

intraday and interday precision of the proposed method was determined by analyzing the

corresponding concentration three times on the same day and on different days,

conditions and changing the wavelength.




S = the slope of the calibration curve



S = the slope of the calibration curve


The system suitability parameters like theoretical plates (N), asymmetry factor (As),

capacity factor (K’), resolution (Rs), retention time (RT) and tailing factor (Tf) reported

in European Pharmacopoeia[2] were calculated by LC solution software. The HPLC

system was equilibrated with the initial mobile phase composition, followed by six

injections of same standard.


The response of sample solution was measured under chromatographic condition as

described above in section 6.1.1.6. The amount of DIC, PCM and MET were determined

by regression equation.






hours. 6ml of the solution was diluted upto 10ml with methanol (60μg/ml) and analysed

by HPLC.




32.5mg of PCM was weighed accurately, transfer into 100ml volumetric flask and


hours (325μg/ml) and the solution was analysed by HPLC.

6.1.1.9.1.3 Acidic degradation of MET

50mg of MET was weighed accurately, transfer into 100ml volumetric flask and


hours. The solution (500μg/ml) was analysed by HPLC.





hours. 6ml of the solution was diluted upto 10ml with methanol (60μg/ml) and analysed

by HPLC.



dissolved in 50ml methanol. 50ml of 0.1 N NaOH was added and refluxed at 80°C for 24

hours (325μg/ml) and the solution was analysed by HPLC.

6.1.1.9.2.3 Alkaline degradation of MET



hours. The solution (500μg/ml) was analysed by HPLC.





for 24 hours. 6.0ml of the solution was diluted upto 10ml with methanol (60μg/ml) and

analysed by HPLC.






for 24 hours (325μg/ml) and the solution was analysed by HPLC.

6.1.1.9.3.3 Oxidative degradation of MET



for 24 hours. The solution (500μg/ml) was analysed by HPLC.



10mg of DIC weighed accurately, kept at 105°C for 8 hours and transferred in 100ml

volumetric flask. The powder was dissolved in 50ml of methanol and diluted upto mark

with methanol. 6ml of the solution was diluted upto 10ml with methanol (60μg/ml) and

analysed by HPLC.


32.5mg of PCM weighed accurately, kept at 105°C for 8 hours and transfered in 100ml


with methanol (325μg/ml) and the solution was analysed by HPLC.

6.1.1.9.4.3 Thermal degradation of MET

50mg of MET weighed accurately, kept at 105°C for 8 hours an transfered in 100ml


with methanol (500μg/ml) and analysed by HPLC.





6ml of the solution was diluted upto 10ml with methanol (60μg/ml) and analysed by

HPLC.





dissolved in 50ml methanol. 50ml of water was added and reflux at 85°C for 24 hour

(325μg/ml) and the solution was analysed by HPLC

6.1.1.9.5.3 Neutral degradation of MET

50mg of MET was weighed accurately, transfer into 10ml volumetric flask and dissolved

in 50ml methanol. 50ml of water was added and reflux at 85°C for 24 hours. The solution

(500μg/ml) was analysed by HPLC.




to 5mg of DIC, 32.5mg of PCM and 50mg of MET was transferred to a 100ml volumetric

flask and dissolved in 50ml methanol. 50ml of 0.1N HCl was added and refluxed at 85°C

for 24 hours and analysed by HPLC.




flask and dissolved in 50ml methanol. 50ml of 0.1N NaOH was added and refluxed at

80°C for 24 hours.




flask and dissolved in 50ml methanol. 50ml of 3% H2O2 was added and kept at room

temperature for 24 hours.



to 5mg of DIC, 32.5mg of PCM and 50mg of MET was kept at 105°C for 8 hours and


and diluted upto mark with methanol.






flask and dissolved in 50ml methanol. 50ml of water was added and refluxed at 80°C for

24 hours.

6.1.2 Results and discussion6.1.2.1 Method optimization

The aim of this study was to develop a stability indicating HPLC method for

simultaneous analysis of DIC, PCM and MET. The review of literature for estimation of

other muscle relaxant drugs either alone or in combination and knowledge of molecule

suggest that reverse phase liquid chromatography (RPLC) is suitable for simultaneous

analysis of DIC, PCM and MET. In case of RP-HPLC various columns are available but

C18 column was preferred over other columns. Different mobile phases containing

acetonitrile, methanol and water were used. The mobile phase methanol: water in

different ratio was tried. Hence forth, changing the composition of mobile phase

optimized the chromatographic condition. It was found that methanol: water in ratio of

80:20, v/v respectively gave acceptable retention time (tR 3.51min for DIC, tR 6.42 min

for PCM and tR 9.90 min for MET) and good resolution for DIC, PCM and MET with

flow rate of 0.8 ml/min at 225 nm.(Table 6.1.1a) The method parameter was optimized to

analyse DIC, PCM and MET in marketed product. A chromatogram is shown in Figure

6.1.2b

Table 6.1.1a Results of optimization of mobile phase

Mobile phase Ratio Result

Acetonitrile: water 50:50 Poor elution of drugs

Acetonitrile: water 70:30 No good shape of drug and

poor resolution of peaks

Methanol: water 50:50 High retention time with bad

shape

Methanol: water 80:20 Less retention time with good

shape and better separation



Figure 6.1.2a: Blank Chromatogram by HPLC

Figure 6.1.2b: Chromatogram of DIC, PCM and MET by HPLC


The calibration curve was plotted over the concentration range of 10-60μg/ml for DIC,

65-390μg/ml for PCM and 100-600μg/ml for MET. A calibration graph was plotted

between the mean peak area Vs respective concentration and regression equation was

derived (Figure 6.1.3, 6.1.4, 6.1.5). The results were shown in Table 6.1.1b.



Figure 6.1.3: Calibration curve of DIC by HPLC

Figure 6.1.4: Calibration curve of PCM by HPLC

y = 5085x - 1345.1R² = 0.9998

0

50000

100000

150000

200000

250000

300000

350000

0 20 40 60 80

Peak

area

y = 4606.9x + 14242R² = 0.9993

0200000400000600000800000

100000012000001400000160000018000002000000

0 100 200 300 400 500


Peak

area




Figure 6.1.5: Calibration curve of MET by HPLC

Table 6.1.1b Linearity and range data for DIC, PCM and MET by HPLC


r2*

Slope* Intercept*DIC 10-60µg/ml 5085 1345.1 0.9998PCM 65-390µg/ml 4606.9 14242 0.9993MET 100-600 µg/ml 4030.6 1086 0.9992



The recovery for DIC, PCM and MET were found between 99% and 101%. The results


Table 6.1.2 Recovery study for DIC, PCM and MET by HPLC

DrugConc. Of

Form.(µg/ml)

Conc. OfStd. added

(µg/ml)

Conc.Recover(µg/ml)

% recovery±SD*

DIC20 20 40.03 100.07%±0.04620 25 44.84 99.64%±0.10920 30 49.89 99.78%±0.046

PCM165 135 299.89 99.96%±0.085165 165 326.94 99.07%±0.16165 195 361.83 100.50%±0.11

MET200 250 450.05 100.02%±0.12200 200 400.87 100.21%±0.12200 250 448.13 99.58%±0.10


y = 4030.6x + 1086R² = 0.9992

0

500000

1000000

1500000

2000000

2500000

3000000

0 100 200 300 400 500 600 700

Peak

area

Conccentration (µg/ml)



6.1.2.2.3 Precision

The %RSD of intraday and interday precision study for DIC, PCM and MET were found


Table 6.1.3 Intra-day & Inter-day precision for DIC, PCM and MET by HPLC


DIC 98.84%±0.30 1.22 98.76%±0.25 1.26PCM 98.29%±1.18 0.26 99.06%±0.36 0.48MET 98.94%±0.12 0.14 99.86%±0.19 0.52






Table 6.1.4 Robustness data for DIC, PCM and MET by HPLC

Chromatographicfactor

levelRetention time* Tailing factor*

DIC PCM MET DIC PCM MET

Flow rate

0.6ml/min

3.72±0.02

6.83±0.01

10.32±0.01

1.06±0.01

1.08±0..01

0.97±0.01

1.2ml/min

3.25±0.03

6.14±0.06

9.23±0.08

1.08±0.08

1.09±0.08

0.96±0.01

Methanol:water

68:32 3.70±0.09

6.82±0.01

10.32±0.01

1.08±0.05

1.08±0.08

0.96±0.08

72:28 3.74±0.08

6.15±0.08

10.23±0.01

1.07±0.08

1.08±0.01

0.97±0.08

Detectionwavelengt

h

268nm 3.71±0.08

6.13±0.01

9.41±0.08

1.07±0.01

1.08±0.01

0.97±0.01

276nm 3.71±0.09

6.13±0.01

9.41±0.08

1.07±0.09

1.09±0.01

0.97±0.02



The LOD for DIC, PCM & MET were found to be 0.15μg/ml, 2.40μg/ml and 1.82μg/ml

respectively, while LOQ were 0.48μg/ml, 7.29μg/ml and 5.53μg/ml respectively.




