PHD 050 2013 Maulik Amin

STABILITY INDICATING SIMULTANEOUS
DETERMINATION OF NEW
MUSCLE RELAXANT DRUGS BY
CHROMATOGRAPHIC METHODS
A thesis submitted in partial fulfillment of the

requirements
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
Certificate
This is to certify that, the thesis entitled Stability indicating
simultaneous determination of new muscle relaxant drugs by
chromatographic methods Submitted by Amin Maulikkumar
Rameshchandra of Kalol Institute of Pharmacy, Kalol is the bonafide
work completed under my supervision and guidance for the award of
Degree of Doctor of Philosophy in the Faculty of Pharmacy, Ganpat
Vidhyanagar. The experimental work included in the thesis was carried
out at Department of Pharmaceutical chemistry, Kalol Institute of
Pharmacy, Kalol and Shree S. K. Patel College of Pharmaceutical
Education and Research, under my supervision and the work is up to
my satisfaction.
Research Guide:
Dr. P. U. PATEL
M. 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. PATEL
M. Pharm., Ph.D.
I/C Principal,
Ganpat University, Ganpat Vidhyanagar-384012
Date:
Place: Ganpat Vidhyanagar
THESIS APPROVAL SHEET

The 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:
Dr. P. U. PATEL
M. Pharm., Ph.D.
Date:
Certificate
This is to certify that the suggestion given by doctoral
committee during pre submission seminar held on 5 th 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:
Dr. P. U. PATEL
M. Pharm., Ph.D.
Professor & Head,

Department of Pharmaceutical Quality Assurance,
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.
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loan such copies at the Universitys 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
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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
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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
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Property Right Policy to facilitate protection of the intellectual property contained in
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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. PATEL
M.Pharm.
Signature of Research student

Date:
M.Pharm., Ph.D.
Signature of Guide
ACKNOWLEDGEMENT
Thesis 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 CHAPTER
Content
Chapter
1
Introduction
Page
No.
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
Review of literature
47-75
Aim of the present work
Chemicals, glasswares and instruments
76
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
Stability indicating method development and validation for
81-128
simultaneous estimation of Diclofenac potassium,

Chlorzoxazone and Paracetamol
5.1
Stability indicating HPLC method development and validation
81-103
for simultaneous estimation of Diclofenac potassium,

Chlorzoxazone and Paracetamol
5.2
Stability indicating HPTLC method for simultaneous estimation
104-126
of diclofenac potassium, Chlorzoxazone and Paracetamol

5.3
Statistical comparison between official and proposed methods
127
5.4
References
128
Stability indicating method development and
validation for
129-173
simultaneous estimation of Diclofenac potassium,

Paracetamol and Methocarbamol
6.1
Stability indicating HPLC method development for simultaneous
129-150
estimation of Diclofenac potassium, Paracetamol and

Methocarbamol
6.2
Stability indicating HPLC method for simultaneous estimation
151-171
of Diclofenac potassium, Paracetamol and Methocarbamol

6.3
Statistical comparison between official and proposed methods
172
6.4
References
173
Summary
174-175
Publications
176
List of Tables
Table
No.
1.3.1
Caption
Page
No.
22
Controlling sample retention by changing solvent strength
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
115
HPTLC
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
127
methods
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
172
methods
List of Figures
Figure No.
Caption
Page
No.
3
1.1.1
Site of action for muscle relaxants
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
39
under acid and alkali conditions

1.6.3
Flow chart for performing stress studies for hydrolytic degradation
40
under neutral condition (in water)

1.6.4
41
5.1.1
Flow chart for performing stress studies for degradation under

oxidative
Conditions
Overlay UV spectra of DIC, CHL and PCM
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
83
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
99
HPLC
5.1.12
Chromatogram of alkaline degradation of marketed product by
100
HPLC
5.1.13
Chromatogram of oxidative degradation of marketed product by
100
HPLC
5.1.14
Chromatogram of thermal degradation of marketed product by
100
HPLC
5.1.15
Chromatogram of neutral degradation of marketed product by
101
HPLC
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
5.2.11
122
HPTLC
5.2.12
123
HPTLC
5.2.13
123
HPTLC
5.2.14
123
HPTLC
5.2.15
124
HPTLC
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
147
6.2.1

HPLC
HPLC
HPLC
HPLC
HPLC
Overlay UV spectra of DIC, PCM and MET
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
6.1.12
6.1.13
6.1.14
6.1.15
147
147
148
148
152
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
6.2.11
167
HPTLC
6.2.12
168
HPTLC
6.2.13
168
HPTLC
6.2.14
169
HPTLC
6.2.15

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
l- 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
- Micro
mg- Milligram
- Lambda
- Beta
- Alpha
&- And
%- Percentage
ng- Nano gram
M- Molar
R2- Coefficient of variance
g/mol- Gram per mole
Rs- Resolution
L- 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
- Standard deviation
DL- Detection limit
QL- Quantification limit
Chapter 1
Introduction
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
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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 gammaamminobutyric 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 nonsteroidal 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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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 Profiles

1.2.1 Chlorzoxazone[5-7]
Systematic (IUPAC) name: 5-Chlorobenzoxazol-2-ol
Synonyms: Chlorobenzoxazolinone
Structure:
O
OH
Cl
Molecular Formula: C7H4 Cl NO2

Mol. mass: 169.565 g/mole
Description: Colourless or white crystalline powder
Melting point: 190 to 194C
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-Acetylpaminophenol
Structure:
HO
O
N
H
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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 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:
Cl
O K+
NH
O
Cl
Molecular Formula: C14H10Cl2KNO2

Mol. mass: 296.2 gm/mole
Description: Yellowish to white crystals or crystalline powder
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 COX2) 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
H2N
O
O
Molecular Formula: C11H15NO5

Mol. mass: 241.2gm/mole
Description: Yellowish to white crystalline powder
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. Chromatography
It 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 1970s, 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 m to 200 m. Fast
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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
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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
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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].
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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,
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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 todays 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.
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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
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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 normalphase 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.
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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 twintrough 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 L can be applied as spots
onto conventional layers without intermediate drying. HPTLC layers take up to 1 L
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
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Chromatography
samples
are
sprayed
onto
the
18
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Introduction
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 L) or as bands or rectangles (0.5 to
>50L) 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
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Introduction
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 chromatographers 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 W 1 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
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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.
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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 Strength
HPLC 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 phase
1.3.3.4.1.1 Solvent -type selectivity
A 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
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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
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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 .
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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 method

A 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
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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.
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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).
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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
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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)
minimum
of
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.
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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 / S
Where = 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 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
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of impurities and/or degradation products. Several approaches for determining the

quantitation limit are possible, depending on whether the procedure is a noninstrumental 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 /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.
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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
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Chapter 1
Introduction
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.
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Chapter 1
Introduction
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 method

1.6.1 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
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Chapter 1
Introduction
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) 10C
increments above the accelerated temperatures (e.g. 50C, 60C, 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 40C and 110C. The reaction time varied, for example, drugs being
kept at 100C 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
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Chapter 1
Introduction
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 30C[59]. At neutral pH, no significant degradation was obtained
when the temperature was 37C 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.
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Chapter 1
Introduction
Table1.6.1 Various degradation conditions described for the stress testing

Decompositi
Recently used
Temperatu
on condition
most
re
Reaction time
Extent of
degradation
frequently
Acidic
Neutral
HCl
(0.1N/1N/5N)
H2SO4
NaOH
(0.1N/1N5N)
Water
Oxidation
H2O2(1%-3%)
Alkaline
Photolytic
UV/Fluorescent
light (4300 to
17000 lux)
RT= room temperature
40C-110C
Few min-2
months
Negligiblecomplete
80C/Reflux
5 min-21days
85C/Reflux
Very long
periods
Few hoursweek
Few hoursmonths
InsignificantExtensive
No significant
degradation
InsignificantExtensive
Nil-Complete
RTRefluxing
RT
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.
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Chapter 1
Introduction
START
1.2 x106 Lux hr

No Degradation
6.0 x106 Lux hr
Total
Degradation
Sufficient
Degradation
Reduce exposure
No Degradation
Sufficient
Degradation
Declare drug to be
practically stable
ACCEPT
Figure 1.6.1 Flow chart for performing stress studies for photolytic degradation
Ganpat University
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Chapter 1
Introduction
START
No Degradation
0.1N HCl/ NaOH,

8 h, Reflux
Total Degradation
Sufficient Degradation
1 N HCl /NaOH,
12 h, Reflux
0.01HCl/ NaOH,
8 h, at 40c
No Degradation
2 N HCl/ NaOH,
24 h, Reflux
Sufficient
Degradation
Total Degradation
ACCEPT
No Degradation
5 N HC l/ NaOH,
24 h, Reflux
Sufficient
Degradation
1N HCl/ NaOH,
2 h at, 25C
Total Degradation
Carry out studies

under milder
conditions
No Degradation
Declare drug to
be practically
stable
Figure 1.6.2: Flow chart for performing stress studies for hydrolytic degradation
under acid and alkali conditions
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Chapter 1
Introduction
START
No Degradation
Water/ 12 h,
Reflux
Total Degradation
Water/ 1 day,
Reflux
No Degradation
Water/ 2 day,
Reflux
Sufficient
D degradation
Water/ 8 hr, at
40C
Total Degradation
ACCEPT
No Degradation
Water/ 5 day
Reflux
Sufficient
Degradation
Water/ 2 hr, at
25C
Total Degradation
Carry out studies

under milder
conditions
No Degradation
Declare drug to
be practically
stable
Figure 1.6.3: Flow chart for performing stress studies for hydrolytic degradation
under neutral condition (in water)
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Chapter 1
Introduction
START
No Degradation
3% H2O2/ 24 h,
RT
3% H2O2/ 6 h,
RT
Sufficient
Degradation
Sufficient
Degradation
No Degradation
10% H2O2/ 24
h, RT
Sufficient
Degradation
1% H2O2/ 3 h,
RT
Total Degradation
ACCEPT
No degradation
30% H2O2/ 24 h,
RT
Total Degradation
Sufficient
Degradation
1% H2O2/ 30 min,
RT
Total Degradation
Carry out studies

under milder
conditions
Declare drug to
be practically
stable
Figure 1.6.4: Flow chart for performing stress studies for degradation under oxidative
conditions.
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Chapter 1
Introduction
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 Dales 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): 10291039.
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. ONeil 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
Ganpat University
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Chapter 1
Introduction
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
th
pharmacotheraputics, 6 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 cyclooxygenase2 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
th
pharmacotheraputics, 6 Ed., Popular prakashan, 1999; 164-166.

