Development and Validation of Analytical Methods for · test. Simple, accurate, precise, robust and reproducible stability indicating RP-HPLC method has been conducted for combined

Development and Validation of Analytical Methods for

the Estimation of Drugs Used in the Treatment of

Arthritis

A Thesis submitted to Gujarat Technological University

for the Award of

Doctor of Philosophy

in

Pharmacy

by

Jignasha M. Derasari

[119997290014]

Under Supervision of

Dr. Vandana B. Patel

GUJARAT TECHNOLOGICAL UNIVERSITY

AHMEDABAD

[April – 2019]

ii

© Jignasha M. Derasari

iii

DECLARATION

I declare that the thesis entitled “Development and Validation of Analytical Methods for

t h e Estimation of Drugs Used in the Treatment of Arthritis” submitted by me for the

degree of Doctor of Philosophy is the record of research work carried out by me during the

period from July 2011 to July 2017 under the supervision of Dr. Vandana B. Patel and this

has not formed the basis for the award of any degree, diploma, associate ship, fellowship, titles

in this or any other University or other institution of higher learning.

I further declare that the material obtained from other sources has been duly acknowledged in

the thesis. I shall be solely responsible for any plagiarism or other irregularities, if noticed in

the thesis.

Signature of the Research Scholar: …………………….………… Date: ….………………

Name of Research Scholar: Jignasha M. Derasari

Place: Vadodara, Gujarat, India.

iv

CERTIFICATE

I certify that the work incorporated in the thesis “Development and Validation of

Analytical Methods for t h e Estimation of Drugs Used in the Treatment of Arthritis”

submitted by Jignasha M. Derasari was carried out by the candidate under my

supervision/guidance. To the best of my knowledge: (i) the candidate has not submitted the

same research work to any other institution for any degree/diploma, Associate ship, Fellowship

or other similar titles (ii) the thesis submitted is a record of original research work done by the

Research Scholar during the period of study under my supervision, and (iii) the thesis

represents independent research work on the part of the Research Scholar.

Signature of Supervisor: ……………………………… Date: ………………

Name of Supervisor: Dr. Vandana B. Patel

Place: ………………………………………….………

v

Originality Report Certificate

It is certified that PhD Thesis titled “Development and Validation of Analytical Methods

for t h e Estimation of Drugs Used in the Treatment of Arthritis” by Jignasha M.

Derasari has been examined by us. We undertake the following:

a. Thesis has significant new work / knowledge as compared already published or is

under consideration to be published elsewhere. No sentence, equation, diagram, table,

paragraph or section has been copied verbatim from previous work unless it is placed

under quotation marks and duly referenced.

b. The work presented is original and own work of the author (i.e. there is no plagiarism).

No ideas, processes, results or words of others have been presented as Author own

work.

c. There is no fabrication of data or results which have been compiled / analysed.

d. There is no falsification by manipulating research materials, equipment or processes, or

changing or omitting data or results such that the research is not accurately represented

in the research record.

e. The thesis has been checked using “turnitin” (copy of originality report attached) and

found within limits as per GTU Plagiarism Policy and instructions issued from time to

time (i.e. permitted similarity index

vii

PhD THESIS Non-Exclusive License to

GUJARAT TECHNOLOGICAL UNIVERSITY

In consideration of being a PhD Research Scholar at GTU and in the interests of the facilitation

of research at GTU and elsewhere, I, Jignasha M. Derasari (Enrollment No. :

119997290014) hereby grant a non-exclusive, royalty free and perpetual license to GTU on the

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d) The Universal Copyright Notice (©) shall appear on all copies made under the authority of

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g) If third party copyrighted material was included in my thesis for which, under the terms of

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h) I retain copyright ownership and moral rights in my thesis, and may deal with the

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copyright in my thesis of the rights granted by me to my University in this non-exclusive

viii

license.

j) I am aware of and agree to accept the conditions and regulations of PhD including all

policy matters related to authorship and plagiarism.

Signature of the Research Scholar:…………………….………

Name of Research Scholar: Jignasha M. Derasari

Date: …………………… Place: ……………………………

Signature of Supervisor: …………………………………………

Name of Supervisor: Dr. Vandana B. Patel

Date: …………………….. Place: …………………………...

Seal:

ix

Thesis Approval Form

The viva-voce of the PhD Thesis submitted by Jignasha M. Derasari (Enrollment No.

119997290014) entitled “Development and Validation of Analytical Methods for t h e

Estimation of Drugs Used in the Treatment of Arthritis” was conducted on

………………………………........………… (Day and date) at Gujarat Technological University.

(Please tick any one of the following option)

The performance of the candidate was satisfactory. We recommend that she be awarded

the PhD degree.

Any further modifications in research work recommended by the panel after 3 months

from the date of first viva-voce upon request of the Supervisor or request of Independent

Research Scholar after which viva-voce can be re-conducted by the same panel again.

(Briefly specify the modifications suggested by the panel)

The performance of the candidate was unsatisfactory. We recommend that she should not be

awarded the PhD degree.

(The panel must give justifications for rejecting the research work)

----------------------------------------------------- ----------------------------------------------

Name and Signature of Supervisor with Seal 1) (External Examiner 1) Name and Signature

---------------------------------------------------- ---------------------------------------------------

2) (External Examiner 2) Name and Signature 3) (External Examiner 3) Name and Signature

x

ABSTRACT

Present research investigation deals with the development of analytical methods and their

validation of drugs used in the treatment of arthritis. Four combinations i.e. 1) Diclofenac

potassium and Febuxostat 2) Febuxostat and Ketorolac tromethamine 3) Febuxostat and

Naproxen 4) Ibuprofen and Tramadol hydrochloride have been chosen for analytical study by

stability indicting RP-HPLC and/or UV spectrophotometric methods.

Two UV spectrophotometric methods viz .simultaneous equation method and first order

derivative method and stability indicating RP-HPLC method have been developed and

validated for combination (1) and (3). These methods (two UV and one stability RP-HPLC) of

these two combinations were compared statistically by one-way ANOVA study with students’t-

test. Simple, accurate, precise, robust and reproducible stability indicating RP-HPLC method

has been conducted for combined dosage form (2) and (4). Stability of all four pharmaceutical

dosage forms was assessed in various degradation conditions like hydrolysis (acid- alkali),

oxidation, thermal and photolytic conditions.

DoE (Design of Experiments) technique, an integral part of QbD has been applied to check the

effect of different variables on acid degradation pattern of both the drugs via Full Factorial

Design (FFD) – 23(three factors at two levels) and kinetic study was also carried out to obtain

degradation rate constant, half-life (t0.5), shelf life (t0.9), activation energy (Ea) and Arrhenius

energy factor for both the drugs of combination (1). Moreover, same degraded samples were

spiked into LC-MS/MS for analytical study.

The calibration plots of all developed methods were linear over the selected concentration

range with a correlation coefficient value nearer to 0.999. % assay values and % recoveries of

drug of combined dosage forms were obtained within the limit specified in ICH guidelines i.e.

98-102%. One-way ANOVA study suggested stability indicating RP-HPLC method is more

accurate and precise than UV spectrophotometric methods for combination 1 and 3. Results of

stability studies in different conditions for combined dosage forms indicated all the drugs were

susceptible to hydrolysis (acidic and alkaline) whereas comparatively stable towards other

degradation conditions.