The parameters like retention time, asymmetric factor, number of theoretical plates and

tailing factors were evaluated for DIC, PCM and MET. The results were shown in Table

6.1.5.

Table 6.1.5 System suitability parameters for DIC, PCM and MET by HPLC

Parameter RT* AUC*No. of

theoreticalplates*

Tailingfactor*

DIC 3.51±0.03 50152.6±503.35 3590.4±90.52 1.04±0.02PCM 6.42±0.04 312120.2±112.54 3437.4±139.80 1.08±0.01MET 9.90±0.05 400120.0 ±3923.11 1263.12±7.005 0.96±0.008



Market formulations containing 50mg DIC, 325mg PCM and 500mg MET was analysed

by HPLC.


Forced degradation studies were performed for bulk drug and marketed product, to

provide an indication of the stability indicating property. The degradation was attempted

to stress conditions like acid hydrolysis, alkaline hydrolysis, oxidative hydrolysis,

thermal treatment and neutral degradation, in order to evaluate the ability of the proposed

method to separate drug from its degradation products[3]. During forced degradation

experiments, more degradation was observed in DIC samples under acidic, alkaline and

oxidative degradation. Mild degradation was observed in PCM sample under oxidative

conditions whereas degradation was not observed under acidic, thermal and neutral

conditions. Moderate degradation was observed in MET acidic, alkaline, oxidative and

neutral samples under stress conditions. Table 6.1.7 and 6.1.8 indicates the extent of

degradation of drug substance and marketed product under various stress conditions.

Figures 6.1.6 to 6.1.15 shows the chromatograms of forced degraded samples. The

degradation products were well resolved from drug, confirming the stability indicating

power of the method.



Figure 6.1.6a: Chromatogram of acidic degradation of DIC by HPLC

Figure 6.1.6b: Chromatogram of acidic degradation of PCM by HPLC

Figure 6.1.6c: Chromatogram of acidic degradation of MET by HPLC



Figure 6.1.7a: Chromatogram of alkaline degradation of DIC by HPLC

Figure 6.1.7b: Chromatogram of alkaline degradation of PCM by HPLC

Figure 6.1.7c: Chromatogram of alkaline degradation of MET by HPLC



Figure 6.1.8a: Chromatogram of oxidative degradation of DIC by HPLC

Figure 6.1.8b: Chromatogram of oxidative degradation of PCM by HPLC

Figure 6.1.8c: Chromatogram of oxidative degradation of MET by HPLC



Figure 6.1.9a: Chromatogram of thermal degradation of DIC by HPLC

Figure 6.1.9b: Chromatogram of thermal degradation of PCM by HPLC

Figure 6.1.9c: Chromatogram of thermal degradation of MET by HPLC



Figure 6.1.10a: Chromatogram of neutral degradation of DIC by HPLC

Figure 6.1.10b: Chromatogram of neutral degradation of PCM by HPLC

Figure 6.1.10c: Chromatogram of neutral degradation of MET by HPLC



Figure 6.1.11: Chromatogram of acidic degradation of marketed product byHPLC

Figure 6.1.12: Chromatogram of alkaline degradation of marketed product byHPLC

Figure 6.1.13: Chromatogram of oxidative degradation of marketed product byHPLC



Figure 6.1.14: Chromatogram of thermal degradation of marketed productby HPLC

Figure 6.1.15: Chromatogram of neutral degradation of marketed productby HPLC



Table 6.1.6 Forced degradation study of DIC, PCM and MET by HPLC

Degradationcondition Drug Conc. Of

drug(µg/ml)

RT ofobserved

peak*AUC* %drug* %

degradation*

Acidic

DIC 50 3.56 241732 99.14%±0.00 0.00%±0.00

PCM 325 6.42 1420526 100%±1.01 0.00%±0.00

MET 500

9.90 1807432 94.86%±1.04 0.00%±0.00

5.49(I) 36123 0.00%±0.00 1.99%±0.98

11.10(II) 84231 0.00%±0.00 4.36%±1.62

Alkaline

DIC 50

4.11 231415 95.34%±0.98 0.00%±0.00

3.52(I) 9329 0.00%±0.00 4.03%±0.99

PCM 325 6.39 1419311 99.21%±1.48 0.00%±0.00

MET 500 9.90 1806415 94.12%±1.35 0.00%±0.008.92(II) 95120 0.00%±0.00 5.5%±0.25

Oxidative

DIC 504.11 231811 95.39%±1.21 0.00%±0.00

3.49(I) 9653 0.00%±0.00 4.16%±0.50

PCM 325 6.39 1419769 91.76%±1.34 0.00%±0.005.62(II) 11981 0.00%±0.00 8.64%±1.21

MET 500 9.90 1806926 99.54%±1.21 0.00%±0.0010.32(III) 91824 0.00%±0.00 5.06%±0.50

ThermalDIC 50 3.51 251830 98.99%±1.09 0.00%±0.00PCM 325 6.42 1500874 99.42%±1.09 0.00%±0.00MET 500 9.90 2007891 99.29%±1.21 0.00%±0.00

Neutral

DIC 50 4.11 24981 98.44%±12 0.00%±0.00

PCM 325 6.39 1465896 98.97%±1.21 0.00%±0.00

MET 500 9.90 1805341 94.76%±1.09 0.00%±0.0010.29(I) 98691 0.00%±0.00 5.46%±0.78




6.1.3 ConclusionThe isocratic RP-HPLC method developed for the analysis of ternary mixtures of DIC,

PCM and MET as bulk drug and in pharmaceutical preparation is simple, accurate,

precise, and repeatable with short run time. In forced degradation the drug degrades as

shown by the decreased areas in peaks when compared with peak areas of the same

concentration of the non-degraded drug, with giving additional degradation peaks at

different retention time. The developed method is stability indicating and separate

degradants and can be used by quality control department to determine the assay of

pharmaceutical preparation and also stability samples.



6.2 STABILITY INDICATING HPTLC METHOD FOR


PARACETAMOL AND METHOCARBAMOL

6.2.1 Experimental6.2.1.1 Solubility

Solubility of Diclofenac potassium (DIC), Paracetamol(PCM) and Methocarbamol

(MET) in different solvent like distilled water, 0.1N HCl, 0.1N NaOH, methanol,

dimethyl formamide, acetonitrile, ethanol and chloroform. All three drugs were soluble in

methanol.


The mobile phase toluene:ethyl acetate:Methanol in the ratio of 4:3:2, v/v/v respectively

was used.








Accurately weighed PCM (10mg) was transferred in 100ml volumetric flask. The drug



6.2.1.3.1.3 Preparation of standard stock solution of MET

Accurately weighed MET (10mg) was transferred in 100ml volumetric flask. The drug





to 50mg of DIC, 325mg of PCM and 500mg of MET was transferred to a 100ml


minutes and volume was made up with methanol.




Solutions of DIC, PCM and MET were scanned between 200 and 400nm. UV spectra of

all three drugs show maximum absorbance at 225nm.

Figure 6.2.1 Overlay UV spectra of DIC, PCM and MET


Chromatography was performed on 10x10cm Precoated Silica Gel G60F254 aluminium

sheets. The samples were applied to the plates as 6mm band, 10 mm apart, by means of

Linomat-V sample applicator with help of 100μl Hamilton syringe. It was developed in

CAMAG TLC chamber (20x10cm, 20x20cm) which was already saturated for 30 min.

with mobile phase at room temperature. The optimum mobile phase consisted of

toluene:ethyl acetate:methanol in the ratio of 4:3:2, v/v/v respectively. After development

the plate was scanned at 225 nm by means of CAMAG TLC Scanner 3 controlled by

WinCATs software.

6.2.1.7 Method validation[1-2]


The calibration curve was plotted over the concentration range of 100-6000ng/spot for

DIC, 150-650ng/spot for PCM and 200-1200ng/spot for MET. All the solutions were

spoted on precoated TLC plate, chromatograms were recorded and it was repeated for six



times. A calibration graph was plotted between the mean peak height Vs respective


6.2.1.7.2 Accuracy

The accuracy of the method was determined by calculating recoveries of DIC, PCM and

MET by standard addition method. Known amount of standard solution of DIC, PCM

and MET (80, 100 and 120% level) were added to preanalysed samples.

6.2.1.7.3 Precision

Precision was evaluated in terms of intra-day and inter-day precision. The intraday

precision was investigated using three different concentrations of sample solutions. The

intraday and interday precision of the proposed method was determined by analyzing the

corresponding concentration three times on the same day and on different days. The TLC

plate was developed and analysed as described under chromatographic condition.



making small changes in the ratio of mobile phase, in the saturation time, changing the

wavelength.