20. Goodman & Gilmans 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. ONeil 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.
Ganpat University
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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: 1107.
36. Snyder LR, Kirkland JJ, Glajch LJ, Getting Started, Practical HPLC Method
Development, 2nd Ed., John Willey and Sons, Inc., Newyork, 1997; 5-1: 351360.
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.
Ganpat University
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Introduction
39. Remington, Chromatography; The Science and Practice of Pharmacy Printed

by the Make Printing Company Eston, pennsylvania, 19th Ed. 1995; 1: 537544.
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.
Ganpat University
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Chapter 1
Introduction
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.
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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
Paracetamol
Method
Description
phase:-
Refere
nce
1
steel
Liquid
Stationary
chromatography
column, 25cm x 4mm long packed with
stainless
octylsilane silica gel

Mobile phase:- Disodium hydrogen
phosphate: methanol (75:25 v/v)
Detection:- 243 nm
Paracetamol
Liquid
Flow rate:- 1.0 mL/min

Stationary phase:- stainless
syrup
chromatography
column, 20cm x 4.6mm long packed with
steel
octadecylsilyl silica gel

Mobile
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phase:-
0.01
Soidumbutane
47
Chapter 2
sulphonate: methanol (85:15 v/v)

Detection:- 243 nm
Paracetamol
Liquid
Stationary
tablets
chromatography
column, 20cm X 4.6mm long packed
method
with octadecylsilane silica gel

Mobile
phase:-
phase:-
butanesuphonate
stainless
0.01
in
85ml
steel
Sodium
water:
methanol and 0.4 volumes of formic acid

Detection:- 243 nm
Paracetamol
Titration

Titrant:- 0.1 M Cerium sulphate
Paracetamol
HPLC
Stationary phase:- Nitrile gp bonded
capsule
2
3
with porus silica (3-10m) 3.9mm x

30cm
Mobile phase:- water: methanol (90:10
v/v)
Detection:- 243 nm
Paracetamol
HPLC
oral solution


30cm
v/v)
Detection:- 243 nm
Paracetamol
UV spectroscopy

Wavelength: 243nm
Paracetamol
HPLC
effervescent
oral solution
30cm
v/v)
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Chapter 2
Detection:- 243 nm
Paracetamol
HPLC
suppository

30cm
v/v)
Detection:- 243 nm
Paracetamol
HPLC
oral
suspension
30cm

v/v)
Detection:- 243 nm
Paracetamol
HPLC
tablet

30cm
v/v)
Detection:- 243 nm
Paracetamol
HPLC
extended
release
30cm

v/v)
Detection:- 243 nm
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Chapter 2
Table 2.1.2 Official methods for Paracetamol in combination

Drug
Method
Paracetamol
HPLC
and Aspirin
Description
Reference

30cm
Mobile
phase:-
Chloroform:
methanol: glacial acetic acid (70:20:1

v/v/v)
Detection:- 280 nm
Paracetamol,
Aspirin
HPLC
and
Caffeine
10cm
Mobile
phase:-
Chloroform:

v/v/v)
Detection:- 275 nm
Paracetamol
HPLC
and Caffeine

10cm
Mobile
phase:-
Chloroform:

v/v/v)
Detection:- 275 nm
Table 2.1.3 Official methods for Diclofenac potassium in combination
Drug
Diclofenac
potassium
Method
Liquid
chromatography
Description
Stationary phase:-
Reference
4
Mobile phase:- Methanol: phosphate

buffer (pH 2.5) (70:30 v/v)
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Chapter 2
Detection:- 254 nm
Diclofenac
potassium
tablets

Stationary phase:-
HPLC

buffer (pH 2.5) (70:30 v/v)
Detection:- 254nm
Diclofenac
potassium
delayed
release
Liquid
chromatography

Stationary phase:-

buffer (pH 2.5) (70:30 v/v)
Detection:- 254 nm
Diclofenac
tablet
Stationary phase:- Plate coated with
TLC
silica gel GF254

Mobile phase:- Toluene: hexane:
formic acid (90:5:5 v/v/v)
Detection:- 254 nm
Diclofenac
sodium
injection
Stationary phase:- Plate coated with
TLC
silica gel GF254 plate

Mobile
phase:-
Chloroform:
acetone: formic acid (90:5:5 v/v/v)

Detection:- 254 nm
Diclofenac
potassium
Potentiometric
Solvent:- Anhyrous acetic acid
Titrant:- 0.1M perchloric acid
Table 2.1.4 Official methods for Chlorzoxazone in combination

Drug
Method
Chlorzoxazone Liquid
tablets
chromatography
Description
Stationary phase:-
Reference
7
Mobile phase:- Water: acetonitrile:

glacial acetic acid (70:30:1 v/v/v)
Detection:- 280 nm
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Chapter 2
Table 2.1.5 Official methods for Methocarbamol

Drug
Method
Description
Reference
Methocarbamol UV
Detection:- 274 nm
Methocarbamol UV
Detection:- 274 nm
Methocarbamol Liquid
Stationary phase:-
tablet
Mobile
injections
chromatography
phase:-
buffer(pH 4.5): methanol
Phospate
(75:25
v/v)
Detection:- 280 nm
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2.2 REPORTED METHODS FOR DRUG UNDER STUDY

Table 2.2.1 Reported methods for Paracetamol
Drug
Paracetamol
Title:-
Method and description
Reference
9
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
Paracetamol
10
Title:Reverse phase HPLC method for determination of

aceclofenac and paracetamol in tablet dosage forms
Description
Stationary phase:- ODS C18
Detection:- 255 nm
Paracetamol
11
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
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Chapter 2
Paracetamol
12
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
Paracetamol
13
Title:Simultaneous
determination
of
paracetamol,
pseudoephedrine, dextrophan and chlorpheniramine in

human plasma by liquid chromatographytandem mass
spectrometry
Description:Stationary phase:- C18 column ODS
Mobile phase:- acetic acid: methanol (20:80 v/v)
Detection:- 275 nm
Paracetamol
Title:-
14
HPLC Method for the Analysis of Paracetamol,

Caffeine and Dipyrone
Description:Stationary phase:- C8 column ( Bondapack)
Mobile phase:- methanol: acetonitrile: isopropyl alcohol
(40:30:30 v/v)
Detection:- 215 nm
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Chapter 2
Paracetamol
15
Title:
Spectrophotometric
Methods
for
Simultaneous
Estimation of Dexibuprofen and Paracetamol

Description:
Solvent:- methanol
Wavelength:- 249.5 nm
Paracetamol
16
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
Paracetamol
17
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
Paracetamol
18
Title:
Sensitive
liquid
chromatographytandem
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
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Chapter 2
Paracetamol
Title:Simultaneous
Determination
of
Paracetamol
and
19
Caffeine in Human Plasma by LCESIMS

Description:
Stationary phase:- C18
Mobile phase:- formic acid: methanol (60:40 v/v)
Detection:- 254 nm
Paracetamol
20
Title:
UV
spectrophotometric
development
for
and
simultaneous
RP-HPLC
method
determination
of
paracetaomol and etodolac in pharmaceutical dosage

form
Description:
Solvent:- methanol
Wavelength:- 226 nm
Paracetamol
Title:
21
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 g/spot
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Chapter 2
Paracetamol
Title:-
22
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)
Paracetamol
Title:-
23
Liquid chromatographic determination of cetrizine

hydrochloride and paracetamol in human plasma and
pharmaceutical formulations
Description:
Detection:- 230 nm
Paracetamol
Title:-
24
Determination of paracetamol and orphenadrine citrate in

dosage formulations and in human plasma
Description:
Detection:- 215 nm
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Chapter 2
Paracetamol
25
Title:A
validated
paracetamol
method
and
its
for
the
determination
glucuronide
and
of
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
Paracetamol
26
Title:Simultaneous
Tolperisone
estimation
of
hydrochloride
in
Paracetamol
tablet
by
and
UV
spectrophotometric methods
Description:
Solvent:- metanol
Wavelength:- 254 nm
Paracetamol
27
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 g/spot
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Chapter 2
Paracetamol
28
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
Paracetamol
Title:Simultaneous
determination
of
aceclofenac,
29
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
Paracetamol
Title:-
30
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
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Chapter 2
Table 2.2.2 Reported methods of Diclofenac potassium

