development and validation of liquid chromatographic

Development and Validation of Liquid Chromatographic Methods for Anti-

Hyperlipidemic Drugs in Binary Combinations

1864

MUHAMMAD ASHFAQ SESSION 2004-2008

13-GCU-PhD-CHEM-04

DEPARTMENT OF CHEMISTRY GC UNIVERSITY LAHORE PAKISTAN



Submitted to GC University Lahore in partial fulfillment of the requirements

for the award of degree of

DOCTOR OF PHILOSOPHY

in

CHEMISTRY

by

Muhammad Ashfaq Session 2004-2008

13-GCU-PhD-Chem-04


DECLARATION

I Muhammad Ashfaq Reg No 13-GCU-PhD-CHEM-04 student of PhD in

the subject of Chemistry Session 2004-2008 hereby declare that the matter

printed in the thesis titled ldquoDevelopment and Validation of Liquid

Chromatographic Methods for Anti-Hyperlipidemic Drugs in Binary

Combinationsrdquo is my own research work and has not been printed published and

submitted as research work thesis publication or in any form in any University

Research Institution etc in Pakistan or abroad

Dated Muhammad Ashfaq

RESEARCH COMPLETION CERTIFICATE Certified that the research work contained in this thesis titled ldquoDevelopment and

Validation of Liquid Chromatographic Methods for Anti-Hyperlipidemic Drugs in

Binary Combinationsrdquo has been carried out and completed by Mr Muhammad

Ashfaq Reg No 13-GCU-PhD-CHEM-04 under my supervision during his PhD

(Chemistry) studies in the laboratories of the Department of Chemistry

______

Dated Supervisor

Prof Dr Islam Ullah Khan

Submitted Through

Prof Dr M Saeed Iqbal Chairperson Department of Chemistry GC University Lahore Controller of Examinations GC University Lahore

CERTIFICATE OF EXAMINERS

Certified that the quantum and quality of the research work contained in this thesis

titled ldquoDevelopment and Validation of Liquid Chromatographic Methods for Anti-

Hyperlipidemic Drugs in Binary Combinationsrdquo is adequate for the award of the

degree of Doctor of Philosophy

Prof Dr Islam Ullah Khan External Examiner Supervisor

Prof Dr M Saeed Iqbal Chairperson Department of Chemistry GC University Lahore

Dedicated

To

My father mother brothers sisters my wife and my son

Whose love is always with me

ACKNOWLEDGEMENTS

All praises to almighty Allah Who endowed the man with intelligence knowledge sight

to observe and mind to think Peace and blessings of Allah almighty be upon the Holy

Prophet Hazrat Muhammad (Salal La Ho Alaihey Wassalam) who exhorted his followers

to seek for knowledge from cradle to grave

My heartful gratitude is to my learned research mentor Dr Islam Ullah Khan

Professor Department of Chemistry GC University Lahore His keen interest scholarly

guidance and encouragement were a great help throughout the course of this research

work

I feel great pleasure in expressing my sincere gratitude and profound thanks to the most

respected honorable Prof Dr Muhammad Saeed Iqbal Chairperson Department of

Chemistry GC University Lahore for providing all facilities and all the necessary

guidance to complete this research work

I am much obliged to Ghulam Mustafa Assistant Prfessor Department of Chemistry

University of Gujrat Gujrat and Mr Nauman Malik my MSc friend (Now a Canadian

immigrant) who always encouraged me throughout the research work and during

compilation of this thesis

My cordial prays are for my father mother brothers sisters and wife for their continuous

encouragement and support Their everlasting love guidance and encouraging passion

will remain with me Insha Allah till my last breath I would not forget to mention my son

Muhammad Aaliyan who was born during my PhD research and my nephew and nice

Their love always guided me in completing my research

My heart-felt thanks are due to all my teachers friends and those who contributed in this

research work in any way especially my PhD fellows Mr Muhammad Nadeem Asghar

Mr Muhammad Nadeem Arshad Mr Muhammad Shafiq Mr Shahzad Sharif and

MPhil fellows Ms Tayyaba Kausar and Mr Sajid Jilani

I am also very much thankful to Mr Syed Shanaz Qutab Mr Naeem Razzaq (Schazoo

Labs) Mr Asim Ms Shazia and Ms Iram (Irza Pharma) They not only encouraged me

during my study but also providing the necessary facilities to carry on some of the work