All the developed methods were able to detect and/or separate the drugs from their potential

related substances. Hence, the proposed methods were found to be simple, sensitive,

xi

economical, precise and robust and can be applicable to the routine analysis of the selected

combinations.

Keywords

UV spectrophotometric methods, stability indicating RP-HPLC, QbD, DoE, Kinetic study,

ANOVA, Validation

xii

ACKNOWLEDGEMENT

Thank you, God, for little things that often come our way-

the things we take for granted and don’t mention when we pray,

Thank You, God for the “miracles”, we are much too blind to see, A hand reached out to help us, in the time of

sudden need.

Thank u God for fulfilling my needs in a very special way!!!

I offer flowers of gratitude to the almighty God for the benediction and grace & for being the source of

strength throughout my life.

Every achievement is a result of committed activities that too when headed & guided by worthy and

knowledgeable persons. Clear paths or hazy roads I always remember the supports I got from the

people whose names I feel privileged to mention here. It is with a sense of pride and pleasure that, I

humbly look back to acknowledge, those who have been a source of encouragement in my entire

endeavor.

At the outset I feel that I may not have reached this stage without the blessings, love & care of my,

loving parents, beloved husband, my sweet little sister & brother in-law, cooperative and caring in-

laws, dear friends & my well-wishers. I also acknowledge Gujarat Technological University for

giving me the opportunity to proceed with the research work and do the PhD.

With a deep sense of respect and gratitude, I would like to express my sincere thanks to my esteemed

guide, Dr. Vandana B. Patel, Principal & Professor in Quality Assurance department, Babaria Institute

of Pharmacy, Vernama, Vadodara, Gujarat for giving me the opportunity to work in the field of

analytical chemistry. Without her perpetual encouragement, constructive guidance, advice and

support throughout my journey of the doctoral research, I would never have succeeded in

accomplishing the work.

With great reverence, I take this opportunity to express my debt of gratitude to the DPC (Doctoral

Progress committee) members, Dr. Sadhana J. Rajput, H O D & Professor, Faculty of Pharmacy,

M . S . University of Baroda and Dr. C. N. Patel, Principal& Professor, Shree Sarvajanik College of

Pharmacy, Mehsana for their critical review, guidance and support for the work.

I bidding my sincere gratitude to Dr. T. Rajmannar, Chairman, SPIL, Vadodara, for providing

infrastructure and research facilities at Sun Pharmaceutical Industries Ltd., Vadodara

xiii

I am immensely thankful to Mr. Pradip Sahi, Head of PK/PD department and his team mates-Mr.

Jitendra Mehta, Mr. Ashish Sohoni, Mr. Harshesh Patel & Mr. Sumit for providing technical and

practical assistance to carry out qualitative analysis on HPLC-MS/MS.

I would like to thank Dr. Dwivedi, Head of Organic Synthesis department and his team members-

Mr. Arun Yadav, Mr. Hiren Shah for conducting research at their lab.

I would like to acknowledge few renowned personalities from pharmacy fraternity (Dr. Shailesh

Shah, Professor, Maliba Pharmacy College, Dr. Purvi A. Shah, Associate professor and Dr. Kalpana

G. Patel, Professor & Head, Anand Pharmacy College, Anand) who have provided their valuable

insights and prompt, timely and helpful guidance for QbD concept and solving technical and

fundamental queries associated with it.

I wish to express my heartiest thanks to Dr. Devanshu Patel and other members of the Managing

trustee of Parul University, Dr. T. Y. Pasha, Principal, Parul Institute of Pharmacy & Research, Dr.

Deepa Patel and all others at Faculty of Pharmacy, Parul University for providing me the

infrastructural and other direct or indirect support for the successful completion of research work.

It would be remiss on my part if I don’t acknowledge the help and support of all my friends; Dr.

Priyanka Patil, Dr. Jignasa Modi, Mr. Nishit Gohel and colleagues for their wonderful company,

unending inspirations, motivations, constant encouragement, technical assistance throughout the

research work.

I am also thankful to ACCUPREC Research Labs and Molecule Laboratory for providing me the

technical and infrastructural support for carrying out the HPLC method development & validation.

I am immensely thankful to Alembic Pharmaceutical Ltd., Vadodara, Unimark Remedies Ltd., Vapi,

Lupin Pharmaceutical Ltd., Vadodara, Dr. Reddy’s Laboratories, Hyderabad, RPG Life Sciences,

Ankleshwar, Mercury Laboratories Ltd., Vadodara, India for providing the gift sample of drugs

required for the study.

I would like to bid special thanks appreciation to Ms. Anjana Bunkar, Mr. Dinesh Patel, Stores in-

charge, Arjun bhai, Ms. Ankita Shinde, Mrs. Darshana Patel and to all office and laboratory staff of the

Faculty of Pharmacy, Parul University for their kind co-operation and assistance during the journey to

this stage.

I would also like to put in record my thanks to Honorable Vice Chancellor Prof. (Dr.) Navin Sheth),

Ex-Vice-Chancellor Dr. Akshai Aggarwal, I/C Registrar, Mr. J.C.Lilani, Research Coordinator, Ms.

xiv

Mona Chaurasiya, Mr. Dhaval Gohil and other Staff Members of Ph.D section for their co-

operative assistance and support.

I express my sincere thanks and apology to all those who have contributed to the success of this

work & helped me in whatsoever manner during this work & whose name I might have missed

inadvertently or those who are like countless stars in numerous galaxies.

Thanks to one & all…

Date: 22n d

March, 2019 Place: Vadodara Jignasha M. Derasari

xv

Table of Content

Sr. No.

I

Content Title Page………………………………………………………………..

P.No.

I

Ii Declaration……………………………………………………………... Iii

Iii Certificate………………………………………………………………. Iv

Iv Originality Report Certificate………………………………………….. V

V Non-Exclusive License Certificate…………………………………….. Vii

Vi Thesis Approval Certificate……………………………………………. Ix

Vii Abstract………………………………………………………………… X

Viii Acknowledgement……………………………………………………… Xii

Ix Table of Contents……………………………………………………….. Xv

X List of Abbreviation……………………………………………………. Xxvi

Xi List of Symbols………………………………………………………… Xxvii

Xii List of Figures………………………………………………………….. Xxix

Xiii List of Tables…………………………………………………………… Xxxvi

Xiv List of Appendices……………………………………………………… X1ii

Chapter 1 Introduction 1-14

1.1 Analytical method development…………………………………………. 1

1.1.1 Need for analytical method development………………………………… 2

1.1.2 Analytical method validation …………………………………………….. 2

1.1.3 Validation parameters…………………………………………………….. 3

1.2 Stability indicating analytical methods (SIAM)…..................................... 5

1.2.1 Forced degradation studies……………………………………………….. 5

1.2.2 Objectives of forced degradation studies………………………………… 6

1.3 Analytical method development and optimization………………………. 6

1.3.1 Selection of chromatographic conditions………………………………… 7

1.3.2 Other analytical methods (AMs) for developing SIAM…………………. 9

xvi

1.4 1.1 Implementation of QbD approach on AM development…………………..