0.8, 1.0 and 1.2μl of the sample solution as described in 6.2.1.4 were spotted on

precoated TLC plate. 1ml of sample solution (6.2.1.4) was diluted upto 10ml with

methanol and 0.8, 1.0 and 1.2μl of this solution were spotted on precoated TLC plate.

The TLC plate was developed and analysed as described under chromatographic

condition. The amount of DIC, PCM and MET were determined by regression equation.











25mg of PCM was weighed accurately, transferred into 100ml volumetric flask and




6.2.1.9.1.3 Acidic degradation of MET

50mg of MET was weighed accurately, transferred into 100ml volumetric flask and















6.2.1.9.2.3 Alkaline degradation of MET


















6.2.1.9.3.3 Oxidative degradation of MET







10mg of DIC was weighed accurately, kept at 105°c for 8 hours and transferred into





25mg of PCM was weighed accurately, kept at 105°C for 8 hours and transferred into






6.2.1.9.4.3 Thermal degradation of MET

50mg of MET was weighed accurately, kept at 105°C for 8 hours and transferred into















6.2.1.9.5.3 Neutral degradation of MET











2.0μl of the solution was spotted on precoated TLC plate (100ng/spot DIC, 650ng/spot

PCM, and 1000ng/spot MET), developed and analysed as described under






to 50 mg of DIC, 325mg of PCM and 500mg of MET was transferred to a 100ml


refluxed at 85°C for 24 hours. 1ml of the solution diluted upto 10ml with methanol then

1.0μl of the solution was spotted on precoated TLC plate (100ng/spot DIC, 650ng/spot

PCM and 1000ng/spot MET) developed and analysed as described under

chromatographic condition



to 50mg of DIC, 325mg of PCM and 500mg of MET was transferred to a 100 ml


at room temperature for 24 hours. 1ml of the solution diluted upto 10ml with methanol

then 2.0μl of the solution was spotted on precoated TLC plate (100ng/spot DIC,

650ng/spot PCM and 1000ng/spot MET) developed and analysed as described under




to 50mg of DIC, 325mg of PCM and 500mg of MET was kept at 105°C for 8 hours and


and diluted upto mark with methanol. 1ml of the solution diluted upto 10ml with

methanol then 2.0μl of the solution was spotted on precoated TLC plate (100ng/spot DIC,

650ng/spot PCM and 1000ng/spot MET) developed and analysed as described under






at 85°C for 24 hours. 1ml of the solution diluted upto 10ml with methanol then 2.0μl of

the solution was spotted on precoated TLC plate (100ng/spot DIC, 650ng/spot PCM and

400ng/spot MET) developed and analysed as described under chromatographic condition.



6.2.2 Results and discussion

6.2.2.1 Method optimization

For optimization, different mobile phases and composition were employed to achieve the

good separation. The method development was initiated with using a mobile phase of

toluene: methanol:ammonium sulphate in various proportions. In the above conditions

only two drugs were separated. All three drugs were separated using mobile phase

consisting of different ratio of toluene: ethylacetate:methanol. Finally mobile phase

consisting mixture of toluene:ethyl acetate:methanol in ratio of 4:3:2, v/v/v respectively

gave reasonable Rf (Rf 0.78 for DIC, 0.63 for PCM and 0.42 for MET) and sharp band

for all three drugs. Saturation of TLC chamber for 30 min assured better reproducibility

and better resolution. All three drugs were detected at 272 nm by means of CAMAG TLC

Scanner 3. A chromatogram is shown in Figure 6.2.2.

Figure 6.2.2: Chromatogram of DIC, PCM and MET by HPTLC



The calibration curve was plotted over the concentration range of 100-600 ng/spot for

DIC, 150-650 ng/spot for PCM and 200-1200 ng/spot for MET. A calibration graph was

plotted between the mean peak height Vs respective concentration and regression

equation was derived (Figure 6.2.3, 6.2.4, 6.2.5). The results were shown in Table 6.2.1.



Figure 6.2.3: Calibration curve for DIC by HPTLC

Figure 6.2.4: Calibration curve of PCM by HPTLC

y = 15.089x + 47.333R² = 0.9997

0100020003000400050006000700080009000

10000

0 100 200 300 400 500 600 700


Peak

Area

y = 22.766x + 53.714R² = 0.9989

0

2000

4000

6000

8000

10000

12000

14000

16000

0 200 400 600 800


Peak

Area



Figure 6.2.5: Calibration curve of MET by HPTLC

Table 6.2.1 Linearity and range data for DIC, PCM and MET by HPTLC


r2*

Slope* Intercept*DIC 100-600ng/spot 15.089 47.33 0.9997PCM 150-650ng/spot 22.76 53.71 0.9989MET 200-1200ng/spot 14.865 196.53 0.9995



The recovery for DIC, PCM and MET were found between 99.0 % and 101%. The results


Table 6.2.2 Recovery study for DIC, PCM and MET by HPTLC

DrugConc. ofForm.

(ng/spot)

Conc. ofStd. added(ng/spot)

Conc.Recover(ng/spot)

% recovery±SD*

DIC300 250 546.20 99.30%±0.047300 300 594.54 99.09%±0.068300 350 650.73 100.11%±0.030

PCM250 150 396.92 99.23%±0.123250 250 496.91 99.38%±0.013250 350 595.22 99.20%±0.067

MET600 400 991.03 99.10%±0.050600 500 1090.1 99.1%±0.055600 600 1188.03 99.00%±0.058


y = 14.865x + 196.53R² = 0.9995

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

0 500 1000 1500Concentration (ng/spot)

Peak

Area

Peak

Area



6.2.2.2.3 Precision

The %RSD of intraday and inter-day precision study for DIC, PCM and MET were found


Table 6.2.3 Intra-day & Inter-day precision for DIC, PCM and MET by HPTLC


DIC 98.97%±0.015 0.010 99.97%±0.019 0.046PCM 99.97%±0.04 0.024 99.98%±0.047 0.036MET 99.90%±0.02 0.016 99.99%±0.01 0.028






Table 6.2.4 Robustness data for DIC, PCM and MET by HPTLC

Chromatographic factor level Amont found*(ng/spot)DIC PCM MET

Mobile phase ratioToluene: ethyl acetate:

methanol

1.2:1:4v/v/v 299.95±0.054

399.95±0.061 599.98±0.057

1:1.2:4v/v/v 299.94±0.044

399.93±0.038 599.96±0.043

Saturation time32 minutes 299.97±0.03

7399.97±0.04

3 599.97±0.047

28 minutes 299.93±0.050

399.93±0.042 599.99±0.037

Detection wavelength275nm 299.93±0.04

9399.95±0.04

3 599.95±0.058

265nm 299.96±0.040 400.00±0.02 600.00±0.033



The LOD for DIC, PCM & MET were found to be 0.005 ng/spot, 3.172 ng/spot and

7.617 ng/spot respectively, while LOQ were 0.875 ng/spot, 9.614 ng/spot and 23.083

ng/spot respectively.

6.2.2.3 Analysis of marketed productThe market formulation containing 50mg DIC, 325mg PCM and 500mg MET was

analysed by HPTLC.




The degradation was attempted to stress conditions like acid hydrolysis, alkaline

hydrolysis, oxidative hydrolysis, thermal treatment and neutral degradation, in order to

evaluate the ability of the proposed method to separate drug from its degradation

products[3]. HPTLC studies of the samples obtained during the stress testing of DIC,

PCM and MET under different conditions using toluene:ethyl acetate:methanol in the

ratio of 4:3:2, v/v/v respectively as the mobile phase shows different degradation peaks as

shown in Figures 6.2.6 to 6.2.15. DIC showed degradation in alkaline and oxidative stress

conditions at Rf of 0.84 and 0.76 respectively. PCM showed degradation oxidation

condtition at Rf value 0.57. MET showed highest degradation in acidic condition and

degradation products appear at Rf of 0.29 and 0.34 and lower degradation in neutral

condition at Rf of 0.37. Table 6.2.6 indicates the extent of degradation of marketed

product under various stress conditions.