Diclofenac
Title:-
31
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
Diclofenac
Title:-
32
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
Diclofenac
Title:-
33
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
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Chapter 2
Diclofenac
34
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
Diclofenac
Title:-
35
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
Diclofenac
Title:-
36
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
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Chapter 2
Diclofenac
Title:-
37
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 g/mL
Diclofenac
Title:-
38
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
Diclofenac
Title:-
39
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
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Chapter 2
Diclofenac
40
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
Diclofenac
41
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
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Chapter 2
Table 2.2.3 Reported methods for Chlorzoxazone

Chlorzoxazone
42
Title:Simultaneous estimation of paracetamol, chlorzoxazone and

diclofenac potassium in pharmaceutical formulation by RPHPLC method
Description
Mobile
phase:-
methanol-0.01M
monobasic
sodium
phosphate (70:30 v/v)

Detection:- 254 nm
Chlorzoxazone
43
Title: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
Chlorzoxazone
44
Title: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
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Chapter 2
Chlorzoxazone
45
Title:Simultaneous estimation of tramadol hydrochloride and

chlorzoxazone by absorbance correction method
Description
Solvent:- methanol
Wavelength:- 225 nm
Chlorzoxazone
46
Title: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
Chlorzoxazone
47
Title: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
Chlorzoxazone
48
Title:Simultaneous determination of paracetamol and chlorzoxazone

using orthogonal functions ratio spectrophotometry
Description
Solvent:- methanol
Wavelength:- 287 nm
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Chapter 2
Table 2.2.4 Reported methods for Methocarbamol

Methocarbamol
49
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
Methocarbamol
50
Title: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
Methocarbamol
Title:-
51
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
Methocarbamol
Title:-
52
Determination of methocarbamol concentration in human

plasma by high performance liquid chromatography
tandem mass spectrometry
Description
Range:- 150-12000 ng/mL
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Chapter 2
Methocarbamol
53
Title:A
stability-indicating
chromatographic
method
high-performance
for
the
liquid
determination
of
methocarbamol in veterinary preparations

Description
Mobile phase:- methanol: water: tetrahydrofuran (25:65:10
v/v/v)
Detection:- 274 nm
Methocarbamol
Title:-
54
Stability indicating HPLC method for simultaneous

determination of methocarbamol and nimesulide from tablet
matrix
Description
Stationary phase:- supercosil LC-8
Detection:- 276 nm
Methocarbamol
Title:-
55
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
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Chapter 2
Methocarbamo
l
Title:-
56
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
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Chapter 2
References
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Chapter 2
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Chapter 2
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Chapter 2
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Chapter 2
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analysis
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Diclofenac
potassium
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estimation of tramadol hydrochloride and chlorzoxazone by absorbance
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Chapter 2
46. Zerillia A, Lucas D, Berthou F, Determination of Cytochrome P450 Activity

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Simultaneous spectrophotometric estimation of ibuprofen and methocarbamol
in tablet dosage form, Indian Journal of Pharmaceutical Sciences, 2004; 16:
810-813.
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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
chromatographytandem
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): 16021605.
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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.
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Aim of the present work
3. AIM OF THE PRESENT WORK

There 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.
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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)
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4.2 Active Pharmaceutical Ingredients (API)

Table 4.2.1: List of Active Pharmaceutical Ingredient
Sr.
Name
Batch No
A.R.No.
Supplier
DFK/09/0018
Zydus Cadila
No.
1
Diclofenac
DFK/19040018
potassium
2
Chlorzoxazone
Ltd
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 (100C)
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 4.6 mm i.d, 5 m particle size)
Injector: Microliter syringe (Rheodyne)
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Detector: Dual wavelength UV Detector

Software: Autochro-3000
2. HPTLC
System: CAMAG
Sample applicator: Linomat-V
Injector: Hamilton syringe (100l)
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.) (0.45m and 0.2m)
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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, A8.
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.
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Stability indicating methods for muscle relaxant drugs
5. STABILITY INDICATING METHOD DEVELOPMENT AND

VALIDATION
DICLOFENAC
FOR
SIMULTANEOUS
POTASSIUM,
ESTIMATION
CHLORZOXAZONE
OF
AND
PARACETAMOL
5.1 STABILITY INDICATING HPLC METHOD DEVELOPMENT AND
VALIDATION FOR SIMULTANEOUS ESTIMATION OF DICLOFENAC
POTASSIUM, CHLORZOXAZONE AND PARACETAMOL
5.1.1 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 0.45 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 500g/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
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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 (500g/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 5-30g/ml solutions.
5.1.1.3.2.2 Preparation of standard working solution of CHL
From stock solution (2500g/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 25-125g/ml solutions.
5.1.1.3.2.3 Preparation of standard working solution of PCM
From stock solution (3250g/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 g/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)
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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 4.6 mm i.d,
5 m 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 l 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 5-30g/ml for DIC, 25125g/ml for CHL and 32.5-182.5g/ml for PCM. All the solution were filtered through
0.2m 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.
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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 / S
Where = the standard deviation of the response, S = the slope of the calibration curve
The LOQ may be expressed as: QL =10 / 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 85C for 24
hours. 0.5ml of the solution was diluted upto 10ml with methanol (5g/ml) and analysed
by HPLC.
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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 85C for 24
hours. 2.5 ml of the solution was diluted upto 10ml with methanol (25g/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 (32.5g/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 80C 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 80C 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 80C for 6
hours. 3.25ml of the solution was diluted upto 10ml with methanol. The solution
(32.5g/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
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for 6 hours. 0.5ml of the solution was diluted upto 10ml with methanol (5g/ml) and
analysed by HPLC.
5.1.1.9.3.2 Oxidative degradation of CHL
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 (32.5g/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 105C 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 (5g/ml) and analysed by HPLC.
5.1.1.9.4.2 Thermal degradation of CHL
Accurately weighed CHL (10mg) was kept at 105C 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 (25g/ml) and analysed by
HPLC.
5.1.1.9.4.3 Thermal degradation of PCM
Accurately weighed PCM (10mg) was kept at 105C for 8 hours and transferred in 10ml
volumetric flask. Dissolved in 5ml of methanol then dilute upto mark with methanol
(32.5g/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 85C for 24 hours.
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0.5ml of the solution was diluted upto 10ml with methanol (5g/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 (25g/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 85C for 24 hours.
The solution (32.5g/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 85C 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 80C for 6 hours. 1ml of the solution was diluted upto 100ml with methanol
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.
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5.1.1.10.4 Thermal degradation of marketed product

to 50mg of DIC, 250mg of CHL and 325mg of PCM was kept at 105C 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
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 85C 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 (t R 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.
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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
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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, 25125g/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.
160000
P 140000
e 120000
a
100000
k
y = 5018.1x + 362
R = 0.9998
80000
a 60000
r 40000
e 20000
a
0
0
10
15
20
Concentration (g/ml)
25
30
35
Figure 5.1.3: Calibration curve of DIC by HPLC
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700000
P
e
a
k
600000
500000
y = 4987.5x - 20588
R = 0.9997
400000
300000
a
r
e
a
200000
100000
0
0
20
40
60
80
100
120
140
Figure 5.1.4: Calibration curve of CHL by HPLC
P 1000000
e 900000
a 800000
700000
k 600000
a
r
e
a
y = 5051.4x - 14453
R = 0.9997
500000
400000
300000
200000
100000
0
0
50
100
150
200
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 range
DIC
5-30 g/ml
CHL
25-125 g/ml
PCM
32.5-182.5 g/ml
*= Average result of six replicate samples
Y=mx+c
Slope*
Intercept*
5018.1
362
4987.5
2058
5051.4
14445
r2*
0.9998
0.9997
0.9997
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.
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Table 5.1.2 Recovery study of DIC, CHL and PCM by HPLC

Drug
DIC
Conc. Of
Form.
(g/ml)
5
5
5
25
CHL
25
25
32.5
PCM
32.5
32.5
Conc. Of
Std. added
(g/ml)
4
Conc.
Recovered
(g/ml) SD*
8.890.12
5
6
24
25
26
34
32.5
36
9.970.30
10.920.02
48.490.07
48.900.71
50.580.05
65.541.51
65.280.08
69.070.16
% recoverySD*
98.78%0.18
99.70%0.75
99.27%1.89
98.96%0.01
98.16%0.18
99.17%1.09
98.56%0.86
100.41%0.15
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
Intra day
Mean %assay*
%RSD*
DIC
99.34%1.16
0.36
CHL
99.83%1.63
0.12
PCM
98.94%0.01
1.16
Drug
Inter day
Mean %assay*
%RSD*
99.15%1.82
1.08
100.05%0.57
1.12
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.
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Table 5.1.4 Robustness data for DIC, CHL and PCM by HPLC
Chromat
ographic
factor
level
0.7ml/
min
1.0ml/
min
Retention time*
DIC
CHL
6.010.0
1
Flow rate
5.040.0
2.950.02
1
6.020.0
Methanol:
75:25 3.910.03
3
phosphate
buffer
5.050.0
85:15 3.960.02
(v/v)
2
5.600.0
Detection 275nm 3.240.04
1
wave5.600.0
length
285nm 3.260.03
1
3.980.02
Tailing factor*
PCM
DIC
9.960.0
1
8.660.0
3
9.950.0
2
0.950.
02
0.960.
01
0.950.
02
0.960.
01
0.950.
01
0.950.
02
8.61.02
9.410.0
1
8.640.0
2
CHL
1.020.01
1.030.01
1.020.01
1.020.01
1.030.01
1.020.01
PCM
0.970.0
1
0.960.0
1
0.960.0
2
0.970.0
1
0.970.0
3
0.970.0
1