10

1.5 Arthritis: An overview…………………………………………………….

12

1.6 Rationale of research work………………………………………………..

12

1.7 Definition of the problem………………………………………………….

13

1.8 Aim of the research work………………………………………………….

13

1.9 Objectives and Scope of work……………………………………………..

13

Chapter 2 Drug Profile 15-22

2.1 Diclofenac potassium (DP)………………………………………………. 15

2.2 Febuxostat (FB)………………………………………………………….. 16

2.3 Ketorolac tromethamine (KT)……………………………………………. 18

2.4 Naproxen (NP)…………………………………………………………… 19

2.5 Ibuprofen (IBU)………………………………………………………….. 20

2.6 Tramadol hydrochloride (TRM)…………………………………………. 21

Chapter 3 Literature Review 23-50

3.1 Official analytical methods for the estimation of DP……………………. 23

3.1.1 Reported analytical methods for the estimation of DP alone and

Gs

in combination with other drugs…………………………………………..

Other Dru

23

3.2 Official analytical methods for the estimation of FB……………………...

26

3.2.1 Reported analytical methods for the estimation of FB alone and

other drugs

in combination with other drugs…………………………………………..

26

3.2.2 Reported analytical methods for the estimation of DP and FB………….. 27

3.3 Official analytical methods for the estimation of KT……………………. 29

3.3.1 Reported analytical methods for the estimation of KT alone and

Gs

in combination with other drugs…………………………………………. 30

3.3.2 Reported analytical methods for the estimation of FB and KT…………...

with other Drugs

32

3.4 Official analytical methods for the estimation of NP……………………. 33

3.4.1 Reported analytical methods for estimation of NP alone and

Gs


xvii

3.5 Official analytical methods for the estimation of IBU…………………… 40

3.5.1 Reported analytical methods for the estimation of IBU alone and

other drugs


3.6 Official analytical methods for the estimation of TRM…………………. 45

3.6.1 Reported analytical methods for the estimation of TRM alone and

in combination with other drugs………………………………………….. 45

3.6.2 Reported analytical methods for the estimation of IBU and TRM combined

In Combined dosage form……………………………………………….. 49

Chapter 4 Experimental 51-93

4.1 Materials and Instruments…………………………………………. 51

4.2 Identification of standards……………………………………………...... 52

DP + FB

4.3 Development of UV spectrophotometric methods……………………….

53

4.3.1 Determination of analytical wavelength for the estimation of DP and FB

53

4.3.2 Preparation of standard stock solutions

54

4.3.2.1 Standard stock solution of DP……………………………………..

54

4.3.2.2 Standard stock solution of FB……………………………………..

54

4.3.3 Simultaneous equation method………………………………………….

54

4.3.4 First order derivative equation method………………………………….

54

4.3.5 Validation of the proposed UV spectrophotometric methods

55

4.3.5.1 Linearity and range……………………………………………….

55

4.3.5.2 Precision…………………………………………………………..

55

4.3.5.3 LOD and LOQ……………………………………………………

56

4.3.5.4 Accuracy………………………………………………………….

56

4.3.6 Analysis of tablet formulation……………………………………………

56

4.4 Development of RP-HPLC method……………………………………..

57

4.4.1 Preparation of standard stock solutions ………………………………….

57

xviii

4.4.1.1 Standard stock solution of DP and FB…………………………..

57

4.4.2 Preparation of working standards of DP and FB………………………..

57

4.4.3 Preparation of sample solutions of DP and FB…………………………..

57

4.4.4 Selection of detection wavelength……………………………………….

58

4.4.5 Preparation and selection of mobile phase…………………………………. 58

4.4.6 Final optimized chromatographic conditions………………………………..

60

4.4.7 System suitability parameters……………………………………………….

60

4.4.8 Forced degradation studies………………………………………………….

60

4.4.8.1 Preparation of standard stock solution and sample solution………….

60

4.4.8.2 Acid degradation……………………………………………………..

61

4.4.8.3 Alkali degradation………………………………………………….

……..

61

4.4.8.4 Oxidative degradation………………………………………………. 61

4.4.8.5 Thermal degradation………………………………………………… 61

4.4.8.6 Photo degradation………………………………………………….. 61

4.4.9 Validation of proposed RP-HPLC method…………………………………

62

4.4.9.1 Linearity……………………………………………………………..

62

4.4.9.2 Specificity……………………………………………………………

62

4.4.9.3 Accuracy…………………………………………………………….

62

4.4.9.4 Precision……………………………………………………………

62

4.4.9.5 Robustness…………………………………………………………..

63

4.4.9.6 LOD and LOQ………………………………………………………

63

4.4.10 Assay procedure for the estimation of DP and FB in tablet dosage form….

63

4.5 Statistical comparison of analytical methods by ANOVA and paired-t-test…

63

4.6 Application of stability indicating RP-HPLC method on HPLC-MS/MS…..

64

4.6.1 Instruments and chemicals…………………………………………………..

64

4.6.2 Preparation of Solutions……………………………………………………..

64

xix

4.6.2.1 Preparation of auto tuning solution for system stabilization…………….

………..

64

4.6.2.2 Preparation of standard and sample solutions of DP and FB…………….

64

4.6.2.2.1 Preparation of standard solution……………………………………..

64

4.6.2.2.2 Preparation of sample solutions of DP and FB…………………………

65

4.6.3 Final optimized chromatographic conditions for HPLC-MS/MS……………….

66

4.7 Implementation of QbD approach on acid degradation for a validated

stability indicating RP-HPLC method of DP and FB and its application to

acid degradation kinetic study…………………………………………………..

66

4.7.1 QbD approach and selection of an experimental design …………………………

67

4.7.2 Acid degradation study by FFD………………………………………………….

67

4.7.3 Acid degradation kinetic study…………………………………………………..

69

FB + KT

4.8 Development of RP-HPLC method………………………………………………

69

4.8.1 Preparation of standard stock solutions ………………………………………….

69

4.8.1.1 Standard stock solution of FB and KT……………………………………

69

4.8.2 Preparation of working standards of FB and KT…………………………………

70

4.8.3 Preparation of sample solutions of FB and KT…………………………………..

70

4.8.4 Selection of detection wavelength………………………………………………..

70

4.8.5 Preparation and selection of mobile phase………………………………………. 71

4.8.6 Final optimized chromatographic conditions…………………………………….

72

4.8.7 System suitability parameters……………………………………………………

73

4.8.8 Forced degradation studies………………………………………………………

73

4.8.8.1 Preparation of standard stock solution and sample solution……………

73

4.8.8.2 Acid degradation…………………………………………………………..

73

4.8.8.3 Alkali degradation………………………………………………………..

73

4.8.8.4 Oxidative degradation…………………………………………………… 73

4.8.8.5 Thermal degradation……………………………………………………… 74

xx

4.8.8.6 Photo degradation………………………………………………………. 74

4.8.9 Validation of proposed RP-HPLC method

74

4.8.9.1 Linearity……………………………………………………………….. 74

4.8.9.2 Specificity……………………………………………………………… 74

4.8.9.3 Accuracy………………………………………………………………. 75

4.8.9.4 Precision……………………………………………………………….. 75

4.8.9.5 Robustness……………………………………………………………… 75

4.8.9.6 LOD and LOQ………………………………………………………… 75

4.8.10 Assay procedure for the estimation of FB and KT in tablet dosage form……..

75

FB + NP

4.9 Development of UV spectrophotometric methods…………………………….

76

4.9.1 Determination of analytical wavelength for the estimation of FB and NP……

76

4.9.2 Preparation of standard stock solutions………………………………………..

76

4.9.2.1 Standard stock solution of FB………………………………………….

76

4.9.2.2 Standard stock solution of NP………………………………………..

76

4.9.3 Simultaneous equation method……………………………………………….

77

4.9.4 First order derivative equation method………………………………………..

77

4.9.5 Validation of the proposed UV spectrophotometric methods

77

4.9.5.1 Linearity and range…………………………………………………….

78

4.9.5.2 Precision……………………………………………………………….

78

4.9.5.3 LOD and LOQ………………………………………………………….

78

4.9.5.4 Accuracy……………………………………………………………….

78

4.9.6 Analysis of synthetic mixture…………………………………………………

79

4.10 Development of RP-HPLC method……………………………………………

79

4.10.1 Preparation of standard stock solutions ………………………………………..

79

4.10.1.1 Standard stock solution of FB ………………………………………..

79

4.10.1.2 Standard stock solution of NP …………………………………….... 79

xxi

4.10.2 Preparation of working standards of FB and NP…………………………….

80

4.10.3 Preparation of sample solutions of FB and NP………………………………

80

4.10.4 Selection of detection wavelength…………………………………………….

80

4.10.5 Preparation and selection of mobile phase…………………………………… 80

4.10.6 Final optimized chromatographic conditions………………………………….

82

4.10.7 System suitability parameters………………………………………………….

……..