Figure 6.2.6a: Chromatogram of acidic degradation of DIC by HPTLC

Figure 6.2.6b: Chromatogram of acidic degradation of PCM by HPTLC



Figure 6.2.6c: Chromatogram of acidic degradation of MET by HPTLC

Figure 6.2.7a: Chromatogram of alkaline degradation of DIC by HPTLC



Figure 6.2.7b: Chromatogram of alkaline degradation of PCM by HPTLC

Figure 6.2.7c: Chromatogram of alkaline degradation of MET by HPTLC

Figure 6.2.8a: Chromatogram of oxidative degradation of DIC by HPTLC



Figure 6.2.8b: Chromatogram of oxidative degradation of PCM by HPTLC

Figure 6.2.8c: Chromatogram of oxidative degradation of MET by HPTLC

Figure 6.2.9a: Chromatogram of thermal degradation of DIC by HPTLC



Figure 6.2.9b: Chromatogram of thermal degradation of PCM by HPTLC

Figure 6.2.9c: Chromatogram of thermal degradation of MET by HPTLC

Figure 6.2.10a: Chromatogram of neutral degradation of DIC by HPTLC



Figure 6.2.10b: Chromatogram of neutral degradation of PCM by HPTLC

Figure 6.2.10c: Chromatogram of neutral degradation of MET by HPTLC

Figure 6.2.11: Chromatogram of acidic degradation of marketed product by

HPTLC



Figure 6.2.12: Chromatogram of alkaline degradation of marketed product by

HPTLC

Figure 6.2.13: Chromatogram of oxidative degradation of marketed product byHPTLC



Figure 6.2.14: Chromatogram of thermal degradation of marketed product by HPTLC

Figure 6.2.15: Chromatogram of neutral degradation of marketed product byHPTLC



Table 6.2.5 Forced degradation study of marketed product by HPTLC

Degradation

conditionDrug

Conc. Ofdrug(ng/sp

ot)

Rf value ofobserved

peak*

Peakarea* %drug* %degradatio

n*

acidic

DIC 100 0.78 3234.5 99.12%±1.08 0.00%±0.00

PCM 250 0.63 5318.9 99.91%±1.01 0.00%±0.00

MET 4000.42 8965.3 84.92%±1.0

5 0.00%±0.00

0.29(I) 692.1 0.00%±0.00 7.71%±0.990.34(II) 746.7 0.00%±0.00 8.32±0.99

alkaline

DIC 1000.78 3102.8

89.51%±0.98 0.00%±0.00

0.84(I) 326.6 0.00%±0.00 10.50%±0.99

PCM 250 0.63 5299.3699.45%±1.0

8 0.00%±0.00

MET 400 0.41 8134.9 94.91%±0.21 0.00%±0.00

0.36(II) 425.6 0.00%±0.00 5.22%±0.55

oxidative

DIC 100 0.78 2894.5 88.39%±0.21 0.00%±0.00

0.76(I) 329.7 0.00%±0.00 11.36%±0.55

PCM 250 0.63 4624.9 90.84%±1.34 0.00%±0.00

0.57(II) 427.4 0.00%±0.00 9.25%±1.26

MET 400 0.42 8128.8 91.92%±0.24 0.00%±0.00

0.35(III) 692.1 0.00%±0.00 8.51%±0.55

Thermal

DIC 100 0.78 3129.66 99.92%±1.09 0.00%±0.00

PCM 250 0.63 5236.34 99.96%±1.09 0.00%±0.00

MET 400 0.42 8631.34 99.95%±0.19 0.00%±0.00

Neutral

DIC 100 0.78 2988.75 98.96%±1.05 4.13%±0.99

PCM 250 0.63 4989.6 99.92%±1.21 0.00%±0.00

MET 400 0.42 7984.199.89%±0.3

4 0.00%±0.00

0.37(I) 536.9 0.00%±0.00 6.71%±0.99*= Average result of six replicate samples



6.2.3 Conclusion

The developed HPTLC technique is simple, accurate, sensitive, precise, rapid and

repeatable. The method was successfully used for determination of DIC, PCM and MET

as bulk drug and in pharmaceutical formulation. After exposing the drugs to different

stress condition, the drug peak area was observed to decrease and also degradant peak

was observed. The developed method is stability indicating and separate degradants and

can be used to determine the assay of pharmaceutical preparation and also stability

samples.



6.3 STATISTICAL COMPARISON BETWEEN OFFICIAL

REPORTED AND PROPOSED METHODS[4-5]

The assay result for DIC, PCM and MET in their dosage form obtained using HPLC and

HPTLC methods were compared with official reported methods by applying the paired t-

test and F-test. The calculated t-value for DIC (0.2201), PCM (0.077) and MET (0.3220)

for HPLC as well as DIC (0.3251), PCM (0.0321), and MET (0.1475) for HPTLC is less

than the tabulated t-value (2.571) at the 95% confidence interval. Moreover, the

calculated F-value for DIC (0.1886), PCM (0.2596) and MET (0.1886) for HPLC and

DIC (0.1850), PCM (0.010) and MET (0.1850) for HPTLC is also less than the tabulated

F-value (4.28). Therefore, there is no significant difference in a determined content of

DIC, CHL and PCM by HPLC and HPTLC methods. Table 6.3.1 indicates statistical

comparison between proposed methods.

Table 6.3.1 Statistical comparison for DIC, PCM and MET between proposed

methods

Drug Methods Mean SD n

Paired t-test F-test

Tabulated

value

Calculated

value

Tabulated

value

Calculated

value

DIC

Official 99.27 0.16 6

2.571 0.2201 4.28 0.1886HPLC 99.71 0.28 6

Official 99.27 0.16 6

0.3251 0.1850HPTLC 99.28 0.14 6

PCM

Official 99.47 0.29 6

2.571 0.077 4.28 0.2596HPLC 99.65 0.14 6

Official 99.47 0.29 6

0.0321 0.010HPTLC 99.15 0.06 6

MET

Official 99.40 0.26 6

2.571 0.3220 4.28 0.1886HPLC 99.36 0.14 6

Official 99.40 0.14 6

0.1475 0.1850HPTLC 99.48 0.15 6



6.4 References1. ICH, Q2(R1) Validation of Analytical Procedure: Text and Methodology,

International Conference on Harmonization, Geneva, October 1994.

2. The United States Pharmacopeia, The National Formulary, USP 30 NF 25, Asian

Edition, Volume 1, 2007; 680-683.

3. Bakshi M, Singh S, Development of validated stability-indicating assay methods

critical review, Journal of Pharmaceutical and Biomedical Analysis, 2002; 28:

1010-1040.

4. Christian GD, Analytical Chemistry, 6th Ed., University of Washington, 2007; 90-

97.

5. Sanford Bolton, Charles Bon, Pharmaceutical Statistics Practical and Clinical

Application, 4th Ed., Revised and Expanded, 2004; 417-436.

Chapter 7 Summary


7. SUMMARY Review was done on muscle relaxant formulations, its cause and drug profile of

the drugs used for the study.

The reported methods for analysis of Diclofenac potassium, Paracetamol,

Chlorzoxazone and Methocarbmol either single or in combination with any one of

mucscle relaxant drugs were reviewed.

Two new simple, precise and accurate methods are described for the

determination of DIC, CHL and PCM in API and in pharmaceutical preparation.

RP-HPLC method was developed and validated using mobile phase consisting of

methanol:phosphate buffer (80:20, v/v respectively), at 0.8ml/min flow rate, C18

column, Autochro-3000 operating software and dual wavelength UV detector.

HPTLC method was developed and validated using mobile phase consisting of

toluene:ethyl acetate:glacial acetic acid(1:2:0.5, v/v/v respectively, at 30 minutes

saturation time, precoated Silica Gel G60F254 aluminium sheets, CAMAG TLC

Scanner 3 controlled by WinCATs software. Forced degradation study on

individual drug substance and drug product was performed under acidic, alkaline,

oxidative, thermal and neutral condition. The developed analytical methods were

statistically compared using paired t-test and F-test.

Two new simple, precise and accurate methods are described for the

determination of DIC, PCM and MET in both raw material and in laboratory

mixture. RP-HPLC method was developed and validated using mobile phase

consisting of methanol:water (80:20, v/v respectively), at 0.8ml/min flow rate, C18

column, Autochro-3000 operating software and dual wavelength UV detector.

HPTLC method was developed and validated using mobile phase consisting of

toluene:ethylacetate:methanol(1:2:4, v/v/v respectively), at 30 minutes saturation

time, precoated Silica Gel G60F254 aluminium sheets, CAMAG TLC Scanner 3

controlled by WinCAT software.

Chapter 7 Summary


Forced degradation study on individual drug substance and drug product was

performed under acidic, alkaline, oxidative, thermal and neutral condition. The

developed analytical methods were statistically compared using paired t-test and

F-test.

Forced degradation study on individual drug substance and drug product was

performed under acidic, alkaline, oxidative, thermal and neutral condition.

Chapter 8 Publication


8. PUBLICATION

TITLE JOURNAL STATUS

Development and Validation of stability indicating

method for determination of Diclodenac potassium,

Chlorzoxazone and Paracetamol in pharmaceutical

dosage form using high performance liquid

chromatography

Inventi Rapid:

Pharm Analysis

& Quality

Assurance

Published

2012(4): 1-

9, 2012.

Stability Indicating HPLC method for simultaneous

determination of Diclofenac potassium, Paracetamol

and Methocarbamol

American

Journal of

Pharmaceutical

Technology &

Research

Accepted

(in Press)

Development and validation of stability indicating

method for the determination of Paracetamol and

Methocarbamol in pharmaceutical dosage form

using HPTLC

Journal of

Chromatography

B

Under

review

Development and validation of simultaneous HPLC

method for estimation of Paracetamol and

Methocarbamol.