The LOD for DIC, CHL & PCM were found to be 0.15g/ml, 1.82g/ml and 2.40g/ml
respectively, while LOQ were 0.47g/ml, 5.53g/ml and 7.29g/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
No. of
Parameter
RT*
AUC*
theoretical
plates*
DIC
3.250.05
25012.20283.43
4204.49.52
CHL
5.600.06
105410.2312.53 3637.4139.80
PCM
9.400.09 150453.123923.11 1263.127.005
*=Average result of six replicate samples
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factor*
0.95.002
1.020.01
0.970.001
93
Chapter 5
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
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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
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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

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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
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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
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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

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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
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Figure 5.1.15: Chromatogram of neutral degradation of marketed product by

HPLC
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Table 5.1.6 Forced degradation study of marketed product by HPLC

Degradatio
n condition
Acidic
Drug
Conc.
Of
drug(
g/ml)
RT of
observed
peak*
AUC*
%drug*
%degradation
*
DIC
3.25
24018
98.14%1.04
0.00%0.00
5.59
101541
91.98%1.31
0.00%0.00
5.19(I)
8931
0.00%0.00
8.791.31
9.40
3.25
149901
24010
99.65%1.45
94.62%1.24
0.00%0.00
0.00%0.00
2.92(I)
1432
0.00%0.00
5.96%1.21
5.62
5.49(II)
6.12(III)
9.41
3.25
94421
3410
1431
149097
23120
91.56%1.57
0.00%0.00
0.00%0.00
99.57%1.34
91.41%1.32
94.10%1.38
3.61%1.41
1.511.30
0.00%0.00
0.00%0.00
2.72(I)
1981
0.00%0.00
8.98%1.54
5.60
5.12(II)
9.41
102384
2398
148320
97.00%1.03
0.00%0.00
93.84%1.09
0.00%0.00
2.64%0.95
0.00%0.00
10.21(III)
9908
0.00%0.00
6.681.41
CHL
25
PCM
32.5
DIC
Alkaline
CHL
Oxidative
Thermal
25
PCM
32.5
DIC
CHL
25
PCM
32.5
DIC
CHL
PCM
5
25
32.5
3.25
5.61
9.41
25320
105908
149959
99.90%1.12
99.88%1.08
99.84%1.24
0.00%0.00
0.00%0.00
0.00%0.00
DIC
3.25
24906
98.45%1.4
0.00%0.00
103956
9327
104903
91.88%1.23
0.00%0.00
99.84%1.21
0.00%0.00
8.64%0.95
0.00%0.00
Neutral
5.60
4.93(I)
PCM
32.5
9.41
CHL
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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.
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5.2
STABILITY
INDICATING
HPTLC
METHOD
FOR
SIMULTANEOUS ESTIMATION OF DICLOFENAC POTASSIUM,

CHLORZOXAZONE AND PARACETAMOL
5.2.1 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.
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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

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 100l 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
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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 12l 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 12l of this solution were spotted on precoated TLC plate. 1ml of above solution
diluted upto 10ml with methanol and 8, 10 and 12l 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.
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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 1.0l 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 8l 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 1.0l of the solution was
hours. 1ml of the solution diluted upto 10ml with methanol then 1.0l of the solution was
5.2.1.9.2.2 Alkaline degradation of CHL
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hours. 1.0l 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 80C for 6
for 6 hours. 1ml of the solution diluted upto 10ml with methanol. 1.0l 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. 1.0l 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 105C 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 1.0l of
the solution was spotted on precoated TLC plate (50ng/spot), developed and analysed as
described under chromatographic condition.
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5.2.1.9.4.2 Thermal degradation of CHL

25mg of CHL was weighed accurately, kept at 105C for 8 hours and transferred into
mark with methanol. 8l 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 105C for 8 hours and transferred into
mark with methanol. 1.0l of the solution was spotted on precoated TLC plate
1ml of the solution diluted upto 10ml with methanol then 1.0l of the solution was
5.2.1.9.5.2 Neutral degradation of CHL
1.0l of the solution was spotted on precoated TLC plate (250ng/spot), developed and
analysed as described under chromatographic condition.
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to 50mg of DIC, 25mg of CHL and 32.5mg of PCM was transferred to a 100ml
refluxed at 85C for 24 hours. 1.0ml of the solution diluted upto 10ml with methanol then
1.0l of the solution was spotted on precoated TLC plate (50ng/spot DIC), developed and
analysed as described under chromatographic condition. Moreover, 1.0l of the solution
was spotted on precoated TLC plate (250ng/spot CHL and 325ng/spot PCM), developed
1.0l 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
volumetric flask and dissolved in 50ml methanol. 50ml of 3% H 2O2 was added and kept
at room temperature for 6 hours. 1.0ml of the solution diluted upto 10ml with methanol
then 1.0l of the solution was spotted on precoated TLC plate (50ng/spot DIC),
developed and analysed as described under chromatographic condition. Moreover, 1.0l
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 105C for 8 hours and
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and diluted upto mark with methanol. 1.0ml of the solution diluted upto 10ml with
methanol then 1.0l of the solution was spotted on precoated TLC plate (50ng/spot DIC),
developed and analysed as described under chromatographic condition. Moreover, 1.0l
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 85C for 24 hours. 1.0ml of the solution diluted upto 10ml with methanol then 1.0l of
the solution was spotted on precoated TLC plate (50ng/spot DIC), developed and

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.
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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.
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7000
P
e
a
k
6000
5000
4000
y = 99.626x + 57.267
R = 0.9994
A
r 3000
e
a 2000
1000
0
0
10
20
30
40
50
60
70
Concentration (ng/spot)
Figure 5.2.3 Calibration curve for DIC by HPTLC
16000
P
e 14000
a 12000
k
y = 40.129x + 3023.7
R = 0.9999
10000
A
r
e
a
8000
6000
4000
2000
0
0
50
100
150
200
250
300
350
Figure 5.2.4 Calibration curve for CHL by HPTLC
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20000
P 18000
e 16000
a
14000
k
y = 41.006x + 2900.1
R = 0.9997
12000
A 10000
r 8000
e 6000
a 4000
2000
0
0
100
200
300
400
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
Drug
Linearity range
DIC
10-60ng/spot
CHL
50-300ng/spot
PCM
100-350ng/spot
Y=mx+c
Slope*
Intercept*
99.62
57.26
40.129
3023.7
41.006
2900.1
r2*
0.9994
0.9999
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).
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Table 5.2.2 Recovery study of DIC, CHL and PCM by HPTLC

Conc. Of
Conc. Of
Form.
Std. added
(ng/spot)
(ng/spot)
50
40
DIC
50
60
50
50
250
240
CHL
250
250
250
260
325
315
PCM
325
325
325
335
Drug
Conc.
Recover
(ng/spot)
89.96
108.95
100.02
489.15
497.11
508.32
638.40
648.83
661.47
% recoverySD*
99.96%0.021
99.04%0.032
100.02%0.019
99.82%0.012
99.42%0.01
99.68%0.04
99.75%0.030
99.82%0.018
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
Intra day
Mean %assay*
%RSD*
DIC
99.98%0.02
0.012
CHL
98.97%0.41
0.014
PCM
99.10%0.04
0.020
*= Average result of six replicate sample
Drug
Inter day
Mean %assay*
%RSD*
97.96%0.048
0.038
98.98%0.047
0.040
99.19%0.01
0.01
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Table 5.2.4 Robustness data for DIC, CHL and PCM by HPTLC
Chromatographic
factor
Mobile phase ratio
level
1.2:1:4v/v/v
1:1.2:4v/v/v
Saturation time
Detection wavelength
32 minutes
28 minutes
275nm
285nm
Amont found*(ng/spot)
DIC
CHL
PCM
199.820.03
39.920.051
249.980.086
2
199.840.02
39.900.062
249.160.034
8
199.970.04
39.940.060
249.970.047
3
199.930.04
39.980.062
249.990.037
2
199.950.04
39.950.038
248.950.058
3
39.960.022
201.000.02 250.100.024

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
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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
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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

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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

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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

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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
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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
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Figure 5.2.12: Chromatogram of alkaline degradation of marketed product

by HPTLC
Figure 5.2.13: Chromatogram of oxidative degradation of marketed product

by HPTLC
Figure 5.2.14: Chromatogram of thermal degradation of marketed product

by HPTLC
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Figure 5.2.15: Chromatogram of neutral degradation of marketed product

by HPTLC
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Table 5.2.5 Result of Forced degradation study of drug products