I express my feelings of gratitude to all the members of non-teaching staff of the

Department especially Mr Hanif Mr Rahmat Mr Mohy-ud-Din Mr Abid and Mr

Abdul Ghafoor for their constant help

Throughout the course of my PhD I have had help from numerous people I have tried to

thank everybody but if I have missed someone I am sorry and it is just down to my

forgetfulness

Muhammad Ashfaq

Abbreviations

Abbreviations

LDL = Low density lipoprotein HDL = High density lipoprotein VLDL = Very Low density lipoprotein WHO = World Health Organization LPL = Lipoprotein Lipase Acetyl CoA = Acetyl Coenzyme A IDL = Intermediate density lipoprotein NCEP = National Cholesterol Education Program HMGR = 3-hydroxy- 3-methylglutaryl-coenzyme A reductase CYP = Cytochrome P-450 SREBP = sterol regulatory element binding proteins PPAR = Peroxisome proliferator activated receptor PPRE = Peroxisome proliferator responsive elements FDA = Food and Drug Administration of the United States LDL-C = Low density lipoprotein cholesterol RP-HPLC = Reverse phase high performance liquid chromatography HPTLC = High performance thin layer chromatography ICH = International Conference on Harmonization LOD = Limits of detection LOQ = Limits of quantitation RSD = Relative standard deviation ODS = Octadecyl Silane ESI = Electrospray Ionization MS = Mass spectrometry MS-MS = Tandem Mass spectrometry IS = Internal standard

THF = Tetrahydrofuran CV = Coefficient of variation CN = Cyano

OD = Optical density SPE = Solid phase extraction DEC = disposable extraction cartridges MRM = Multiple reactions monitoring DW = Distilled Water ACN = Acetonitrile

LIST OF TABLES

xiv

List of Tables

TAB DESCRIPTION PAGE 41 Recovery experiments of the proposed HPLC method 97

42 Within-day and Between-day precision of the proposed HPLC method 97

43 Selectivity of the proposed HPLC method 98

44 Stability study of atorvastatin calcium and ezetimibe in solution 99

45 Robustness study of Atorvastatin 100

46 Robustness study of Ezetimibe 100

47 Analysis of atorvastatin calcium and ezetimibe in tablets 102

48 Results of recovery experiments of the proposed HPLC method 107

49 Within and Between-day precision of the proposed HPLC method 107


411 Stability study of ezetimibe and simvastatin in solution 108


413 Robustness study of Simvastatin 110

414 Results of analysis of ezetimibe and simvastatin in tablets 111

415 Accuracy of the proposed HPLC method 116

416 Precision of the proposed HPLC method 116


418 Stability study of gemfibrozil and simvastatin in solution 119

419 Robustness study of Gemfibrozil 121


LIST OF TABLES

xv


422 Within-day and between day precision of the proposed HPLC method 126


424 Stability study of Ezetimibe and Fenofibrate in solution 128


426 Robustness study of Fenofibrate 129

427 Analysis of Ezetimibe and Fenofibrate in tablets 131




431 Stability study of Ezetimibe and Lovastatin in solution 140


433 Robustness study of Lovastatin 141




437 Stability study of Atorvastatin and Gemfibrozil in solution 150






443 Stability study of Rosuvastatin and ezetimibe in solution over 72 hours 159

LIST OF TABLES

xvi

444 Robustness study of Rosuvastatin 161


446 Results of analysis of Rosuvastatin and ezetimibe in tablets 162

LIST OF FIGURES

xvii

List of Figures

FIG DESCRIPTION PAGE 11 Chemical structure of atorvastatin calcium 17 12 Chemical structure of simvastatin 18 13 Chemical structure of lovastatin 20 14 Chemical structure of rosuvastatin calcium 21 15 Chemical structure of gemfibrozil 22 16 Chemical structure of Fenofibrate 24 17 Chemical structure of ezetimibe 25 41 Chromatograms of atorvastatin calcium and ezetimibe 96

reference substance

42 Chromatograms of atorvastatin calcium and ezetimibe Tablets 96 43 Chromatograms of ezetimibe and simvastatin reference substance 105 44 Chromatograms of ezetimibe and simvastatin Tablets 105 45 Chromatograms of Gemfibrozil and simvastatin reference substance 115 46 Chromatograms of Gemfibrozil and simvastatin in a synthetic mixture 117 47 Chromatogram of ezetimibe and fenofibrate reference substance 125 48 Chromatogram of ezetimibe and fenofibrate Tablets 125 49 Chromatogram of ezetimibe and lovastatin reference substance 135 410 Chromatogram of ezetimibe and lovastatin in synthetic mixture form 137 411 Chromatogram of Atorvastatin and gemfibrozil reference substance 145