Arabian Journal

of Chemistry

Under

review

Inventi Rapid: Pharm Analysis & Quality Assurance Vol. 2012, Issue 4 [ISSN 0976-3813]

2012 ppaqa 543, CCC: $10 © Inventi Journals (P) Ltd Published on Web 08/10/2012, www.inventi.in

RESEARCH ARTICLE

INTRODUCTION Chlorzoxazone a centrally acting acting agent for painful musculoskeletal condition it primarily acts at the level of the spinal cord and subcortical area of brain. It inhibits degranulation of mast cells, subsequently preventing the release of histamine and slow-reacting substance of anaphylaxis, mediators of type I allergic reactions (1). It is absorbed orally and undergoes first pass metabolism and is excreted by kidney. t1/2 is 2-3 hrs. It is indicated in spasticity due to neurological disorders and in painful muscle spasms of spinal origin (2). Diclofenac potassium [2-[(2,6-dichlorophenyl)amino] benzeneacetic acid, mono potassium salt], is an analgesic which acts by non-steroidal anti-inflammatory drug (NSAID) that exhibits anti-inflammatory, analgesic, and antipyretic activities in animal models. The mechanism of action of diclofenac potassium tablets, like that of other NSAIDs, is not completely understood but may be related to prostaglandin synthetase inhibition (3-6). It is official in Indian Pharmacopoeia and United States Pharmacopoeia. It is official in Indian Pharmacopoeia and United States Pharmacopoeia. Paracetamol [N-(4-Hydroxyphenyl) acetamide], an analgesic and antipyretic action with weak anti inflammatory activity. These effects are related to inhibition of prostaglandin synthesis (7,8). It is official in Indian Pharmacopoeia and United States Pharmacopoeia. It is official in United States Pharmacopoeia (Figure 1 Structure of diclofenac potassium, chlorzoxazone and

1Kalol Institute of Pharmacy, Kalol-Mehsana Highway, Kalol -382721, Gujarat, India. E-mail: [email protected] *Corresponding author 2Shree S. K. Patel College of Pharmaceutical Education & Research, Ganpat University, Kherva, Mehsana-382711, Gujarat, India.

3Faculty of Pharmacy, D. D. University College Road, Nadiad-387001, Gujarat, India.

4Shankersinh Vaghela Bapu Institute of Pharmacy, Gandhinagar-Mansa Road, PO. Vasan, Gandhinagar-382650, Gujarat, India.

paracetamol). Several UV (9-11) and HPLC (12-16) methods either single or in combination have been reported for estimation of chlorzoxazone (CHL), diclofenac potassium(DIC) and paracetamol (PCM). Few HPTLC (17-20) methods are also reported for estimation of DIC and PCM. The International Conference on Harmonization (ICH) guideline entitled ‘stability testing of new drug substances and products’ requires that stress testing be carried out to elucidate the inherent stability characteristics of the active substances. An ideal stability indicating method is one that resolves the drug and its degradation products efficiently. Consequently, the implementation of an analytical methodology to determine CHL, DIC and PCM simultaneously in presence of its degradation products is rather a challenge for pharmaceutical analyst. Thus, thought necessary to study the stability of CHL, DIC and PCM under acidic, alkaline, neutral hydrolysis, oxidative and dry heat conditions. This work describes validated stability-indicating HPLC method for simultaneous estimation of CHL, DIC, and PCM. Reversed-phase chromatographic method with UV detection has shown to be sensitive, accurate and suitable for analyzing a large support bioavailability and stability studies. EXPERIMENTAL Reagents and Chemicals Pure chlorzoxazone, diclofenac potassium and paracetamol were obtained as a gift samples from Zydus Cadila Ltd (Ahmedabad, India) with purity of 98.99, 99.87 and 99.93% respectively. The formulation of the tablet with combination of diclofenac potassium 50mg, chlorzoxazone 250mg, and paracetamol 325mg is available in market by brand name Dan-MR. Tablet was purchased from the local market. Methanol and acetonitrile of HPLC grade were procured by Merck Ltd., India. Disodium hydrogen phosphate was procured from Merck Ltd., India. Sodium hydroxide, hydrochloric acid, disodium hydrogen phosphate, hydrogen peroxide and glacial acetic acid of analytical reagent were procured from Merck Ltd., India.

Development and Validation of Stability Indicating Method for Determination of Chlorzoxazone, Diclofenac Potassium and Paracetamol in Pharmaceutical Dosage form using High Performance Liquid Chromatography

Maulikkumar R Amin1*, Paresh U Patel2, B N Suhagia3, Madhabhai M Patel4

Abstracts: A stability-indicating HPLC method has been developed and validated for simultaneous estimation chlorzoxazone, diclofenac potassium and paracetamo in bulk drug and pharmaceutical dosage forms. Stress studies were conducted for all three drugs under ICH prescribed stress conditions viz. hydrolysis, oxidation, thermal and neutral stress. An isocratic RP-HPLC method was achieved on younglin HPLC system using Varian C18 (250x4.6 mm i.d, 5 μm particle size) column for separation of drug from its degradation products using mobile phase containing mixture of methanol: phosphate buffer (pH 3.0, adjusted with glacial acetic acid) (70:30, v/v). The flow rate was 0.8ml/min and the eluent was monitored at 280nm. Linearity was found in the range of 5-25μg/ml for diclofenac potassium (DIC), 25-125μg/ml for chlorzoxazone (CHL) and 32.5-182.5μg/ml for paracetamol (PCM). The limit of detection for DIC, CHL & PCM was found to be 0.15μg/ml, 1.82μg/ml and 2.40μg/ml respectively, while limit of quantification was 0.47μg/ml, 5.53μg/ml and 7.29μg/ml respectively. The developed method was found to be fast, accurate, precise, reproducible and suitable for analysis of all three drugs in bulk and pharmaceutical dosage forms.

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Instrumentation and Chromatographic Conditions The HPLC system used was Younglin equipped with binary solvent managers, manual, dual wavelength UV detector operated at 280nm. HPLC column incorporated solvent delivery model LC-10ATVP. Moreover, HPLC column is Varian C-18 (250x4.6) mm i.d. 5 μm particle size with microliter syringe (rheodyne) injector. The

HPLC was connected to personal computer with Class- Autochro-3000 software. The isocratic mobile phase consisted of methanol : water (80:20 v/v) and was delivered at a flow rate of 0.8 ml min-1. Detection was carried out using a UV detector at 280 nm. The column was maintained at ambient temperature and injection volume of 20μl was used.

Table 1: Results of System Suitability Parameters

Parameter RT* AUC* No. of Theoretical Plates* Tailing Factor* DIC 3.25±0.05 25012.20±283.43 4204.4±9.52 0.95±.002 CHL 5.60±0.06 105410.2±312.53 3637.4±139.80 1.02±0.01 PCM 9.40±0.09 150453.12±3923.11 1263.12±7.005 0.97±0.001


Table 2: Linearity and Calibration Curve

Drug Linearity Range Y=mx+c r2* Slope* Intercept* DIC 5-30 μg/ml 5018.1 362 0.9998 CHL 25-125 μg/ml 4987.5 2058 0.9997 PCM 32.5-182.5 μg/ml 5051.4 14445 0.9997


Table 3: Results of Recovery Studies

Drug Conc. of Form.(μg/ml) Conc. of Std. Added(μg/ml) Conc. Recovered(μg/ml) ±SD* % Recovery±SD*

DIC 5 4 8.89±0.12 98.78%±0.18 5 5 9.97±0.30 99.70%±0.75 5 6 10.92±0.02 99.27%±1.89

CHL 25 24 48.49±0.07 98.96%±0.01 25 25 48.90±0.71 98.16%±0.18 25 26 50.58±0.05 99.17%±1.09

PCM 32.5 34 65.54±1.51 98.56%±0.86 32.5 32.5 65.28±0.08 100.41%±0.15 32.5 36 69.07±0.16 100.83%±0.11


Table 4: Results of Intraday & Inter Day Precision

Drug Intra day Inter day Mean % Assay* %RSD* Mean % Assay* % RSD*

DIC 99.34%±1.16 0.36 99.15%±1.82 1.08 CHL 99.83%±1.63 0.12 100.05%±0.57 1.12 PCM 98.94%±0.01 1.16 98.86%±0.12 1.52


Table 5: Robustness Data for DIC, CHL and PCM

Chromatographic Factor level Retention Time* Tailing Factor*

DIC CHL PCM DIC CHL PCM

Flow rate 0.7ml/min 3.98±0.02 6.01±0.01 9.96±0.01 0.95±0.02 1.02±0.01 0.97±0.01 1.0ml/min 2.95±0.02 5.04±0.01 8.66±0.03 0.96±0.01 1.03±0.01 0.96±0.01

Methanol: phosphate buffer (v/v)