Degradatio
n condition
Conc.
of
Drug
drug(ng
/spot)
DIC
Acidic
50
Thermal
Peak
area*
%of drug*
0.73
2041.2
99.91%0.21
0.29
4694.94
91.89%1.01
0.21(I)
464.1
0.00%0.00
0.51
0.72
6392.9
2064.32
99.92%1.05
89.42%0.94
0.00%0.00
0.00%0.00
0.89(I)
210.6
0.00%0.00
10.17%0.91
0.29
0.19(II)
0.51
0.72
4487.52
208.39
6219.9
2138.71
95.27%0.77
0.00%0.00
99.89%0.34
95.39%1.22
0.00%0.00
4.63%0.29
0.00%0.00
0.00%0.00
0.84(I)
108.8
0.00%0.00
5.05%0.69
4599.6
196.45
5999.34
210.1
2014.56
4689.87
6382.98
96.00%1.03
0.00%0.00
96.95%0.19
0.00%0.00
99.93%0.82
99.87%1.08
99.92%0.24
0.00%0.00
4.26%0.96
0.00%0.00
3.50%0.96
0.00%0.00
0.00%0.00
0.00%0.00
1996.75
98.92%1.03
0.00%0.00
4499.12
291.3
6779.99
99.93%0.63
0.00%0.00
99.91%0.21
0.00%0.00
6.46%0.96
0.00%0.00
CHL
250
PCM
325
DIC
50
CHL
250
PCM
325
DIC
50
CHL
250
PCM
325
DIC
CHL
PCM
50
250
325
0.29
0.38(II)
0.32
0.24(III)
0.72
0.29
0.51
DIC
50
0.71
Alkaline
Oxidative
Rf value
of
observed
peak*
Neutral
0.29
0.21(I)
PCM
325
0.52
CHL
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%of
degradation
*
0.00%0.00
0.00%0.00
9.880.91
125
Chapter 5
5.2.3 Conclusion
The 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.
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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
Paired t-test
Drug Methods Mean SD
n Tabulated Calculated Tabulated Calculated

value
DIC
CHL
PCM
Official
99.11
0.06 6
HPLC
99.64
0.19 6
Official
99.11
0.06 6
HPTLC
99.29
0.20 6
Official
99.78
0.27 6
HPLC
99.56
0.12 6
Official
99.78
0.27 6
HPTLC
99.55
0.07 6
Official
99.65
0.24 6
HPLC
99.25
0.14 6
Official
99.65
0.24 6
HPTLC
99.67
0.15 6
Ganpat University
F-test
value
value
0.0011
2.571
0.096
0.0252
4.28
0.030
2.571
0.213
2.571
0.1854
0.3567
0.1711
4.28
0.0057
value
4.28
0.011
0.1741
0.1854
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Chapter 5
5.4 References
1. 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; 9097.
5. Sanford Bolton, Charles Bon, Pharmaceutical Statistics Practical and Clinical
Application, 4th Ed., Revised and Expanded, 2004; 417-436.
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6. STABILITY INDICATING METHOD DEVELOPMENT AND

VALIDATION
FOR
DICLOFENAC
SIMULTANEOUS
POTASSIUM,
ESTIMATION
PARACETAMOL
OF
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 0.45 filter paper to remove particulate matter and
then degassed by sonication.
Accurately weighed PCM (32.5mg) was transferred in 100ml volumetric flask. The drug
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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 (500g/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 10-60g/ml solutions.
6.1.1.3.2.2 Preparation of standard working solution of PCM
From the stock solution (325g/ml), an accurately measured 2, 4, 6, 8, 10 and 12ml was
transferred into separate 10ml volumetric flask and final volume was adjusted with
6.1.1.3.2.3 Preparation of standard working solution of MET
From the stock solution (1000g/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 100-600g/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)
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Figure 6.1.1 Overlay UV spectra of DIC, PCM and MET

The chromatographic separations were performed using Varian C-18 (250 4.6 mm i.d,
5 m particle size) column at room temperature. The optimum mobile phase consisted of
methanol: water in the ratio of 80:20, v/v respectively. 20 l 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 10-60g/ml for DIC,
65-390g/ml for PCM and 100-600g/ml for MET. All the solution were filtered through
0.2m 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.
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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 (60g/ml) and analysed
by HPLC.
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32.5mg of PCM was weighed accurately, transfer into 100ml volumetric flask and
hours (325g/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 (500g/ml) was analysed by HPLC.
hours. 6ml of the solution was diluted upto 10ml with methanol (60g/ml) and analysed
by HPLC.
dissolved in 50ml methanol. 50ml of 0.1 N NaOH was added and refluxed at 80C for 24
hours (325g/ml) and the solution was analysed by HPLC.
6.1.1.9.2.3 Alkaline degradation of MET
hours. The solution (500g/ml) was analysed by HPLC.
analysed by HPLC.
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for 24 hours (325g/ml) and the solution was analysed by HPLC.
6.1.1.9.3.3 Oxidative degradation of MET
for 24 hours. The solution (500g/ml) was analysed by HPLC.
10mg of DIC weighed accurately, kept at 105C 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 (60g/ml) and
analysed by HPLC.
32.5mg of PCM weighed accurately, kept at 105C for 8 hours and transfered in 100ml
with methanol (325g/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 105C for 8 hours an transfered in 100ml
with methanol (500g/ml) and analysed by HPLC.
6ml of the solution was diluted upto 10ml with methanol (60g/ml) and analysed by
HPLC.
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dissolved in 50ml methanol. 50ml of water was added and reflux at 85C for 24 hour
(325g/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 85C for 24 hours. The solution
(500g/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 85C
for 24 hours and analysed by HPLC.
flask and dissolved in 50ml methanol. 50ml of 0.1N NaOH was added and refluxed at
80C 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 105C for 8 hours and
and diluted upto mark with methanol.
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flask and dissolved in 50ml methanol. 50ml of water was added and refluxed at 80C for
24 hours.

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
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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 10-60g/ml for DIC,
65-390g/ml for PCM and 100-600g/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.
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350000
P
e 300000
a 250000
k
y = 5085x - 1345.1
R = 0.9998
200000
a 150000
r
100000
e
a 50000
0
20
40
60
80
Figure 6.1.3: Calibration curve of DIC by HPLC
2000000
P 1800000
e 1600000
a 1400000
k 1200000
a
r
e
a
y = 4606.9x + 14242
R = 0.9993
1000000
800000
600000
400000
200000
0
0
100
200
300
400
500
Figure 6.1.4: Calibration curve of PCM by HPLC
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P
e
a
k
a
r
e
a
3000000
2500000
2000000
y = 4030.6x + 1086
R = 0.9992
1500000
1000000
500000
0
0
100
200
300
400
500
600
700
Conccentration (g/ml)
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
Drug
Linearity range
Y=mx+c
Slope*
DIC
10-60g/ml
5085
PCM
65-390g/ml
4606.9
MET
100-600 g/ml
4030.6
Intercept*
1345.1
14242
1086
r2*
0.9998
0.9993
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
Conc. Of
Conc. Of
Drug
Form.
Std. added
(g/ml)
(g/ml)
20
20
DIC
20
25
20
30
165
135
PCM
165
165
165
195
200
250
MET
200
200
200
250
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Conc.
Recover
(g/ml)
40.03
44.84
49.89
299.89
326.94
361.83
450.05
400.87
448.13
% recoverySD*
100.07%0.046
99.64%0.109
99.78%0.046
99.96%0.085
99.07%0.16
100.50%0.11
100.02%0.12
100.21%0.12
99.58%0.10
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Chapter 6
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
Intra day
Mean %assay*
%RSD*
DIC
98.84%0.30
1.22
PCM
98.29%1.18
0.26
MET
98.94%0.12
0.14
Drug
Inter day
Mean %assay*
%RSD*
98.76%0.25
1.26
99.06%0.36
0.48
99.86%0.19
0.52
Table 6.1.4 Robustness data for DIC, PCM and MET by HPLC
Chromato
graphic
factor
level
Retention time*
DIC
PCM
0.6ml/m
in
1.2ml/m
in
3.720. 6.830.
02
01
Flow rate
3.250. 6.140.
03
06
3.700. 6.820.
68:32
09
01
Methanol:
water
3.740. 6.150.
72:28
08
08
3.710. 6.130.
268nm
Detection
08
01
wavelengt
3.710. 6.130.
h
276nm
09
01
Tailing factor*
MET
DIC
PCM
MET
10.320.
01
9.230.0
8
10.320.
01
10.230.
01
9.410.0
8
9.410.0
8
1.060.
01
1.080.
08
1.080.
05
1.070.
08
1.070.
01
1.070.
09
1.080..
01
1.090.0
8
1.080.0
8
1.080.0
1
1.080.0
1
1.090.0
1
0.970.
01
0.960.
01
0.960.
08
0.970.
08
0.970.
01
0.970.
02

The LOD for DIC, PCM & MET were found to be 0.15g/ml, 2.40g/ml and 1.82g/ml
respectively, while LOQ were 0.48g/ml, 7.29g/ml and 5.53g/ml respectively.
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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
theoretical
plates*
Tailing
factor*
DIC
3.510.03
50152.6503.35 3590.490.52 1.040.02
PCM
6.420.04
312120.2112.54 3437.4139.80 1.080.01
MET
9.900.05
400120.0 3923.11 1263.127.005 0.960.008
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.
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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
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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