82

4.10.8 Forced degradation studies…………………………………………………….

83


83

4.10.8.2 Acid degradation………………………………………………………

83

4.10.8.3 Alkali degradation……………………………………………………..

83

4.10.8.4 Oxidative degradation………………………………………………… 83

4.10.8.5 Thermal degradation………………………………………………….. 83

4.10.8.6 Photo degradation…………………………………………………….. 84


…………..

84

4.10.9.1 Linearity……………………………………………………………….. 84

4.10.9.2 Specificity……………………………………………………………… 84

4.10.9.3 Accuracy………………………………………………………………. 85

4.10.9.4 Precision………………………………………………………………. 85

4.10.9.5 Robustness……………………………………………………………. 85

4.10.9.6 LOD and LOQ……………………………………………………….. 85

4.10.10 Assay procedure for the estimation of FB and NP in synthetic mixture……….

85

4.11 Statistical comparison of analytical methods by ANOVA and paired-t-test……

86

4.12 Development of RP-HPLC method…………………………………………….

86

4.12.1 Preparation of standard stock solutions…………………………………………

86

4.12.1.1 Standard stock solution of IBU ……………………………………….

86

4.12.1.2 Standard stock solution of TRM …………………………………….. 86

4.12.2 Preparation of working standards of IBU and TRM…………………………..

87

xxii

4.12.3 Preparation of sample solutions of IBU and TRM…………………………….

87


87

4.12.5 Preparation and selection of mobile phase…………………………………… 87

4.12.6 Final optimized chromatographic conditions………………………………….

89

4.12.7 System suitability parameters…………………………………………………..

90

4.12.8 Forced degradation studies……………………………………………………

90


90

4.12.8.2 Acid degradation……………………………………………………..

90

4.12.8.3 Alkali degradation…………………………………………………….

90

4.12.8.4 Oxidative degradation………………………………………………… 91

4.12.8.5 Thermal degradation…………………………………………………… 91

4.12.8.6 Photo degradation……………………………………………………… 91


91

4.12.9.1 Linearity………………………………………………………………. 91

4.12.9.2 Specificity…………………………………………………………….. 91

4.12.9.3 Accuracy……………………………………………………………… 92

4.12.9.4 Precision……………………………………………………………… 92

4.12.9.5 Robustness……………………………………………………………. 92

4.12.9.6 LOD and LOQ………………………………………………………… 92

4.12.10 Assay procedure for the estimation of IBU and TRM on tablet

Formulation……………………………………………………………………

92

Chapter 5 Results and Discussion 94-233

5.1 Identification of standards………………………………………………………

94

5.1.1 Melting point determination……………………………………………………

94

5.1.2 Solubility study………………………………………………………………… 94

5.1.3 FT-IR spectra identification…………………………………………………… 95

DP + FB

xxiii

5.2 Development and validation of UV spectrophotometric methods……………..

101

5.2.1 Simultaneous equation method…………………………………………………

101

5.2.2 First order derivative method………………………………………………….

108

5.3 Development of RP-HPLC method…………………………………………….

113

5.3.1 Selection of detection wavelength………………………………………………

113

5.3.2 Preparation and selection of mobile phase……………………………………..

114

5.3.3 Identification of API peaks from the chromatogram at optimized

ditions

Chromatographic conditions…………………………………………………..

Con

117


118

5.3.5 Validation of proposed RP-HPLC method…………………………………….. 127

5.4 Statistical comparison of assay results by ANOVA and paired t-test…………

133

5.5 Application of stability indicating RP-HPLC method on HPLC-

MS/MS for degradation product identification……………………………… 135

5.6 QbD approach on optimization of acid degradation study by full factorial

design………………………………………………………………………….

146

5.6.1 Graphical interpretation……………………………………………………….

147

5.6.2 Optimization of critical factors by numerical settings………………………….

150

5.6.3 Acid Degradation kinetic study of DP and FB…………………………………

154

FB + KT

5.7 Development of RP-HPLC method……………………………………………..

155


155

5.7.2 Preparation and selection of mobile phase……………………………………...

156


ditions

Chromatographic conditions……………………………………………………

159


161

5.7.5 Validation of proposed RP-HPLC method……………………………………. 170

FB + NP

5.8 Development and validation of UV spectrophotometric methods

176

xxiv

5.8.1 Simultaneous equation method………………………………………………

176

5.8.2 First order derivative method………………………………………………….

184


189


189


190


Chromatographic conditions…………………………………………………. 193

5.9.4 Forced degradation studies……………………………………………………..

194


5.9.6 Statistical comparison of assay results by ANOVA and paired t-test………….. 209

IBU + TRM


211


211


211


Chromatographic conditions…………………………………………………

215


217


5.10.6 Assay……………………………………………………………………………

…

233

Chapter 6 Summary and Conclusion 234-247

6.1 DP + FB………………………………………………………………………… 234

6.1.1 UV spectrophotometric methods……………………………………………….. 234

6.1.1.1 Simultaneous equation method…………………………………………

234

6.1.1.2 First order derivative method………………………………………….

235

6.1.2 Development of stability indicating RP-HPLC method………………………..

236

6.1.2.1 Forced degradation study of DP and FB in various stressed conditions

237

6.1.3 Statistical comparison of developed methods by ANOVA……………………

238

xxv

6.1.4 Application of stability indicating RP-HPLC method on HPLC-MS/MS for

degradation product identification……………………………………………..

product identification

238

6.1.5 Optimization of acid degradation by FFD …………………………………….

239

6.1.5.1 Acid degradation kinetic study of DP and FB…………………………..

239

6.2 FB + KT 240

6.2.1 Development of stability indicating RP-HPLC method ……………………….

240

6.2.2 Forced degradation study of FB and KT in various stressed conditions………. 241

6.3 FB + NP

242

6.3.1 UV spectrophotometric methods……………………………………………… 242

6.3.1.1 Simultaneous equation method……………………………………….

242

6.3.1.2 First order derivative method………………………………………….

243

6.3.2 Development of stability indicating RP-HPLC method ……………………….

244

6.3.2.1 Forced degradation study of FB and NP in various stressed conditions

245

6.3.3 Statistical comparison of developed methods by ANOVA……………………

245

6.4 IBU + TRM 245

6.4.1 Development of stability indicating RP-HPLC method………………………..

245

6.4.1.1 Forced degradation study of IBU and TRM in various stressed

conditions……………………………………………………………………

….