412 Chromatograms of Atorvastatin and gemfibrozil in synthetic mixture form 147

LIST OF FIGURES

xviii

413 Scheme showing degradation of atorvastatin in the presence of hydrogen peroxide 152

414 X-Ray structure of atorvastatin degradation product produced

under oxidative stress 152

415 Chromatograms of rosuvastatin and Ezetimibe under basic stress 155 416 Chromatograms of rosuvastatin and Ezetimibe under oxidative stress 155

LIST OF PUBLICATIONS

xiii

List of Publications 1 SS Qutab S N Razzaq I U Khan M Ashfaq and Z A Shuja Simultaneous

determination of Atorvastatin Calcium and Ezetimibe in pharmaceutical formulations using liquid Chromatography Journal of Food and Drug Analysis (Taiwan) 2007 15 139-144

(Impact Factor 0568)

2 M Ashfaq I U Khan M N Asghar Development and validation of liquid chromatographic method for gemfibrozil and simvastatin in binary combination Journal of Chilean Chemical Society 2008 53(3) 1617-1619


3 M Ashfaq M N Tahir I U Khan M S Iqbal M N Arshad Degradation of

atorvastatin (1R2S4S5S)-4-(4-fluorophenyl)- 2-hydroperoxy-4-hydroxy-2-isopropyl-N5-diphenyl-36- dioxabicyclo[310]hexane-1-carboxamide Acta Cryst E 2008 E64 o1548


4 M Ashfaq I U Khan S S Qutab S N Razzaq HPLC determination of ezetimibe and simvastatin in pharmaceutical formulations Journal of Chilean Chemical Society 2007 52 1220-1223


ABSTRACT

i

ABSTRACT

In the present dissertation stress was applied to determine anti-hyperlipidemic drugs in

combination form especially in binary combinations using simple sensitive and

economic HPLC methods Seven HPLC methods have been developed for Atorvastatin-

Ezetimibe Ezetimibe-Simvastatin Gemfibrozil-Simvastatin Ezetimibe-Fenofibrate

Ezetimibe-Lovastatin Atorvastatin-Gemfibrozil and Rosuvastatin-Ezetimibe dual

formulations

The first HPLC method was developed for the simultaneous determination of atorvastatin

and ezetimibe in tablet formulations Chromatographic separation was achieved on a 250

times 46 mm 5micro Hypersil phenyl-2 column at 242 nm using a mixture of 01 M ammonium

acetate (pH 65) and acetonitrile in the ratio of 2872 (vv) as a mobile phase The method

was linear in the concentration range of 12-52 microgml for both atorvastatin and ezetimibe

with correlation coefficient between 09966 and 09993 The total run time was less than

5 min

The second method which was developed was for the simultaneous determination of

ezetimibe and simvastatin in pharmaceutical formulations Chromatographic separation

was performed on a Merck C18 column at a wavelength of 240 nm using a mixture of

01M ammonium acetate buffer pH 50 and acetonitrile in the ratio of (3070 vv) The

method results in excellent separation with good resolution between the two analytes

The within day variation was between 028 and 110 and between day variation was

between 056 and 132 The recovery was greater than 9912 with RSD less than

138

In the third method conditions were optimized to develop a simple sensitive and

validated HPLC method to determine gemfibrozil and simvastatin simultaneously in

synthetic mixture form Chromatographic separation was achieved on a C-18 column

using a mixture of 01 M ammonium acetate pH 50 and acetonitrile in the ratio of 1585

(vv) at a wavelength of 237 nm Linearity of the method was found to be in the

concentration range of 60-420 microgml for gemfibrozil and 1-7 microgml for simvastatin with

correlation coefficient greater than 09999

The fourth method developed for available binary combination was the simultaneous

ABSTRACT

ii

determination of ezetimibe and fenofibrate in tablets Isocratic chromatography was

performed on a Merck C-18 column using a mixture of 01 M ammonium acetate pH 50

and acetonitrile in the ratio of (2575 vv) at a flow rate of 15 mlmin The detection was

carried out at a wavelength of 240 nm using a photodiode array detector The method was

linear in the concentration range of 08-40 microgml for ezetimibe and 128-640 microgml for

fenofibrate

The fifth method developed was for the simultaneous determination of ezetimibe and

lovastatin in synthetic mixture form Chromatographic separation was performed on a C-