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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

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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
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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
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HPLC

HPLC

HPLC
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Figure 6.1.14: Chromatogram of thermal degradation of marketed product

by HPLC
Figure 6.1.15: Chromatogram of neutral degradation of marketed product

by HPLC
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Table 6.1.6 Forced degradation study of DIC, PCM and MET by HPLC
RT of
Degradation
Conc. Of
Drug
observed
condition
drug(g/ml)
peak*
DIC
PCM
Alkaline
Oxidative
Thermal
Neutral
%drug*
%
degradation*
50
3.56
241732
99.14%0.00
0.00%0.00
325
6.42
1420526
100%1.01
0.00%0.00
9.90
1807432 94.86%1.04
Acidic
MET
AUC*
500
DIC
50
PCM
325
MET
500
DIC
50
PCM
325
MET
500
DIC
PCM
MET
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
4.11
231415
95.34%0.98
0.00%0.00
3.52(I)
9329
0.00%0.00
4.03%0.99
6.39
1419311 99.21%1.48
0.00%0.00
9.90
8.92(II)
4.11
1806415 94.12%1.35
95120
0.00%0.00
231811 95.39%1.21
0.00%0.00
5.5%0.25
0.00%0.00
3.49(I)
9653
0.00%0.00
4.16%0.50
50
325
500
6.39
5.62(II)
9.90
10.32(III)
3.51
6.42
9.90
1419769
11981
1806926
91824
251830
1500874
2007891
91.76%1.34
0.00%0.00
99.54%1.21
0.00%0.00
98.99%1.09
99.42%1.09
99.29%1.21
0.00%0.00
8.64%1.21
0.00%0.00
5.06%0.50
0.00%0.00
0.00%0.00
0.00%0.00
DIC
50
4.11
24981
98.44%12
0.00%0.00
PCM
325
6.39
9.90
MET
500
10.29(I)
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1465896 98.97%1.21
1805341 94.76%1.09
98691
0.00%0.00
0.00%0.00
0.00%0.00
5.46%0.78
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Chapter 6
6.1.3 Conclusion
The 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.
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6.2
STABILITY
INDICATING
HPTLC
METHOD
FOR

PARACETAMOL AND METHOCARBAMOL
6.2.1 Experimental
6.2.1.1 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.
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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 G60F 254 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 100l 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
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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 1.2l 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 1.2l 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.
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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
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6.2.1.9.3.3 Oxidative degradation of MET
10mg of DIC was weighed accurately, kept at 105c for 8 hours and transferred into
25mg of PCM was weighed accurately, kept at 105C for 8 hours and transferred into
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6.2.1.9.4.3 Thermal degradation of MET

50mg of MET was weighed accurately, kept at 105C for 8 hours and transferred into
6.2.1.9.5.3 Neutral degradation of MET
2.0l 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
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to 50 mg of DIC, 325mg of PCM and 500mg of MET was transferred to a 100ml
refluxed at 85C for 24 hours. 1ml of the solution diluted upto 10ml with methanol then
1.0l 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
volumetric flask and dissolved in 50ml methanol. 50ml of 3% H 2O2 was added and kept
at room temperature for 24 hours. 1ml of the solution diluted upto 10ml with methanol
then 2.0l 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 105C for 8 hours and
and diluted upto mark with methanol. 1ml of the solution diluted upto 10ml with
methanol then 2.0l 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 85C for 24 hours. 1ml of the solution diluted upto 10ml with methanol then 2.0l 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.
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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.
Ganpat University
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Chapter 6

10000
9000
8000
7000
6000
5000
4000
3000
2000
1000
0
P
e
a
k
A
r
e
a
y = 15.089x + 47.333
R = 0.9997
100
200
300
400
500
600
700
Figure 6.2.3: Calibration curve for DIC by HPTLC

16000
P 14000
e
a 12000
k 10000
A
r
e
a
y = 22.766x + 53.714
R = 0.9989
8000
6000
4000
2000
0
0
200
400
600
800
Figure 6.2.4: Calibration curve of PCM by HPTLC
Ganpat University
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Chapter 6
P
Pe
ea
ak
k
A
Ar
re
ea
a
20000
18000
16000
14000
y = 14.865x + 196.53
R = 0.9995
12000
10000
8000
6000
4000
2000
0
0
500
1000
1500
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
Drug
Linearity range
DIC
100-600ng/spot
PCM
150-650ng/spot
MET
200-1200ng/spot
Y=mx+c
Slope*
Intercept*
15.089
47.33
22.76
53.71
14.865
196.53
r2*
0.9997
0.9989
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
Conc. of
Conc. of
Form.
Std. added
(ng/spot)
(ng/spot)
300
250
DIC
300
300
300
350
250
150
PCM
250
250
250
350
600
400
MET
600
500
600
600
Drug
Ganpat University
Conc.
Recover
(ng/spot)
546.20
594.54
650.73
396.92
496.91
595.22
991.03
1090.1
1188.03
% recoverySD*
99.30%0.047
99.09%0.068
100.11%0.030
99.23%0.123
99.38%0.013
99.20%0.067
99.10%0.050
99.1%0.055
99.00%0.058
160
Chapter 6
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
Intra day
Mean %assay*
%RSD*
DIC
98.97%0.015
0.010
PCM
99.97%0.04
0.024
MET
99.90%0.02
0.016
Drug
Inter day
Mean %assay*
%RSD*
99.97%0.019
0.046
99.98%0.047
0.036
99.99%0.01
0.028
Table 6.2.4 Robustness data for DIC, PCM and MET by HPTLC
Chromatographic factor
level
Mobile phase ratio

Toluene: ethyl acetate:
methanol
1.2:1:4v/v/v
Saturation time
Detection wavelength
1:1.2:4v/v/v
32 minutes
28 minutes
275nm
265nm
Amont found*(ng/spot)
DIC
PCM
MET
299.950.05 399.950.06
599.980.057
4
1
299.940.04 399.930.03
599.960.043
4
8
299.970.03 399.970.04
599.970.047
7
3
299.930.05 399.930.04
599.990.037
0
2
299.930.04 399.950.04
599.950.058
9
3
299.960.04
400.000.02 600.000.033
0

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.

The market formulation containing 50mg DIC, 325mg PCM and 500mg MET was
analysed by HPTLC.
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Chapter 6

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

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162
Chapter 6
Figure 6.2.6c: Chromatogram of acidic degradation of MET by HPTLC
Figure 6.2.7a: Chromatogram of alkaline degradation of DIC by HPTLC
Ganpat University
163
Chapter 6
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

Ganpat University
164
Chapter 6
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
Ganpat University
165
Chapter 6
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

Ganpat University
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Chapter 6
Figure 6.2.10b: Chromatogram of neutral degradation of PCM by HPTLC
Figure 6.2.10c: Chromatogram of neutral degradation of MET by HPTLC

HPTLC
Ganpat University
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Chapter 6

HPTLC

HPTLC
Ganpat University
168
Chapter 6
Figure 6.2.14: Chromatogram of thermal degradation of marketed product by HPTLC
Figure 6.2.15: Chromatogram of neutral degradation of marketed product by

HPTLC
Ganpat University
169
Chapter 6
Table 6.2.5 Forced degradation study of marketed product by HPTLC

Degradat
ion
condition
acidic
alkaline
oxidative
Thermal
Neutral
Drug
Conc. Of
drug(ng/sp
ot)
Rf value of
observed
peak*
Peak
area*
%drug*
%degradatio
n*
DIC
100
0.78
3234.5
99.12%1.0
8
0.00%0.00
PCM
250
0.63
5318.9
0.42
8965.3
0.29(I)
0.34(II)
692.1
746.7
MET
400
DIC
100
PCM
250
MET
400
DIC
100
PCM
250
MET
400
DIC
0.78
0.84(I)
0.63
3102.8
326.6
5299.36
0.41
8134.9
0.36(II)
425.6
0.78
2894.5
0.76(I)
329.7
0.63
4624.9
0.57(II)
427.4
0.42
8128.8
0.35(III)
692.1
100
0.78
3129.66
PCM
250
0.63
5236.34
MET
400
0.42
8631.34
DIC
100
0.78
2988.75
PCM
250
0.63
4989.6
MET
400
0.42
0.37(I)
Ganpat University
7984.1
536.9
99.91%1.0
1
84.92%1.0
5
0.00%0.00
0.00%0.00
89.51%0.9
8
0.00%0.00
99.45%1.0
8
94.91%0.2
1
0.00%0.00
88.39%0.2
1
0.00%0.00
90.84%1.3
4
0.00%0.00
91.92%0.2
4
0.00%0.00
99.92%1.0
9
99.96%1.0
9
99.95%0.1
9
98.96%1.0
5
99.92%1.2
1
99.89%0.3
4
0.00%0.00
0.00%0.00
0.00%0.00
7.71%0.99
8.320.99
0.00%0.00
10.50%0.99
0.00%0.00
0.00%0.00
5.22%0.55
0.00%0.00
11.36%0.55
0.00%0.00
9.25%1.26
0.00%0.00
8.51%0.55
0.00%0.00
0.00%0.00
0.00%0.00
4.13%0.99
0.00%0.00
0.00%0.00
6.71%0.99
170
Chapter 6
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.
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Chapter 6
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 ttest 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
Paired t-test
Drug
DIC
PCM
MET
Methods
Mean
SD
n Tabulated
Official
99.27
0.16 6
HPLC
99.71
0.28 6
Official
99.27
0.16 6
HPTLC
99.28
0.14 6
Official
99.47
0.29 6
HPLC
99.65
0.14 6
Official
99.47
0.29 6
HPTLC
99.15
0.06 6
Official
99.40
0.26 6
HPLC
99.36
0.14 6
Official
99.40
0.14 6
99.48
0.15 6
HPTLC
Ganpat University
F-test
Calculated
Tabulated
Calculated
value
value
value
value
2.571
0.2201
4.28
0.1886
0.3251
2.571
0.077
0.1850
4.28
0.0321
2.571
0.3220
0.1475
0.2596
0.010
4.28
0.1886
0.1850
172
Chapter 6
6.4 References
1. 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; 9097.
5. Sanford Bolton, Charles Bon, Pharmaceutical Statistics Practical and Clinical
Application, 4th Ed., Revised and Expanded, 2004; 417-436.
Ganpat University
173
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 G60F 254 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, C 18
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.
Ganpat University
174
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.
Ganpat University
175
Chapter 8
Publication
8. PUBLICATION
TITLE
JOURNAL
STATUS
Development and Validation of stability indicating Inventi Rapid:
Published
method for determination of Diclodenac potassium, Pharm Analysis
2012(4): 1-
Chlorzoxazone and Paracetamol in pharmaceutical & Quality