246

List of References……………………………………………………… 248-268

List of Publications…………………………………………………….. 269-270

List of Appendices……………………………………………………. 271

Appendix A : A let ter of t raining and successful

completion of research work at Sun Pharmaceutical Industries Ltd.

Vadodara, GUJARAT………………………………………..

xxvi

List of abbreviations

Abbreviations Full Form

AMV Analytical Method Validation

GMP Good Manufacturing Practice

GLP Good Laboratory Practice

ICH International Conference on Harmonization

IP Indian Pharmacopeia

BP British Pharmacopeia

USP-NF United States of Pharmacopeia-National Formulary

SIAM Stability Indicating Analytical Method

API Active Pharmaceutical Ingredient

RSD Relative Standard Deviation

LOD Limit of Detection

LOQ Limit of Quantitation

RP-HPLC Reverse Phase High Performance Liquid Chromatography

CAN Acetonitrile

MeOH Methanol

UV Ultra Violet

LC-MS/MS Liquid Chromatography-Mass Spectroscopy

PDA Photo Diode Array

QbD Quality by Design

AQbD Analytical Quality by Design

DoE Design of Expert

RA Rheumatoid Arthritis

DMARDs Disease Modifying Anti Rheumatic Drugs

NSAIDs Non-Steroidal Anti Inflammatory Drugs

ANOVA Analysis of Variance

BA/BE Bio Availability/ Bio Equivalence

RT Room Temperature

FTIR Fourier Transform Infrared

xxvii

Abbreviations Full Form

NaOH Sodium Hydroxide

HCl Hydrochloric acid

KH2PO4 Potassium Dihydrogen Phosphate

H2O2 Hydrogen Peroxide

KBr Potassium Bromide

ZCP Zero Crossing Point

Conc. Concentration

Min. Minute

Obs. Observed

Abs. Absorbance

Hr. Hour

Fcal. F Calculated

Fcri. F Critical

SD Standard Deviation

xxviii

List of Symbols

Symbol Meaning

Pka Degree of Ionization

pH Negative logarithmic Hydrogen Ion Concentration

Log P Log Partition Coefficient

λmax Maximum absorbance

µg Microgram

°C Degree Centigrade

µL Micro Liter

mL Milli Liter

Mg Milligram

g/moL Gram per Mole

K Degradation Constant

A Arrhenius Energy Factor

Rt Retention Time

Rs Resolution between Peaks

Tf Tailing Factor

N Theoretical Plate Number

Ea Energy of Activation

K Asymmetric Factor

T0.5 Half Life

T0.9 Shelf Life

R Gas Constant

% Percent

xxix

List of Figures

Figure No. Legend Page

No.

Chapter 1 Introduction

1.1 AQbD tools and life cycle………………………………………………………. 11

1.2 Normal and Arthritic joints of Human being……………………………………... 12

Chapter 2 Drug Profile

2.1 Structure of DP……………………………………………………………………. 15

2.2 Structure of FB……………………………………………………………………. 17

2.3 Structure of KT…………………………………………………………………… 18

2.4 Structure of NP……………………………………………………………………. 19

2.5 Structure of IBU………………………………………………………………… 20

2.6 Structure of TRM………………………………………………………………… 21

Chapter 5 Results and Discussion

5.1 Reference FTIR Spectra of DP…………………………………………………… 95

5.2 Sample FTIR Spectra of DP………………………………………………………. 96

5.3 Reference FTIR Spectra of FB……………………………………………………. 96

5.4 Sample FTIR Spectra of FB………………………………………………………. 97

5.5 Reference FTIR Spectra of KT…………………………………………………… 97

5.6 Sample FTIR Spectra of KT……………………………………………………… 97

5.7 Reference FTIR Spectra of NP…………………………………………………… 98

5.8 Sample FTIR Spectra of NP………………………………………………………. 98

5.9 FTIR spectra of IBU………………………………………………………………. 99

5.10 Sample FTIR spectra of IBU……………………………………………………… 99

5.11 Reference FTIR spectra of TRM ….. ……………………………………………. 100

5.12 Sample FTIR Spectra of TRM……………………………………………………. 100

5.13 Overlain spectra of DP and FB………………………………………………. 101

5.14 Calibration curve of DP……………………………………………………….. 102

5.15 Calibration curve of FB……………………………………………………….. 102

5.16 Calibration curve of DP……………………………………………………….. 103

xxx


5.18 Linearity overlay of DP and FB……………………………………………… 104

5.19 Overlay spectra of DP and FB……………………………………………… 108

5.20 Calibration curve of DP………………………………………………………….. 109

5.21 Calibration curve of FB………………………………………………………….. 109

5.22 UV overlay spectrum of DP and FB……………………………………………… 114

5.23 Chromatogram of standard solution of DP (45 µg/mL) and FB (18 µg/mL) using

mobile phase of methanol: water (60: 40)…………………………………………

114


mobile phase of methanol: water (80: 20)…………………………………………

115


mobile phase of methanol: KH2PO4 buffer (90: 10 v/v), pH=5.5…………………

115


mobile phase of methanol: KH2PO4 buffer (70: 30 v/v), pH=5.5…………………

115


mobile phase of acetonitrile: ammonium acetate buffer (80:20), pH=4…………..

116


mobile phase of acetonitrile: ammonium acetate buffer (53:47), pH=4…………..

116

5.29 Chromatogram of standard solution of DP (45 µg/mL) at optimized

chromatographic conditions……………………………………………………….

117

5.30 Chromatogram of standard solution of FB (18 µg/mL) at optimized

chromatographic conditions……………………………………………………….

117

5.31 Chromatogram of standard solution of DP (45 µg/mL) FB (18 µg/mL) at

optimized chromatographic conditions……………………………………………

118

5.32 Chromatogram of sample solution of DP (45 µg/mL) FB (18 µg/mL) under

normal condition at optimized chromatographic conditions………………………

119

5.33 Chromatogram of (a) blank, (b) acid degraded sample of DP (45 µg/mL), (c) FB

(18 µg/mL) and (d) sample solution of tablet. ……………………………………

120

5.34 Chromatogram of (a) blank, (b) alkali degraded sample of DP (45 µg/mL), (c)

FB (18 µg/mL) and (d) sample solution of tablet....................................................

121

5.35 Chromatogram of (a) blank, (b) oxidative degraded sample of DP (45 µg/mL),

(c) FB (18 µg/mL) and (d) sample solution of tablet..............................................

123

5.36 Chromatogram of (a) blank, (b) thermal degraded sample of DP (45 µg/mL), (c)

xxxi

FB (18 µg/mL) and (d) sample solution of tablet. 124

5.37 Chromatogram of (a) blank, (b) photo degraded sample of DP (45 µg/mL), (c)

FB (18 µg/mL) and (d) sample solution of tablet....................................................

126

5.38 Overlay chromatogram of DP and FB for linearity………………………………. 127

5.39 Linearity graph for DP……………………………………………………………. 127

5.40 Linearity graph for FB……………………………………………………………. 128

5.41 Standard chromatogram of (a) DP (45 µg/mL) (b) FB (18 µg/mL) and (C)

sample mixture under normal condition…………………………………………..

136

5.42 Chromatogram of (a) placebo for DP (b) standard DP (45 µg/mL), (c) Placebo of

FB (d) standard of FB (18 µg/mL) and (e) Placebo chromatogram of DP and FB

(f) sample mixture in (0.1N HCl) acidic condition………………………………..