18 column using a mixture of 01M ammonium acetate buffer pH 50 and acetonitrile in

the ratio of (2872 vv) The detection was carried out at a wavelength of 240 nm using a

photodiode array detector The method was linear in the concentration range of 02-100

microgml for ezetimibe and 04-200 microgml for lovastatin The within day variation was

between 032 and 122 and between day variation was between 098 and 163 The

recovery was greater than 102 with RSD less than 15 Later the method was also

applied for the determination of these two drugs in spiked human plasma No plasma

peaks interfered with the peaks of active anaytes which means it can also be used for the

determination in human plasma

The separation procedure for the simultaneous determination of atorvastatin and

gemfibrozil in synthetic mixture form was also developed Chromatographic separation

was achieved on a C-18 column using a mixture of 01 M ammonium acetate pH 50 and

acetonitrile in the ratio of 4555 (vv) at a wavelength of 240 nm Linearity of the method

was found to be in the concentration range of 01-20 microgml for atorvastatin and 6-1200

microgml for gemfibrozil with correlation coefficient 09997 for atorvastatin and 09976 for

gemfibrozil The elution time for the two components was less than twelve minutes

Forced degradation study was also applied to both the drugs individually and in

combination form During the forced degradation study under oxidative stress a novel

degradation product was also isolated in crystalline form Later the developed method

under the same chromatographic conditions was also applied for the determination of

these two drugs in spiked human plasma No plasma peaks interfered with the peaks of

active anaytes which means it can also be used for the determination in human plasma

ABSTRACT

iii

The pair for the simultaneous quantification of rosuvastatin and ezetimibe was also

proceeded Chromatographic separation was performed on a C18 column at a wavelength

of 240 nm using a mixture of 1 phosphoric acid solution and acetonitrile in the ratio of

(4060 vv) The method was linear in the concentration range of 08 to 160 microgml for

rosuvastatin and 02 to 40 microgml for ezetimibe with correlation coefficient equal to

09993 for rosuvastatin and 09996 for ezetimibe The within day precision was between

095 and 151 and between day precision was between 128 and 205

All the developed methods were validated in terms of linearity accuracy recovery

precision robustness specificity and LODLOQ values The total eluting time for every

method was less than twelve minutes The results obtained for each method indicate that

they can be reliably used for the simultaneous determination of dual components present