dosage
form
using
high
performance
9, 2012.
liquid Assurance
chromatography
Stability Indicating HPLC method for simultaneous American
Accepted
determination of Diclofenac potassium, Paracetamol Journal of
(in Press)
and Methocarbamol
Pharmaceutical
Technology &
Research
Development and validation of stability indicating Journal of
Under
method for the determination of Paracetamol and Chromatography
review
Methocarbamol in pharmaceutical dosage form B

using HPTLC
Development and validation of simultaneous HPLC Arabian Journal
Under
method
review
for
estimation
of
Paracetamol
and of Chemistry
Methocarbamol.
Ganpat University
176
RESEARCH ARTICLE
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 m 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 5-25g/ml for diclofenac potassium (DIC), 25-125g/ml for chlorzoxazone (CHL) and 32.5-182.5g/ml for
paracetamol (PCM). The limit of detection for DIC, CHL & PCM was found to be 0.15g/ml, 1.82g/ml and 2.40g/ml
respectively, while limit of quantification was 0.47g/ml, 5.53g/ml and 7.29g/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.
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.
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 antiinflammatory, 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
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.
Kalol Institute of Pharmacy, Kalol-Mehsana Highway, Kalol -382721,

Gujarat, India.
E-mail: amin_maulik2003@yahoo.co.in
*Corresponding author
1
Shree S. K. Patel College of Pharmaceutical Education & Research, Ganpat

University, Kherva, Mehsana-382711, Gujarat, India.
Faculty of Pharmacy, D. D. University College Road, Nadiad-387001,

Gujarat, India.
Shankersinh Vaghela Bapu Institute of Pharmacy, Gandhinagar-Mansa

Road, PO. Vasan, Gandhinagar-382650, Gujarat, India.
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
Table 1: Results of System Suitability Parameters
Parameter
DIC
CHL
PCM
RT*
3.250.05
5.600.06
9.400.09
AUC*
25012.20283.43
105410.2312.53
150453.123923.11
No. of Theoretical Plates*

4204.49.52
3637.4139.80
1263.127.005
Tailing Factor*
0.95.002
1.020.01
0.970.001
Table 2: Linearity and Calibration Curve

Drug
Linearity Range
DIC
CHL
PCM
5-30 g/ml
25-125 g/ml
32.5-182.5 g/ml
Y=mx+c
Slope*
5018.1
4987.5
5051.4
r2*
Intercept*
362
2058
14445
0.9998
0.9997
0.9997
Table 3: Results of Recovery Studies

Drug
Conc. of Form.(g/ml)
5
5
5
25
25
25
32.5
32.5
32.5
DIC
CHL
PCM
Conc. of Std. Added(g/ml)

4
5
6
24
25
26
34
32.5
36
Conc. Recovered(g/ml) SD*

8.890.12
9.970.30
10.920.02
48.490.07
48.900.71
50.580.05
65.541.51
65.280.08
69.070.16
% RecoverySD*
98.78%0.18
99.70%0.75
99.27%1.89
98.96%0.01
98.16%0.18
99.17%1.09
98.56%0.86
100.41%0.15
100.83%0.11
Table 4: Results of Intraday & Inter Day Precision

Intra day
Drug
Mean % Assay*
99.34%1.16
99.83%1.63
98.94%0.01
DIC
CHL
PCM
Inter day
%RSD*
0.36
0.12
1.16
Mean % Assay*
99.15%1.82
100.05%0.57
98.86%0.12
% RSD*
1.08
1.12
1.52
Table 5: Robustness Data for DIC, CHL and PCM

Chromatographic
Factor
level
Flow rate
Methanol:
phosphate buffer (v/v)
Detection wave-length
0.7ml/min
1.0ml/min
75:25
85:15
275nm
285nm
DIC
3.980.02
2.950.02
3.910.03
3.960.02
3.240.04
3.260.03
Retention Time*
CHL
6.010.01
5.040.01
6.020.03
5.050.02
5.600.01
5.600.01
PCM
9.960.01
8.660.03
9.950.02
8.61.02
9.410.01
8.640.02
DIC
0.950.02
0.960.01
0.950.02
0.960.01
0.950.01
0.950.02
Tailing Factor*
CHL
1.020.01
1.030.01
1.020.01
1.020.01
1.030.01
1.020.01
PCM
0.970.01
0.960.01
0.960.02
0.970.01
0.970.03
0.970.01
Table 6: Limit of Detection and Limit of Quantification

Drugs
DIC
CHL
PCM
Standard Deviation
238.7
2759.5
3684.4
Slope of Calibration Curve

5018.1
4987.
5051.4
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 m particle
size with microliter syringe (rheodyne) injector. The
[ISSN 0976-3813]
LOD(g/ml)
0.15
1.82
2.40
LOQ(g/ml)
0.47
5.53
7.29
HPLC was connected to personal computer with ClassAutochro-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 20l was used.

RESEARCH ARTICLE
Table 7: Summary of Validation Parameters
Parameter
Wavelength
Range
Linearity
Intercept
Slope
Intraday precision
Interday precision
LOD
LOQ
DIC
280nm
5-30g/ml
0.9998
362
5018.1
0.36
1.08
0.15
0.47
CHL
280nm
25-125g/ml
0.9997
2058
4987.5
0.12
1.12
1.82
5.53
PCM
280nm
32.5-182.5g/ml
0.9997
14445
5051.4
1.16
1.52
2.40
7.29
Table 8: Analysis of Marketed Formulation of the Product

Brand Name
Drug
DIC
CHL
PCM
DAN-MR
Amount Taken (g/ml)

50
250
325
Amount Found (g/ml)

40.987
249.89
324.26
%Amount found
99.740.22
99.560.11
99.260.21
Table 9: Results of Forced Degradation Study

Degradation Condition
Drug
DIC
Conc. of Drug(g/ml)
5
Acidic
CHL
25
PCM
32.5
DIC
CHL
25
PCM
32.5
DIC
CHL
25
PCM
32.5
DIC
CHL
PCM
DIC
5
25
32.5
5
CHL
25
PCM
32.5
Alkaline
Oxidative
Thermal
Neutral
RTof Observed Peak*

3.25
5.59
5.19(I)
9.40
3.25
2.92(I)
5.62
5.49(II)
6.12(III)
9.41
3.25
2.72(I)
5.60
5.12(II)
9.41
10.21(III)
3.25
5.61
9.41
3.25
5.60
4.93(I)
9.41
% Drug*
% Degradation*
98.14%1.04
0.00%0.00
91.98%1.31
0.00%0.00
0.00%0.00
8.791.31
99.65%1.45
0.00%0.00
94.62%1.24
0.00%0.00
0.00%0.00
5.96%1.21
91.56%1.57
94.10%1.38
0.00%0.00
3.61%1.41
0.00%0.00
1.511.30
99.57%1.34
0.00%0.00
91.41%1.32
0.00%0.00
0.00%0.00
8.98%1.54
97.00%1.03
0.00%0.00
0.00%0.00
2.64%0.95
93.84%1.09
0.00%0.00
0.00%0.00
6.681.41
99.90%1.12
0.00%0.00
99.88%1.08
0.00%0.00
99.84%1.24
0.00%0.00
98.45%1.4
0.00%0.00
91.88%1.23
0.00%0.00
0.00%0.00
8.64%0.95
99.84%1.21
0.00%0.00
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
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 500g/ml stock
solution.
Preparation of Standard Working Solutions

Preparation of Standard Working Solution of DIC
From the stock solution (500g/ml), an aliquot quantity 1,
2, 3, 4, and 5ml were transferred into separate 100ml
volumetric flask and final volume was adjusted with
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
Preparation of Standard Working Solution of CHL