138



(f) sample mixture in (1N HCl) acidic condition………………………………….

140



(f) sample mixture in (0.1N NaOH) alkaline condition…………………………..

142

5.45 Chromatogram of (a) standard DP (45 µg/mL), (b) standard FB (18 µg/mL) and

(c) sample mixture in oxidative degradation condition……………………………

143

5.46 Chromatogram of (a) standard DP (45 µg/mL), (b) standard FB (18 µg/mL) and

(c) sample mixture in thermal degradation condition……………………………..

144

5.47 Chromatogram of (a) standard DP (45 µg/mL) (b) standard FB (18 µg/mL) and

(c) sample mixture in photolytic degradation condition…………………………..

145

5.48 Perturbation plots displaying effect of conc. of HCl on % degradation of (a) DP

and (b) FB…………………………………………………………………………

148

5.49 3D response surface plots showing the effect of critical factors on % acid

degradation of DP…………………………………………………………………

148

5.50 3D response surface plots showing the effect of critical factors on % acid

degradation of FB………………………………………………………………….

149

5.51 3D Pareto charts showing the effect of critical factors on % acid degradation of

(a) DP and of (b) FB……………………………………………………………….

150

5.52 Acid degradation chromatogram of (a) Predicted condition I and of (b) Predicted

condition IX……………………………………………………………………….

152

xxxii

5.53 Individual chromatograms for predicted condition I of DP (a), and FB (b) and for

predicted condition IX of DP (c) and FB (d)………………………………….....

153

5.54 Design space overlay plot………………………………………………………… 154

5.55 First order plots for degradation of DP (I) and FB (II) in acidic stressed

conditions………………………………………………………………………….

154

5.56 Arrhenius plots for the degradation of DP (I) and FB (II) in acidic media (0.12N

HCl) and its extrapolation for prediction degradation at RT (25 °C±2°C)………

155

5.57 UV overlay spectra of FB and KT………………………………………………... 156

5.58 Chromatogram of standard solution of FB (100 µg/mL) and KT (37.5 µg/mL)

using mobile phase of water: acetonitrile (50: 50 v/v)…………………………….

156


using mobile phase of Methanol: Buffer (60:40), KH2PO4, pH=2.8 adjusted with

OPA……………………………………………………………………………….

157


using mobile phase of Buffer: ACN (50:50), pH=1.4 adjusted with OPA………

157


using mobile phase of Buffer: ACN (60:40), pH=5 adjusted with OPA…………

158


using mobile phase of ACN: Buffer (60:40), Ammonium acetate, pH=4………..

158


using mobile phase of Buffer: ACN (60:40), KH2PO4, pH=4 adjusted with OPA.

159

5.64 Chromatogram of standard solution of FB (100 µg/mL) at optimized

chromatographic conditions………………………………………………………

160

5.65 Chromatogram of standard solution of KT (37.5 µg/mL) at optimized

chromatographic conditions………………………………………………………

160

5.66 Chromatogram of standard solution of FB (100 µg/mL) and KT (37.5 µg/mL) at

optimized chromatographic conditions……………………………………………

160

5.67 Chromatogram of sample solution of FB (100 µg/mL) and KT (37.5 µg/mL)

under normal condition at optimized chromatographic conditions……………….

161

5.68 Chromatogram of (a) blank, (b) acid degraded sample of FB (100µg/mL), (c) KT

(37.5 µg/mL) and (d) sample mixture……………………………………………..

163

5.69 Chromatogram of (a) blank, (b) alkali degraded sample of FB (100 µg/mL), (c)

KT (37.5 µg/mL) and (d) sample mixture…………………………………………

164

xxxiii

5.70 Chromatogram of (a) blank, (b) oxidative degraded sample of FB (100 µg/mL),

(c) KT (37.5 µg/mL) and (d) sample mixture……………………………………

166

5.71 Chromatogram of (a) blank, (b) degraded sample of FB (100 µg/mL)…………. 167

5.72 Chromatogram of (a) blank, (b) degraded sample of FB (100 µg/mL), …………. 169

5.73 Overlay chromatogram of FB and KT for linearity……………………………… 170

5.74 Linearity graph for FB…………………………………………………………… 171

5.75 Linearity graph for KT……………………………………………………………. 171

5.76 UV overlay spectrum of FB and NP……………………………………………… 177

5.77 Calibration curve of FB…………………………………………………………… 178

5.78 Calibration curve of NP…………………………………………………………... 178

5.79 Calibration curve of FB…………………………………………………………… 179

5.80 Calibration curve of NP…………………………………………………………... 179

5.81 Linearity overlay spectrum of FB and NP………………………………………... 180

5.82 Overlaid spectra of FB and NP………………………………………………. 184


5.84 Calibration curve of NP……………………………………………………….. 185

5.85 UV overlay spectra of FB and NP…………………………………………… 189

5.86 Chromatogram of standard solution of FB (10 µg/mL) and NP (62.5 µg/mL)

using mobile phase of Methanol: KH2PO4 buffer (90: 10 v/v), pH=5.5, flow rate

0.9 mL/min………………………………………………………………………...

190


using mobile phase of Methanol: KH2PO4 buffer (70: 30 v/v), pH=5.5, flow rate

0.9 mL/min………………………………………………………………………..

190


using mobile phase of Methanol: ACN: Buffer (10:10:80), Phosphate buffer,

pH=5, flow rate 1mL/min........................................................................................

191


using mobile phase of Methanol: ACN: Buffer (35:30:35), Ammonium acetate,

pH=5, flow rate 0.9 mL/min.

191


using mobile phase of Methanol: ACN: Buffer (35:20:45), Phosphate buffer,

pH=5, flow rate 09 mL/min………………………………………………………

192

5.91 Chromatogram of standard solution of FB (10 µg/mL) …………………………. 193

xxxiv

5.92 Chromatogram of standard solution of NP (62.5 µg/mL) ………………………... 193

5.93 Chromatogram of standard solution of FB (10 µg/mL) and NP (62.5 µg/mL)…... 193

5.94 Chromatogram of sample solution of FB (10 µg/mL) and NP (62.5 µg/mL)

under normal condition at optimized chromatographic conditions………………..

194

5.95 Chromatogram of (a) blank, (b) acid degraded sample of FB (10 µg/mL), (c) NP

(62.5 µg/mL) and (d) synthetic mixture of FB and NP……………………………

196

5.96 Chromatogram of (a) blank, (b) alkali degraded sample of FB (10 µg/mL), (c)

NP (62.5 µg/mL) and (d) synthetic mixture of FB and NP……………………….

197

5.97 Chromatogram of (a) blank, (b) oxidative degraded sample of FB (10 µg/mL),

(c) NP (62.5 µg/mL) and (d) synthetic mixture of FB and NP……………………

199

5.98 Chromatogram of (a) blank, (b) thermal degraded sample of FB (10 µg/mL), (c)

NP (62.5 µg/mL) and (d) synthetic mixture of FB and NP………………………..

200

5.99 Chromatogram of (a) blank, (b) photo degraded sample of FB (10 µg/mL), (c)

NP (62.5 µg/mL) and (d) synthetic mixture of FB and NP………………………

202

5.100 Overlay chromatogram of FB and NP for linearity………………………………. 203

5.101 Linearity graph for FB……………………………………………………………. 203

5.102 Linearity graph for NP……………………………………………………………. 204

5.103 UV overlay spectra of IBU and TRM……………………………………….. 211

5.104 Chromatogram of standard solution of IBU (48 µg/mL) and TRM (6 µg/mL)

using mobile phase of methanol: water (50: 50)…………………………………

212


using mobile phase of methanol: water (80:20)…………………………………...