75:25 3.91±0.03 6.02±0.03 9.95±0.02 0.95±0.02 1.02±0.01 0.96±0.02 85:15 3.96±0.02 5.05±0.02 8.61±.02 0.96±0.01 1.02±0.01 0.97±0.01

Detection wave-length 275nm 3.24±0.04 5.60±0.01 9.41±0.01 0.95±0.01 1.03±0.01 0.97±0.03 285nm 3.26±0.03 5.60±0.01 8.64±0.02 0.95±0.02 1.02±0.01 0.97±0.01

Table 6: Limit of Detection and Limit of Quantification

Drugs Standard Deviation Slope of Calibration Curve LOD(μg/ml) LOQ(μg/ml)

DIC 238.7 5018.1 0.15 0.47 CHL 2759.5 4987. 1.82 5.53 PCM 3684.4 5051.4 2.40 7.29

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Preparation of Stock Solutions Preparation of Standard Stock Solution of DIC Accurately weighed 50mg DIC was transferred in 100ml volumetric flask. The drug was dissolve in methanol with sonication and final volume was adjusted with methanol upto mark to prepare a 500μg/ml stock solution. Preparation of Standard Stock Solution of CHL Accurately weighed 50mg CHL was transferred in 25ml volumetric flask. The drug was dissolve in methanol with sonication and final volume was adjusted with methanol upto mark to prepare a 2500μg/ml stock solution. Preparation of Standard Stock Solution of PCM

Accurately weighed 32.5mg PCM was transferred in 10ml volumetric flask. The drug was dissolve in methanol with sonication and final volume was adjusted with methanol upto mark to prepare a 3250μg/ml stock solution. Preparation of Standard Working Solutions Preparation of Standard Working Solution of DIC From the stock solution (500μg/ml), an aliquot quantity 1, 2, 3, 4, and 5ml were transferred into separate 100ml volumetric flask and final volume was adjusted with methanol upto mark to prepare 5-25μg/ml solutions. Preparation of Standard Working Solution of CHL From stock solution (2500μg/ml), an aliquot quantity 1, 2, 3, 4, and 5 ml were transferred into 100 ml volumetric flask

Table 7: Summary of Validation Parameters

Parameter DIC CHL PCM Wavelength 280nm 280nm 280nm

Range 5-30μg/ml 25-125μg/ml 32.5-182.5μg/ml Linearity 0.9998 0.9997 0.9997 Intercept 362 2058 14445

Slope 5018.1 4987.5 5051.4 Intraday precision 0.36 0.12 1.16 Interday precision 1.08 1.12 1.52

LOD 0.15 1.82 2.40 LOQ 0.47 5.53 7.29

Table 8: Analysis of Marketed Formulation of the Product

Brand Name Drug Amount Taken (μg/ml) Amount Found (μg/ml) %Amount found

DAN-MR

DIC 50 40.987 99.74±0.22 CHL 250 249.89 99.56±0.11 PCM 325 324.26 99.26±0.21

Table 9: Results of Forced Degradation Study

Degradation Condition Drug Conc. of Drug(μg/ml) RTof Observed Peak* AUC* % Drug* % Degradation*

Acidic

DIC 5 3.25 24018 98.14%±1.04 0.00%±0.00

CHL 25 5.59 101541 91.98%±1.31 0.00%±0.00

5.19(I) 8931 0.00%±0.00 8.79±1.31 PCM 32.5 9.40 149901 99.65%±1.45 0.00%±0.00

Alkaline

DIC 5 3.25 24010 94.62%±1.24 0.00%±0.00

2.92(I) 1432 0.00%±0.00 5.96%±1.21

CHL 25 5.62 94421 91.56%±1.57 94.10%±1.38

5.49(II) 3410 0.00%±0.00 3.61%±1.41 6.12(III) 1431 0.00%±0.00 1.51±1.30

PCM 32.5 9.41 149097 99.57%±1.34 0.00%±0.00

Oxidative

DIC 5 3.25 23120 91.41%±1.32 0.00%±0.00

2.72(I) 1981 0.00%±0.00 8.98%±1.54

CHL 25 5.60 102384 97.00%±1.03 0.00%±0.00

5.12(II) 2398 0.00%±0.00 2.64%±0.95

PCM 32.5 9.41 148320 93.84%±1.09 0.00%±0.00

10.21(III) 9908 0.00%±0.00 6.68±1.41

Thermal

DIC 5 3.25 25320 99.90%±1.12 0.00%±0.00 CHL 25 5.61 105908 99.88%±1.08 0.00%±0.00

PCM 32.5 9.41 149959 99.84%±1.24 0.00%±0.00

Neutral

DIC 5 3.25 24906 98.45%±1.4 0.00%±0.00

CHL 25 5.60 103956 91.88%±1.23 0.00%±0.00

4.93(I) 9327 0.00%±0.00 8.64%±0.95

PCM 32.5 9.41 104903 99.84%±1.21 0.00%±0.00

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and final volume was adjusted with methanol upto mark to prepare 25-125μg/ml solutions. Preparation of Standard Working Solution of PCM From stock solution (3250μg/ml), an aliquot quantity 1, 2, 3, 4, and 5 ml were transferred into 100 ml volumetric flask and final volume was adjusted with methanol upto mark to prepare 32.5-162.5 μg/ml solutions.

Preparation of Sample Solution Twenty tablets were weighed accurately and one tablet was calculated. The tablets were crushed to furnish a homogenous powder and quantity equivalent to 50 mg of DIC, 250 mg of CHL and 325 mg of PCM was transferred to a 100 ml volumetric flask. The powder was dissolved in 60 ml of methanol with sonication for 15 minutes and volume was made up with methanol. 1ml of the solution was

O

O

NH

Cl

Cl

K+

HO

NH

CH3

O

Diclofenac Potassium Paracetamol

N

O

Cl

OH

Chlorzoxazone

Figure 1: Structure of diclofenac potassium, chlorzoxazone and paracetamol

Figure 2: Overlay UV spectra of DIC, CHL and PCM

Figure 3: A typical Chromatogram for DIC, CHL and PCM

Figure 4: Calibration curve of DIC

Figure 5: Calibration curve of CHL

y = 5018.1x + 362 R² = 0.9998

0

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transferred into 10ml volumetric flask and diluted upto mark with methanol. Selection of Detection Wavelength Solutions of DIC, CHL and PCM were scanned between 200 and 400nm. UV spectra of all three drugs show maximum absorbance at 280nm (Figure 2 Overlay UV spectra of DIC, CHL and PCM). Selection of Mobile Phase Different mobile phases were tried and 20μL of mixed standard solution was injected. The mobile phase consisted of methanol: phosphate buffer pH 4.0(pH adjusted with glacial acetic acid) in the ratio of 70:30, v/v was selected respectively was selected as it gave well resolved sharp peaks with reasonable retention time for all the drugs. The analysis was carried out using Younglin series LC-10ATVP binary gradient system, Varian 5μm C-18 column (250x4.6 mm) with flow rate 0.8 mL/min. (Figure 3 A typical Chromatogram for DIC, CHL and PCM).

Method Validation System Suitability Parameters System suitability parameter was established to ensure that the validity of the analytical method was maintained whenever used. Typical variations are the stability of analytical solution, different equipment, and different analyzer. In case of liquid chromatography typical variations are composition of mobile phase, different lots or supplier of columns, temperature and flow rate. The parameters like retention time, asymmetric factor, number of theoretical plates and tailing factors were evaluated for DIC, CHL and PCM. The results of system suitability parameters are shown in Table 1. Linearity and Calibration Graph The linearity was evaluated by linear regression analysis. The series of dilution ranging from 5-25μg/ml for DIC, 25-125μg/ml for CHL and 32.5-182.5μg/ml for PCM was prepared and performed for linearity. All the solutions were filtered through 0.2 μm membrane filter and injected, chromatograms were recorded and it was repeated for six

Figure 6: Calibration curve of PCM

Figure 7: Forced degradation under acidic condition

Figure 8: Forced degradation under alkaline condition

Figure 9: Forced degradation under oxidative condition

Figure 10: Forced degradation under thermal condition

Figure 11: Forced degradation under neutral condition

y = 5051.4x - 14453 R² = 0.9997

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times. The results of linearity and calibration curve are shown in Table 2. (Figure 4 Calibration curve of DIC, Figure 5 Calibration curve of CHL, and Figure 6 Calibration curve of PCM). Recovery Studies The recovery studies were performed to validate the accuracy of developed method. To a pre analysed sample solution, a definite concentration of standard drug wasadded and recovery was studied. The results of recovery studies are shown in Table 3.