in each study

TABLE OF CONTENTS

iv

Table of Contents

DESCRIPTION PAGE

Abstract i-iii

List of Publications xiii

List of Tables xiv-xvi

List of Figures xvii-xviii

CHAPTER 1 INTRODUCTION 1-34

11 What is Hyperlipidemia 01

12 Causes of hyperlipidemia 01

13 Symptoms and diagnoses of Hyperlipidemia 02

14 Classes of Lipoprotein 03

141 Chylomicrons 03

142 Very-Low-Density Lipoproteins (VLDL) 03

143 Low-Density Lipoproteins (LDL) 03

144 High-Density Lipoproteins (HDL) 04

15 Classification of hyperlipidemia 04

151 Hyperlipoproteinemia Type-I 04

152 Hyperlipoproteinemia Type-II 04

1521 Hyperlipoproteinemia Type-IIa 05

1522 Hyperlipoproteinemia Type-IIb 05

153 Hyperlipoproteinemia Type-III 05

154 Hyperlipoproteinemia Type-IV 05

155 Hyperlipoproteinemia Type-V 05

16 Classification of Antihyperlipidemic Drugs 06

161 Statins 06

1611 Mechanism of Action of Statins 08

1612 Adverse effects of statin therapy 08

162 Fibrates 09

1621 Mechanism of Action of Fibrates 09

TABLE OF CONTENTS

v

1622 Adverse effects of Fibrate therapy 11

163 Cholesterol absorption Inhibitors 11

1631 Mechanism of Action of Ezetimibe 11

1632 Adverse Effects of Ezetimibe 12

17 Combination therapy for Hyperlipidemia 12

171 Statin and ezetimibe combination therapy 13

172 Statin and fibrate combination therapy 14

173 Ezetimibe and fibrate combination therapy 15

18 Antihyperlipidemic Drugs 16

181 Atorvastatin Calcium 16

182 Simvastatin 18

183 Lovastatin 19

184 Rosuvastatin Calcium 20

185 Gemfibrozil 22

186 Fenofibrate 23

187 Ezetimibe 24

19 High Performance Liquid Chromatography (HPLC) 26

191 Types of Detectors Used In HPLC 26

192 Chromatographic Terms 27

1921 Chromatogram 27

1922 Column 27

1923 Column Performance 27

1924 Eluent 27

1925 Flow Rate 27

1926 Peak 27

1927 Resolution 27

1928 Retention Factor 27

1929 Retention Time 28

19210 Tailing 28

193 Method Validation on HPLC 28

TABLE OF CONTENTS

vi

110 Quantitative Analysis 28

1101 Quantitative Instrumental Analysis 29

111 Statistics 30

1111 Average 30

1112 Standard Deviation 30

1113 Relative Standard Deviation 30

1114 Linear Regression Analysis 31

1115 Correlation Coefficients 31

112 Manufacturing Process of Tablet Dosage form 32

1121 What is a Tablet 32

1122 Manufacturing Process 32

11221 Granulation 32

112211 Wet granulation 33

112212 Dry granulation 33

11222 Tablet Compression 33

11223 Tablet coating 33

113 Aims and objective of the research work 34

CHAPTER 2 LITERATURE SURVEY 35-62

21 Analytical Methods for Atorvastatin 35

22 Analytical Methods for Simvastatin 41

23 Analytical Methods for Lovastatin 46

24 Analytical Methods for Rosuvastatin 50

25 Analytical Methods for Gemfibrozil 52

26 Analytical Methods for Fenofibrate 55

27 Analytical Methods for Ezetimibe 59

CHAPTER 3 EXPERIMENTAL WORK 63-92

31 Solvents 63

32 Chemicals 63

33 Analytical equipments 64

34 Glass Apparatus 64

TABLE OF CONTENTS

vii

35 Atorvastatin calcium and Ezetimibe 66

351 Preparation of mobile phase 66

352 Preparation of standard solution 66

353 Linearity 66

354 Limits of detection and Limits of quantitation (LOD and LOQ) 66

355 Accuracy 67

356 Precision 67

357 Selectivity 67

358 Robustness 68

359 Forced Degradation study 68

3510 Stability of Solutions 68

3511 Application of the method 68

3512 HPLC Set Up 69

36 Ezetimibe and Simvastatin 70



363 Linearity 70

364 Limit of detection and Limits of quantitation 70

365 Accuracy 70

366 Precision 71

367 Selectivity 71

368 Robustness 71

369 Forced degradation study 71

3610 Stability of solutions 72

3611 Application of the Method 72

3612 HPLC Set Up 73

37 Gemfibrozil and Simvastatin 74



373 Linearity 74


TABLE OF CONTENTS

viii

375 Accuracy 74

376 Precision 75

377 Selectivity 75

378 Robustness 75



3711 HPLC Set Up 76

38 Ezetimibe and Fenofibrate 77


382 Preparation of standard solutions 77

383 Linearity 77

384 Limit of detection and limit of quantitation 77

385 Accuracy 77

386 Precision 78

387 Selectivity 78

388 Robustness 78




3812 HPLC Set Up 80

39 Ezetimibe and Lovastatin 81



393 Linearity 81

394 Limits of detection and Limits of quantitation 81

395 Accuracy 81

396 Precision 82

397 Selectivity 82

398 Robustness 82

399 Forced Degradation Study 83


TABLE OF CONTENTS

ix

3911 HPLC Set Up 84

310 Atorvastatin and Gemfibrozil 85



3103 Linearity 85


3105 Accuracy 86

3106 Precision 86

3107 Selectivity 86

3108 Robustness 86



31011 HPLC Set Up 88

311 Rosuvastatin and Ezetimibe 89



3113 Preparation of sample solution 89

3114 Linearity 89


3116 Accuracy 90

3117 Precision 90

3118 Selectivity 90

3119 Robustness 91




CHAPTER 4 RESULTS AND DISCUSSION 93-164


411 Method Development and Optimization 93

412 Method validation 93

4121 Linearity 93

TABLE OF CONTENTS

x


4123 Accuracy 94

4124 Precision 94