212


using mobile phase of methanol: water (80: 20)………………………………….

213


using mobile phase of methanol: water (30: 70)………………………………….

213


using mobile phase of methanol: 0.05 M KH2PO4 buffer (70: 30 v/v), pH=3 (1%

o-phosphoric acid)…………………………………………………………………

214


using mobile phase of methanol: 0.05 M KH2PO4 buffer (90: 10 v/v), pH=5 (1%


214


xxxv

using mobile phase of methanol: 0.05 M KH2PO4 buffer (70:30 v/v), pH=5 (1%


215

5.111 Chromatogram of standard solution of IBU (48 µg/mL)…………………………. 216

5.112 Chromatogram of standard solution of TRM (6 µg/mL)…………………………. 216

5.113 Chromatogram of standard solution of IBU (48 µg/mL) TRM…………………... 216

5.114 Chromatogram of sample solution of IBU (48 µg/mL) TRM (6 µg/mL) under

normal condition at optimized chromatographic conditions………………………

217

5.115 Chromatogram of (a) blank, (b) acid degraded sample of IBU (48 µg/mL), (c)

TRM (6 µg/mL) and (d) Sample solution of IBU (48 µg/mL) and TRM (6

µg/mL)…………………………………………………………………………….

219

5.116 Chromatogram of (a) blank, (b) alkali degraded sample of IBU (48 µg/mL), (c)


µg/mL)…………………………………………………………………………….

220

5.117 Chromatogram of (a) blank, (b) oxidative degraded sample of IBU (48 µg/mL),

(c) TRM (6 µg/mL) and (d) Sample solution of IBU (48 µg/mL) and TRM (6

µg/mL)…………………………………………………………………………….

222

5.118 Chromatogram of (a) blank, (b) thermal degraded sample of IBU (48 µg/mL), (c)


µg/mL)………………………………………………………………………….....

224

5.119 Chromatogram of (a) blank, (b) photo degraded sample of IBU (48 µg/mL), (c)


µg/mL)…………………………………………………………………………….

225

5.120 Overlay chromatogram of IBU and TRM for linearity…………………………… 227

5.121 Linearity graph for IBU…………………………………………………………... 227

5.122 Linearity graph for TRM………………………………………………………….. 227

xxxvi

List of Tables

Table No. Caption Page

No.

Chapter 2 Drug Profile

2.1 Drug Profile – DP……………………………………………………….. 16

2.2 Drug Profile – FB……………………………………………………….. 17

2.3 Drug Profile – KT……………………………………………………….. 18

2.4 Drug Profile – NP……………………………………………………….. 19

2.5 Drug Profile – IBU……………………………………………………… 20

2.6 Drug Profile – TRM…………………………………………………….. 21

Chapter 4 Experimental

4.1 List of Materials used…………………………………………………… 51

4.2 List of Instruments used………………………………………………… 52

4.3 Trials of different mobile phases and their observation for DP and FB.. 58

4.4 Details of instrument and chemicals used for HPLC-MS/MS………… 64

4.5 Optimized Chromatographic Conditions for HPLC-MS/MS…………. 66

4.6 Observed responses in FFD for eight analytical trials………………….. 68

4.7 Trials of different mobile phases and their observation for FB and KT.. 71

4.8 Trials of different mobile phases and their observations for FB and NP.. 81

4.9 Trials of different mobile phases and their observation for IBU and

TRM……………………………………………………………………..

88

Chapter 5 Results and Discussion

5.1 Melting Point Determination……………………………………………. 94

5.2 Results of solubility test………………………………………………… 95

5.3 FTIR Absorption characteristic bands for DP………………………….. 96

5.4 FTIR absorption characteristic bands of FB……………………………. 97

5.5 FTIR Absorption characteristic bands of KT .............................................. Error! Bookmark not defined. 97

5.6 FTIR absorption characteristic bands of NP……………………………. 98

5.7 FTIR absorption characteristic bands of IBU…………………………… 99

5.8 FTIR absorption characteristic bands of TRM…………………………. 100

xxxvii

5.9 Calibration readings of DP and FB at their respective wavelength ……. 101

5.10 Calibration readings of DP and FB at 314 nm and 276 nm…………….. 102

5.11 Repeatability data of DP and FB………………………………………... 104

5.12 Intraday Precision data of DP and FB…………………………………... 105

5.13 Inter-day Precision data of DP and FB………………………….. 105

5.14 LOD and LOQ data for DP and FB…………………………………….. 106

5.15 % Recovery of DP………………………………………………………. 106

5.16 % Recovery of FB………………………………………………………. 107

5.17 Assay data of DP and FB……………………………………………….. 107

5.18 Calibration readings of DP and FB at their respective wavelength 109

5.19 Repeatability data of DP and FB……………………………………….. 110

5.20 Intraday Precision data of DP and FB………………………………….. 110

5.21 Inter-day Precision data of DP and FB…………………………………. 111


5.23 % Recovery of DP……………………………………………………… 112

5.24 % Recovery of FB………………………………………………………. 112

5.25 %Assay data of DP and FB…………………………………………… 112

5.26 Final Optimized Chromatographic Conditions for the estimation of DP

and FB………………………………………………………………….

117

5.27 System suitability parameters of the proposed method………………… 118

5.28 Result of acid degradation of tablet formulation……………………… 120

5.29 Result of acid degradation of tablet formulation……………………… 121

5.30 Result of oxidative degradation of tablet formulation………………… 123

5.31 Result of thermal degradation of tablet formulation………………….. 124

5.32 Result of photo degradation of tablet formulation……………………… 126

5.33 Summary of % forced degradation of DP and FB……………………… 126

5.34 Summary of the forced degradation product of DP and FB…………….. 126

5.35 Linearity data for DP and FB…………………………………………… 128

5.36 Specificity data of DP and FB…………………………………………... 128

5.37 % Recovery data of DP…………………………………………………. 129

5.38 % Recovery data of FB…………………………………………………. 129

5.39 Repeatability data of DP and FB………………………………………... 130

5.40 Intraday precision data of DP and FB…………………………………... 130

xxxviii

5.41 Inter-day precision data of DP and FB…………………………………. 131

5.42 Robustness data of DP and FB…………………………………………. 132


5.44 % Assay data of DP and FB……………………………………………. 133

5.45 Statistical comparisons of data by ANOVA-single factor……………… 133

5.46 Paired t-test summaries of three methods for FB………………………. 134

5.47 Summary of % degradation of DP and FB in different forced

degradation conditions…………………………………………………..

145

5.48 Range of factors selected for FFD………………………………………. 146

5.49 ANOVA results on acid degradation of DP…………………………….. 146

5.50 ANOVA results on acid degradation of FB ................................................ Error! Bookmark not defined. 146

5.51 Numerical setting for constraints……………………………………… 150

5.52 Numerical setting of various constraints for optimization……………… 151

5.53 Result data of predicted conditions and performed trials……………….. 153

5.54 Degradation rate constant, half-life and shelf life for DP and FB………. 155

5.55 Degradation kinetic profiles in acidic media at RT±2°C……………….. 155

5.56 Final Optimized Chromatographic Conditions for the estimation of FB

and KT…………………………………………………………………...