Table 1 Results of system suitability parameters, Table 2 Linearity and calibration curve, Table 3 Results of recovery studies. Precision The precision of the procedure was determined by repeatability (intraday). Intraday precision was evaluated by assaying same concentration and during the same day. Repeatability of sample measurement was carried out in six different sample preparations from same homogenous blend of sample. Another replicate determination on three different days to estimate interday precision. Limit of Detection and Limit of Quantification For HPLC method, the limit of detection (LOD) and limit of quantification (LOQ) were calculated based on the standard deviation of the response and the slope by using calibration curves. Robustness For the HPLC method, robustness was determined by analysis of the samples under a variety of conditions making small changes in the percentage of mobile phase compounds (phosphate buffer: methanol in the ratios 68:32 and 42:58), in the flow rate (0.6 and 1.2 ml/min), in the temperature conditions (35 and 45°C), and changing the wavelength (268 and 276 nm). Forced Degradation Study (21-26) Forced degradation for all three drugs was carried out under conditions of acid/base/neutral hydrolysis, oxidation, and dry heat. For each study, six samples were prepared and injected then study was extended up to the formulation. Acidic Degradation Acidic Degradation of DIC Accurately weighed (10mg) of DIC was, transferred into 100ml volumetric flask and dissolved in 50ml methanol. 50ml of 0.1N HCl was added and refluxed at 80°C for 6 hours. 0.5ml of the solution was diluted upto 10ml with methanol (5μg/ml) and analysed by HPLC. Acidic Degradation of CHL Accurately weighed CHL (10mg) was transferred into 100ml volumetric flask and dissolved in 50ml methanol. 50ml of 5N HCl was added and refluxed at 85°C for 24 hours. 2.5 ml of the solution was diluted upto 10ml with methanol (25μg/ml) and analysed by HPLC.

Acidic Degradation of PCM Accurately weighed PCM (10mg) was transferred into 100ml volumetric flask and dissolved in 5ml methanol. 50ml of 5N HCl was added and refluxed at 85°C for 24 hours. 3.25ml of the solution was diluted upto 10ml with methanol (32.5μg/ml) was analysed by HPLC. Alkaline Degradation Alkaline Degradation of DIC Accurately weighed DIC (10mg) was transferred into 100ml volumetric flask and dissolved in 50ml methanol. 50ml of 0.1N NaOH was added and refluxed at 80°C for 6 hours. 0.5ml of the solution was diluted upto 10ml with methanol (5μg/ml) and analysed by HPLC. Alkaline Degradation of CHL Accurately weighed CHL (10mg) was transferred into 100ml volumetric flask and dissolved in 50ml methanol. 50ml of 0.1 N NaOH was added and refluxed at 85°C for 2 hours. 2.5ml of the solution was diluted upto 10ml with methanol (25μg/ml) and analysed by HPLC. Alkaline Degradation of PCM Accurately weighed PCM (10mg) was transferred into 100ml volumetric flask and dissolved in 50ml methanol. 50 ml of 5N NaOH was added and refluxed at 85°C for 24 hours. 3.25ml of the solution was diluted upto 10ml with methanol. The solution (32.5μg/ml) was analysed by HPLC. Oxidative Degradation Oxidative Degradation of DIC Accurately weighed DIC (10mg) was transferred into 100ml volumetric flask and dissolved in 50ml methanol. 50ml of 3% H2O2 was added and kept at room temperature for 6 hours. 0.5ml of the solution was diluted upto 10ml with methanol (5μg/ml) and analysed by HPLC. Oxidative Degradation of CHL Accurately weighed CHL (10mg) was transferred into 100ml volumetric flask and dissolved in 50ml methanol. 50ml of 30% H2O2 was added and kept at room temperature for 4 hours. 2.5ml of the solution was diluted upto 10ml with methanol (25μg/ml) and analysed by HPLC. Oxidative Degradation of PCM Accurately weighed PCM (10mg) was, transferred into 10ml volumetric flask and dissolved in 5ml methanol. 3.25 ml of 30% H2O2 was added and kept at room temperature for 24 hours. The solution (32.5μg/ml) was analysed by HPLC. Thermal Degradation Thermal Degradation of DIC Accurately weighed DIC (10mg) was kept at 105°C for 8 hours and transferred in 100ml volumetric flask. Dissolved in 50ml of methanol then dilute upto mark with methanol. 0.5 ml of the solution was diluted upto 10ml with methanol (5μg/ml) and analysed by HPLC.

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Thermal Degradation of CHL Accurately weighed CHL (10mg) was kept at 105°C for 8 hours and transferred in 100ml volumetric flask. Dissolved in 50ml of methanol then dilute upto mark with methanol. 2.5 ml of the solution was diluted upto 10ml with methanol (25μg/ml) and analysed by HPLC. Thermal Degradation of PCM Accurately weighed PCM (10mg) was kept at 105°C for 8 hours and transferred in 100ml volumetric flask. Dissolved in 5ml of methanol then dilute upto mark with methanol (32.5μg/ml) and analysed by HPLC. Neutral Degradation Neutral Degradation of DIC Accurately weighed DIC (10mg) was transferred into 100ml volumetric flask and dissolved in 50ml methanol. 50ml of water was added and refluxed at 80°C for 6 hours. 0.5ml of the solution was diluted upto 10ml with methanol (5μg/ml) and analysed by HPLC. Neutral Degradation of CHL Accurately weighed CHL (10mg) was transferred into 100ml volumetric flask and dissolved in 50ml methanol. 50ml of water was added and refluxed at 85°C for 24 hours. 2.5ml of the solution was diluted upto 10ml with methanol (25μg/ml) and analysed by HPLC. Neutral Degradation of PCM Accurately weighed PCM (10mg) was transferred into 100ml volumetric flask and dissolved in 50ml methanol. 50ml of water was added and reflux at 85°C for 24 hours. The solution (32.5μg/ml) was analysed by HPLC. Forced Degradation Study of Marketed Product Acidic Degradation of Marketed Product Twenty tablets were weighed accurately and finely powdered. Powder exactly equivalent to 50mg of DIC, 250mg of CHL and 325mg of PCM were transferred to a 100ml volumetric flask and dissolved in 50ml methanol. 50ml of 0.1N HCl was added and refluxed at 80°C for 6 hours. 1ml of the solution was diluted upto 100ml with methanol and analysed by HPLC. Alkaline Degradation of Marketed Product Twenty tablets were weighed accurately and finely powdered. Powder exactly equivalent to 50mg of DIC, 250mg of CHL and 325mg of PCM was transferred to a 100ml volumetric flask and dissolved in 50ml methanol. 50ml of 0.1N NaOH was added and refluxed at 80°C for 6 hours. 1ml of the solution was diluted upto 100ml with methanol and analysed by HPLC. Oxidative Degradation of Marketed Product Twenty tablets were weighed accurately and finely powdered. Powder exactly equivalent to 50mg of DIC, 250mg of CHL and 325mg of PCM was transferred to a 100ml volumetric flask and dissolved in 50ml methanol. 50ml of 3% H2O2 was added and kept at room temperature

for 6 hours. 1ml of the solution was diluted upto 100ml with methanol and analysed by HPLC. Thermal Degradation of Marketed Product Twenty tablets were weighed accurately and finely powdered. Powder exactly equivalent to 50mg of DIC, 250mg of CHL and 325mg of PCM was kept at 105°C for 8 hours and transferred in 100ml volumetric flask. The powder was dissolved in 50ml of methanol and diluted upto mark with methanol. 1ml of the solution was diluted upto 100ml with methanol and analysed by HPLC. Neutral Degradation of Marketed Product Twenty tablets were weighed accurately and finely powdered. Powder exactly equivalent to 50mg of DIC, 250mg of CHL and 325mg of PCM was transferred to a 100ml volumetric flask and dissolved in 50ml methanol. 50ml of water was added and refluxed at 80°C for 6 hours. 1ml of the solution was diluted upto 100ml with methanol and analysed by HPLC. RESULTS AND DISCUSSION The mobile phase consisting of methanol: water (80:20, v/v) at 0.8ml/min flow rate which gave sharp, well-resolved peak with minimum tailing factor for DIC, CHL and PCM. The retention time for all three drugs was tR 3.25min for DIC, tR 5.60 min for CHL and tR 9.40 min for PCM at wavelength 280nm. The calibration curve for DIC, CHL and PCM was found to be linear over the range of 5-25�g/ml for DIC, 25-125μg/ml for CHL and 32.5-162.5 �g/ml for PCM. The linearity was found to be 0.9998, 0.9997 and 0.9997 for DIC, CHL and PCM respectively. The proposed method was successfully applied to the determination of DIC, CHL and PCM in market formulation. The %RSD of intraday and interday precision study for DIC, CHL and PCM were found to be <2 as shown in Table 4. The LOD for DIC, CHL & PCM was found to be 0.15μg/ml, 1.82μg/ml and 2.40μg/ml respectively, while LOQ were 0.47μg/ml, 5.53μg/ml and 7.29μg/ml respectively as shown in Table 5. The summary of all validated parameters is shown in Table 6. The summary of method validation and analysis of marketed formulation is shown in Table 7 and Table 8 respectively.

Table 4 Results of Intraday & Inter day precision, Table 5 Robustness data for DIC, CHL and PCM, Table 6 Limit of detection and limit of quantification, Table 7 Summary of validation parameters, Table 8 Analysis of marketed formulation of the product.