From stock solution (2500g/ml), an aliquot quantity 1, 2,
3, 4, and 5 ml were transferred into 100 ml volumetric flask
Preparation of Standard Stock Solution of PCM
[ISSN 0976-3813]
AUC*
24018
101541
8931
149901
24010
1432
94421
3410
1431
149097
23120
1981
102384
2398
148320
9908
25320
105908
149959
24906
103956
9327
104903

RESEARCH ARTICLE
Cl
HO
O K+
NH
O
Cl
N
H
Diclofenac Potassium
CH3
Paracetamol
O
OH
N
Cl
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

700000
160000
P
e
a
k
a
r
e
a
P
e
a
k
140000
120000
100000
80000
a
r
e
a
y = 5018.1x + 362
R = 0.9998
60000
600000
500000
400000
300000
y = 4987.5x - 20588
R = 0.9997
200000
40000
100000
20000
0
0
0
10
20
30
40
50
100
150
Figure 4: Calibration curve of DIC
Figure 5: Calibration curve of CHL
and final volume was adjusted with methanol upto mark to

prepare 25-125g/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
Preparation of Standard Working Solution of PCM

From stock solution (3250g/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 g/ml solutions.
[ISSN 0976-3813]

RESEARCH ARTICLE
P
e
a
k
a
r
e
a
1000000
900000
800000
700000
600000
500000
400000
300000
200000
100000
0
y = 5051.4x - 14453
R = 0.9997
50
100
150
200
Concentration
Figure 7: Forced degradation under acidic condition
Figure 6: Calibration curve of PCM
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
transferred into 10ml volumetric flask and diluted upto

mark with methanol.
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.
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 20L 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 5m C-18 column (250x4.6 mm) with flow rate
0.8 mL/min. (Figure 3 A typical Chromatogram for DIC,
CHL and PCM).
[ISSN 0976-3813]
Linearity and Calibration Graph

The linearity was evaluated by linear regression analysis.
The series of dilution ranging from 5-25g/ml for DIC, 25125g/ml for CHL and 32.5-182.5g/ml for PCM was
prepared and performed for linearity. All the solutions
were filtered through 0.2 m membrane filter and injected,
chromatograms were recorded and it was repeated for six

RESEARCH ARTICLE
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 85C for 24
hours. 3.25ml of the solution was diluted upto 10ml with
methanol (32.5g/ml) was analysed by HPLC.
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 was
added 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.
Alkaline Degradation
Alkaline Degradation of DIC
Accurately weighed DIC (10mg) was transferred into
50ml of 0.1N NaOH was added and refluxed at 80C for 6
methanol (5g/ml) and analysed by HPLC.
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.
Alkaline Degradation of CHL

Accurately weighed CHL (10mg) was transferred into
50ml of 0.1 N NaOH was added and refluxed at 85C for 2
Alkaline Degradation of PCM
50 ml of 5N NaOH was added and refluxed at 85C for 24
methanol. The solution (32.5g/ml) was analysed by
HPLC.
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 45C), and changing
the wavelength (268 and 276 nm).
Oxidative Degradation
Oxidative Degradation of DIC
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 (5g/ml) and analysed by HPLC.
Oxidative Degradation of CHL
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 (25g/ml) and analysed by HPLC.
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
50ml of 0.1N HCl was added and refluxed at 80C for 6
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 (32.5g/ml) was analysed by
HPLC.
Thermal Degradation
Thermal Degradation of DIC
Accurately weighed DIC (10mg) was kept at 105C 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
(5g/ml) and analysed by HPLC.
Acidic Degradation of CHL

50ml of 5N HCl was added and refluxed at 85C for 24
hours. 2.5 ml of the solution was diluted upto 10ml with
[ISSN 0976-3813]

RESEARCH ARTICLE
for 6 hours. 1ml of the solution was diluted upto 100ml
with methanol and analysed by HPLC.
Thermal Degradation of CHL

Accurately weighed CHL (10mg) was kept at 105C for 8
in 50ml of methanol then dilute upto mark with methanol.
2.5 ml of the solution was diluted upto 10ml with methanol
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 105C 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.
Thermal Degradation of PCM

Accurately weighed PCM (10mg) was kept at 105C for 8
in 5ml of methanol then dilute upto mark with methanol
(32.5g/ml) and analysed by HPLC.
Neutral Degradation of Marketed Product

250mg of CHL and 325mg of PCM was transferred to a
50ml of water was added and refluxed at 80C for 6 hours.
1ml of the solution was diluted upto 100ml with methanol
Neutral Degradation
Neutral Degradation of DIC
0.5ml of the solution was diluted upto 10ml with methanol
RESULTS AND DISCUSSION

The mobile phase consisting of methanol: water (80:20,
v/v) at 0.8ml/min flow rate which gave sharp, wellresolved 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 5-25Pg/ml for
DIC, 25-125g/ml for CHL and 32.5-162.5 Pg/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 0.15g/ml, 1.82g/ml and
2.40g/ml respectively, while LOQ were 0.47g/ml,
5.53g/ml and 7.29g/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 80c for 10hr condition and chromatogram showed no
additional peak.
Figure 7 Forced degradation under acidic condition,
Figure 8 Forced degradation under alkaline condition.
Neutral Degradation of CHL

2.5ml of the solution was diluted upto 10ml with methanol
Neutral Degradation of PCM
50ml of water was added and reflux at 85C for 24 hours.
The solution (32.5g/ml) was analysed by HPLC.
Forced Degradation Study of Marketed Product
Acidic Degradation of Marketed Product
250mg of CHL and 325mg of PCM were transferred to a
50ml of 0.1N HCl was added and refluxed at 80C for 6
hours. 1ml of the solution was diluted upto 100ml with
Alkaline Degradation of Marketed Product
50ml of 0.1N NaOH was added and refluxed at 80C for 6
hours. 1ml of the solution was diluted upto 100ml with
Oxidative Degradation of Marketed Product
50ml of 3% H2O2 was added and kept at room temperature
[ISSN 0976-3813]

RESEARCH ARTICLE
6. Gan TJ. Diclofenac: an update on its mechanism of action and
safety profile. Curr Med Res Opin., 26(7): 1715-1731, 2010.
7. ONeil 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, 654:287292, 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):6367, 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. RPHPLC 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
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2010.
21. ICH, Q2B Validation of Analytical Procedure: Methodology, in:
Proceeding of the International Conference on Harmonization,
Geneva, March 1996.
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):109114, 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.
[ISSN 0976-3813]

RESEARCH ARTICLE
Acknowledgments
The authors are greaful to Zydus Cadila Ltd for providing gift
samples of diclofenac potassium, chlorzoxazone and
paracetamol respectively.
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 stabilityindicating 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.
[ISSN 0976-3813]
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): 19, 2012.


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STABILITY INDICATING SIMULTANEOUS

A thesis submitted in partial fulfillment of the

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

THESIS APPROVAL SHEET

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

Professor & Head,

simultaneous determination of new muscle relaxant drugs by

which is submitted herewith to Ganpat

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

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

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

Name of Research student

Signature of Research student

Development of new analytical method

Stability indicating analytical method

Aim of the present work

Chemicals, glasswares and instruments

Active Pharmaceutical Ingredients

Stability indicating method development and validation for

simultaneous estimation of Diclofenac potassium,

Stability indicating HPLC method development and validation

for simultaneous estimation of Diclofenac potassium,

Stability indicating HPTLC method for simultaneous estimation

of diclofenac potassium, Chlorzoxazone and Paracetamol

Statistical comparison between official and proposed methods

Stability indicating method development and

simultaneous estimation of Diclofenac potassium,

Stability indicating HPLC method development for simultaneous

estimation of Diclofenac potassium, Paracetamol and

Stability indicating HPLC method for simultaneous estimation

of Diclofenac potassium, Paracetamol and Methocarbamol

Statistical comparison between official and proposed methods

Controlling sample retention by changing solvent strength

Various degradation conditions described for the stress testing

Official methods for paracetamol

Official methods for paracetamol in combination

Official methods for diclofenac potassium in combination

Official methods for chlorzoxazone in combination

Official methods for methocarbamol

Reported methods for paracetamol

Reported methods for diclofenac potassium

Reported methods for chlorzoxazone

Reported methods for methocarbamol

List of active pharmaceutical ingredients

Results of optimization of mobile phase

Recovery study of DIC, CHL and PCM by HPLC

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

Robustness data for DIC, CHL and PCM by HPLC

System suitability parameters for DIC, CHL and PCM by HPLC

Forced degradation study of marketed product by HPLC

Recovery study of DIC, CHL and PCM by HPTLC

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

Robustness data for DIC, CHL and PCM by HPTLC

Result of Forced degradation study of drug products

Statistical comparison for DIC, CHL and PCM between proposed

Results of optimization of mobile phase

Recovery study for DIC, PCM and MET by HPLC

Robustness data for DIC, PCM and MET by HPLC

System suitability parameters for DIC, PCM and MET by HPLC

Forced degradation study of DIC, PCM and MET by HPLC

Recovery study for DIC, PCM and MET by HPTLC

Robustness data for DIC, PCM and MET by HPTLC

Forced degradation study of marketed product by HPTLC