159

5.57 System suitability parameters of the proposed method…………………. 161

5.58 Result of acidic degradation on sample mixture……………………… 163

5.59 Result of alkali degradation on sample mixture………………………… 164

5.60 Result of oxidative degradation on sample mixture…………………….. 166

5.61 Result of thermal degradation on sample mixture………………………. 167

5.62 Result of photo degradation on sample mixture………………………… 169

5.63 Summary of the forced degradation of FB and KT……………………... 169

5.64 Summary of the forced degradation product of FB and KT in sample

mixture ………………………………………………………………….

169

5.65 Linearity data for FB and KT…………………………………………… 171

5.66 Specificity data of FB and KT………………………………………….. 172

5.67 % Recovery data of FB………………………………………………… 172

5.68 % Recovery data of KT………………………………………………… 173

5.69 Repeatability data of FB and KT………………………………………... 173

5.70 Intraday precision data of FB and KT………………………………….. 174

xxxix

5.71 Inter-day precision data of FB and KT………………………………… 174

5.72 Robustness study of FB and KT………………………………………… 175

5.73 LOD and LOQ data of FB and KT……………………………………… 176

5.74 Assay of tablet formulation…………………………………………… 176

5.75 Calibration readings of FB and NP at their respective wavelength…….. 177

5.76 Calibration readings of FB and NP at each other’s wavelength………… 178

5.77 Repeatability data of FB and NP………………………………………... 180

5.78 Intraday Precision data of FB and NP…………………………………... 181

5.79 Inter-day Precision data of FB and NP .......................................................... Error! Bookmark not defined. 181

5.80 LOD and LOQ data for FB and NP……………………………………... 182

5.81 % Recovery of FB………………………………………………………. 182

5.82 % Recovery of NP………………………………………………………. 183

5.83 % Assay of FB and NP…………………………………………………. 183

5.84 Calibration readings of FB and NP at their respective wavelength….. 184

5.85 Repeatability data of FB and NP……………………………………….. 186

5.86 Intraday Precision data of FB and NP………………………………….. 186

5.87 Inter-day Precision data of FB and NP…………………………………. 187

5.88 LOD and LOQ data for FB and NP……………………………………………………… 187

5.89 % Recovery of FB………………………………………………………. 188

5.90 % Recovery of NP………………………………………………………. 188

5.91 Analysis of synthetic mixture…………………………………………… 189

5.92 Final Optimized Chromatographic Conditions for the proposed method 192

5.93 System suitability parameters of the proposed method………………… 194

5.94 Result of acid degradation of synthetic mixture ............................................ Error! Bookmark not defined. 196

5.95 Result of alkali degradation of synthetic mixture………………………. 197

5.96 Result of oxidative degradation of synthetic mixture…………………... 199

5.97 Result of thermal degradation of synthetic mixture……………………. 200

5.98 Result of photo degradation of synthetic mixture .......................................... Error! Bookmark not defined. 202

5.99 Summary of % forced degradation of FB and NP in synthetic mixture ... 202

5.100 Summary of the forced degradation product of FB and NP……………. 202

5.101 Linearity data for FB and NP…………………………………………… 204

5.102 Specificity data of FB and NP ....................................................................... Error! Bookmark not defined. 204

xl

5.103 Recovery data of FB…………………………………………………….. 205

5.104 Recovery data of NP……………………………………………………. 205

5.105 Repeatability data of FB and NP……………………………………….. 206

5.106 Intraday precision data of FB and NP…………………………………... 206

5.107 Inter-day precision data of FB and NP…………………………………. 207

5.108 Robustness study of FB and NP……………………………………….. 208

5.109 LOD and LOQ data for FB and NP……………………………………... 208

5.110 Assay of synthetic mixture……………………………………………… 209

5.111 Statistical comparisons of data by ANOVA-single factor……………… 209

5.112 Paired t-test summaries of three methods for NP………………………. 210

5.113 Final Optimized Chromatographic Conditions…………………………. 215

5.114 System suitability parameters…………………………………………… 217

5.115 Result of acid degradation in sample mixture of IBU and TRM ………. 219

5.116 Result of alkali degradation in sample mixture of IBU and TRM ……... 221

5.117 Result of oxidative degradation in sample mixture of IBU and TRM …. 222

5.118 Result of thermal degradation in sample mixture of IBU and TRM …… 224

5.119 Result of photo degradation in sample mixture of IBU and TRM …….. 225

5.120 Summary of % forced degradation of IBU and TRM…………………. 226

5.121 Summary of the forced degradation product of IBU and TRM in sample

mixture………………………………………………………………….

226

5.122 Linearity data for IBU and TRM……………………………………….. 227

5.123 Specificity data of IBU and TRM………………………………………. 228

5.124 % Recovery data of IBU………………………………………………... 229

5.125 % Recovery data of TRM……………………………………………….. 229

5.126 Repeatability data of IBU and TRM .............................................................. Error! Bookmark not defined. 230

5.127 Intraday precision data of IBU and TRM……………………………… 230

5.128 Inter-day precision data of IBU and TRM……………………………… 231

5.129 Robustness studies of IBU and TRM…………………………………… 232

5.130 LOD and LOQ data for IBU and TRM…………………………………. 232

5.131 Assay of tablet formulation……………………………………………... 233

Chapter 6 Summary and Conclusions

6.1 Summary of validation parameters……………………………………… 235

xli




6.5 Summary of % forced degradation of DP and FB……………………... 237

6.6 Summary of % degradation of DP and FB in different forced

degradation conditions…………………………………………………..

238

6.7 Degradation rate constant, half-life and shelf life for FB and DP 240

6.8 Degradation kinetic profiles in acidic media at RT±2°C………….. 240



6.11 Summary of % forced degradation of FB and KT…………………….. 242

6.12 Summary of validation parameters of simultaneous equation method…. 242



6.15 Summary of validation parameters ……………………………………... 244

6.16 Summary of % forced degradation of FB and NP…………………….. 245



6.19 Summary of % forced degradation of IBU and TRM…………………. 246

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List of Appendices

Appendix A:

A letter of training and successful completion of research work at Sun Pharmaceutical Industries

Ltd., Vadodara, Gujarat......................................................................................... 271

CHAPTER 1 Introduction

119997290014 /Gujarat Technological University Page 1

CHAPTER 1

Introduction

Analytical chemistry is one of the divisions of chemistry that deals with separation,

identification and quantification of chemical constituents in natural and synthetic

materials. It is mainly divided into two categories, qualitative analysis that confirms the

quality of sample whereas quantitative analysis assesses the amount of certain elements in

the sample.

Pharmaceutical analysis [1-3] plays a very significant role in the investigation of

pharmaceutical formulations and bulk drugs. Quick growth in pharmaceutical industries

[4] and manufacturing of drugs in and around the world bring forward a rise in inevitable

demand to seek innovative and efficient analytical techniques in the pharmaceutical

industries. As a result, analytical method (AM) development has become the basic activity

of analysis.

Progress in technical and concrete AM‘s has been ensued by the use of modern analytical

instruments. They are the key element to attain high quality and consistent analytical data

in the process. Advancements in the field of analytical chemistry have reduced the cost

and time of analysis [5]. It also enhanced exactness and accurateness of developed

methods. Systematic procedures are established and validated for active pharmaceutical

ingredients, excipients, rela

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Development and Validation of Analytical Methods for · test. Simple, accurate, precise, robust and reproducible stability indicating RP-HPLC method has been conducted for combined