The degradation study indicated that DIC was susceptible to base and H2O2 under experimental conditions. Moreover, CHL was not stable towards acidic, oxidative and neutral degradation conditions. It was noticed that except oxidation PCM found to stable for all the experimental conditions. The study revealed that DIC and PCM showed no degradation in 0.1 N HCl when reflux at 80°c for 10hr condition and chromatogram showed no additional peak.

Figure 7 Forced degradation under acidic condition, Figure 8 Forced degradation under alkaline condition.

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In alkaline hydrolysis DIC and CHL degraded as observed by the decreased area in the peak of the drug when compared with peak area of the same concentration of the non-degraded drug, with giving additional degradation peak. In oxidative degradation all the three drugs were observed to be labile and 9% of degradation for DIC was found. There was no degradation found under thermal and neutral conditions for DIC, CHL and PCM. (Figure 9 Forced degradation under oxidative condition, Figure 10 Forced degradation under thermal condition & Figure 11 Forced degradation under neutral condition). Percent degradation was calculated by comparing the areas of the degraded peaks in each degradation condition with the corresponding areas of the peaks of the drug under non degradation condition. The summary of degradation studies is given in Table 9. CONCLUSION It was possible in this study to develop a stability indicating LC assay method for simultaneous estimation of DIC, CHL and PCM by subjecting the drugs to ICH recommended stress conditions. The drugs and degradation products got well separated from each other in an isocratic mode using a reversed phase. The developed method was found to be accurate, precise sensitive, selective and repeatable for analysis of diclofenac potassium, chlorzoxazone paracetamol in market formulation without any interference from the excipients. The method was successfully used for determination of drugs in a pharmaceutical formulation. It was possible in this study to develop a stability indicating assay method for the drugs by subjecting ICH recommended stress conditions. The drugs and degradation products got well separated from each other in isocratic mode using a reversed phase C18 column and mobile phase composed of methanol: phosphate buffer (70:30, v/v) at 0.8ml/min flow rate and the eluent was monitored at 280nm.The results indicated suitability of this method to study stability of three drugs under various forced degradation conditions like acid, base, dry heat and oxidative degradation. There was no interference observed due to excipients or other components present in tablet dosage form. The developed method is stability indicating and separate degradants and can be used to determine the stability of samples. REFERENCES AND NOTES 1. Wan J, Ernstgard L, Song BJ, Shoaf SE. Chlorzoxazone

metabolism is increased in fasted Sprague-Dawley rats. J Pharm Pharmacology, 58(1): 51-61, 2006.

2. Park JY, Kim KA, Park PW, Ha JM. Effect of high-dose aspirin on CYP2E1 activity in healthy subjects measured using Chlorzoxazone as a probe. J Clinical Pharmacology, 46(1):109-114, 2006.

3. Sweetman SC. Martindale: The Complete Drug Reference, 37th ed. London: Pharmaceutical Press; 32-33, 2011.

4. Solomon DH, Avorn J, Sturmer T, Glynn RJ, Mogun H, Schneeweiss S. Cardiovascular outcomes in new users of coxibs and nonsteroidal antiinflammatory drugs: high-risk subgroups and time course of risk. Arthritis Rheum., 54(5):1378-1389, 2006.

5. FitzGerald GA, Patrono C. The coxibs selective inhibitors of cyclooxygenase-2. N Engl J Med., 345(6):433-442, 2000.

6. Gan TJ. Diclofenac: an update on its mechanism of action and safety profile. Curr Med Res Opin., 26(7): 1715-1731, 2010.

7. O’Neil MJ. The Merck Index: An Encyclopedia of chemicals, drugs and biological, 14th ed., Merck Research Laboratories, 1033, 2006.

8. Weng N. HPLC and CE Methods for Pharmaceutical Analysis. Journal of Chromatography B Biomedical Analysis, 654:287-292, 1994.

9. Patel MG, Parmar RR, Nayak PP, Shah SA. Simultaneous estimation of Paracetamol and Tolperisone hydrochloride in tablet by UV spectrophotometric methods. Journal of Pharmaceutical Science and Bioscientific Research, 2(2):63-67, 2012.

10. Girolamo O, Neill M, Wainer IW. A Validated Method for the Determination of Paracetamol and its Glucuronide and Sulphate Metabolites in the Urine of HIV/AIDS Patients Using Wavelength-Switching UV Detection. Journal of Pharmaceutical and Biomedical Analysis, 17:1191-1197, 1998.

11. Jadi MK, Narayan UL. UV spectrophotometric and RP-HPLC method development for simultaneous determination of paracetaomol and etodolac in pharmaceutical dosage form, 56(1): 169-174, 2009.

12. Yadav AH, Kothapalli LP, Barhate AN, Pawar I, Pawar CR. RP-HPLC method for determination of aceclofenac, chlorzoxazone and paracetamol in bulk and pharmaceutical formulation. International Journal of Pharma Research and Development, 1(10):1-7, 2009.

13. Biswas A, Basu A. Simultaneous estimation of paracetamol, chlorzoxazone and diclofenac potassium in pharmaceutical formulation by RP-HPLC method. International Journal of Pharma and Bio Sciences, 1(2):1-5, 2010.

14. Dhaneshwar SR, Bhusari VK. Validated HPLC method for simultaneous quantitation of diclofenac sodium and misoprostol in bulk drug and formulation, Pelagia Research Library Der Chemica Sinica, 1(2):110-18, 2010.

15. Panda SS, Patnaik d, Ravikumar B, New Stability-Indicating RP-HPLC method for determination of diclofenac potassium and metaxalone from their combined dosage form, Scientia Pharmaceutica, Under Press, 2012.

16. Shinde VM, Desai BS, Tendolkar NM. Simultaneous determination of paracetamol, diclofenac sodium and chlorzoxazone by HPLC from tablet. Indian Journal of Pharmaceutical Sciences, 57(1):35-37, 1995.

17. Rao JR, Mulla TS, Bharekar VV, Yadav SS, Rajput MP. Simultaneous HPTLC determination of paracetamol and dexketoprofen trometamol in pharmaceutical dosage form. Der Pharma Chemica, 3(3): 32-38, 2011.

18. Rajput MP, Bharekar VV, Yadav SS, Mulla TS, Rao JR. Validated HPTLC method for simultaneous estimation of diclofenac potassium and metaxalone in bulk drug and formulation, Pharmacie Globale International Journal of Comprehensive Pharmacy, 12(4):1-4, 2011.

19. Potawale SE, Nanda RK, Bhagwat VV. Hamane SC, Deshmukh S, Puttamsetti S, Development and validation of a HPTLC method for simultaneous densitometric analysis of Diclofenac potassium and Dicyclomine hydrochloride as the bulk drugs and in the tablet dosage form, Journal of Pharmacy Research, 4(9):3116-18, 2011.

20. Yuvaraj G, Pavan Kumar P, Elangovan N, The Simultaneous Estimation of Aceclofenac, Paracetamol and Chlorzozazone in Pharmaceutical Dosage Forms by HPTLC, International Journal of Chemical and Pharmaceutical Sciences, 1(2):24-27, 2010.

21. ICH, Q2B Validation of Analytical Procedure: Methodology, in: Proceeding of the International Conference on Harmonization, Geneva, March 1996.

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22. ICH, Guidance on Analytical Method Validation, in: Proceedings of International Convention on Quality for the Pharmaceutical Industry, Toronto, Canada, September 2002.

23. Bakshi M, Singh S, Development of validated stability-indicating assay methods-critical review, Journal of Pharmaceutical and Biomedical Analysis, 28:1010-1040, 2002.

24. Rhodes CT, Introductory overview, Drug Stability: Principle and practices, Marcel Dekker, New York, 1-12, 2000.

25. Matthews BR, Regulatory aspects of Stability Testing in Europe in: Drug Stability: Principles and Practices, Carstensen JT and Rhodes C T, 3rd ed., Marcel Dekker, New York, 107: 580, 2000.

26. Hong DD, Shah M, Development and validation of HPLC stability-indicating assays.in: Drug Stability: Principles and Practices, J.T. Carstensen and C.T. Rhodes, 3rd ed., Marcel Dekker, New York, 107:329-384, 2000.

Acknowledgments The authors are greaful to Zydus Cadila Ltd for providing gift samples of diclofenac potassium, chlorzoxazone and paracetamol respectively.

Cite this article as: Maulikkumar R Amin, Paresh U Patel, B N Suhagia, Madhabhai M Patel. Development and Validation of Stability Indicating Method for Determination of Chlorzoxazone, Diclofenac Potassium and Paracetamol in Pharmaceutical Dosage form using High Performance Liquid Chromatography. Inventi Rapid: Pharm Analysis & Quality Assurance, 2012(4): 1-9, 2012.

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Phd 050 2013 Maulik Amin