4125 Selectivity 95


4127 Robustness 95






4221 Linearity 104

4222 Limit of detection and Limit of quantitation 104

4223 Accuracy 106

4224 Precision 106

4225 Selectivity 106


4227 Robustness 109






4321 Linearity 113

4322 Limits of detection and Quantitation 113

4323 Accuracy 113

4324 Precision 114



4327 Robustness 120


TABLE OF CONTENTS

xi


441 Method development and Optimization 122

442 Method Validation 122

4421 Linearity 122


4423 Accuracy 123

4424 Precision 123



4427 Robustness 124

4428 Forced degradation Study 124





4521 Linearity of the method 133

4522 Limit of detection and quantitation 133

4323 Accuracy 134

4524 Precision 134



4527 Robustness 139





4621Linearity 143

4622Limit of detection and quantitation 143

4623 Accuracy 144

4624 Precision 144

TABLE OF CONTENTS

xii



4627 Robustness 149





4721 Linearity 153


4723 Accuracy 156

4724 Precision 156



4727 Robustness 160



48 Conclusion 163

CHAPTER 5 REFERENCES 165-181

CHAPTER 1 INTRODUCTION

1

1 INTRODUCTION

11 What is Hyperlipidemia

Hyperlipidemia a broad term also called hyperlipoproteinemia is a common

disorder in developed countries and is the major cause of coronary heart disease It

results from abnormalities in lipid metabolism or plasma lipid transport or a

disorder in the synthesis and degradation of plasma lipoproteins [1-4] The term

ldquodyslipidaemiardquo now a days is increasingly being used to describe abnormal

changes in lipid profile replacing the old term hyperlipidaemia [5] Hyperlipidemia

means abnormally high levels of fats in the blood These fats include cholesterol

and triglycerides These are important for our bodies to function but when they are

high they can cause heart disease and stroke Hyperlipidemia is manifested as

hypercholesterolemia andor hypertriglycerolemia However hypercholesterolemia

is the most common hyperlipidemia The lipids that are involved in

hypercholesterolemia are cholesterol an essential component of cell membrane and

a precursor of steroid hormone synthesis and triglycerides an important energy

source They are transported in blood as lipoproteins [1] The consequence of

hyperlipidaemia is that with time it can cause atherosclerosis and thus the risk of

coronary heart disease and stroke is increased However according to the newer

scientific view the cholesterol level alone is not the whole story The risk of heart

disease in future also depends on many other factors that influence the health of a

personrsquos blood vessels and circulation [6]

12 Causes of hyperlipidemia

Mostly hyperlipidemia is caused by lifestyle habits or treatable medical conditions

Lifestyle habits include obesity not exercising and smoking Medical diseases that

may result in hyperlipidemia are diabetes kidney disease pregnancy and an under

active thyroid gland One can also inherit hyperlipidemia The cause may be

genetic if a patient has a normal body weight and other members of hisher family


2

have hyperlipidemia One has a greater chance of developing hyperlipidemia if

heshe is a man older than age 45 or a woman older than age 55 If a close relative

had early heart disease there is also an increased risk of this disease [7] Common

secondary causes of hypercholesterolemia are hypothyroidism pregnancy and

kidney failure Common secondary causes of hypertriglyceridemia are diabetes

excess alcohol intake obesity and certain prescription medications [8]

13 Symptoms and diagnoses of Hyperlipidemia

Hyperlipidemia in general has no apparent symptoms and it is discovered and

diagnosed during routine examination or evaluation for atherosclerotic

cardiovascular disease However deposits of cholesterol may be formed under the

skin in individuals with familial forms of the disorder or in persons with very high

levels of cholesterol in the blood In individuals with hypertriglyceridemia several

pimple-like lesions may be developed across their bodies Pancreatitis a severe

inflammation of the pancreas that may be life-threatening can also be developed

due to extremely high levels of triglycerides [9] For diagnosis of hyperlipidemia

levels of total cholesterol low density lipoprotein cholesterol high density

lipoprotein cholesterol and triglycerides are measured in a blood sample It is

important to note that the lipid profile should be measured in all adults 20 years and

older and the measurement should be repeated after every 5 years Food or

beverages may increase triglyceride levels temporarily so people must fast at least

12 hours before giving their blood samples Special blood tests are carried out to

identify the specific disorder when lipid levels in the blood are very high Specific

disorders may include several hereditary disorders which produce different lipid

abnormalities and have different risks [10]


3

14 Classes of Lipoprotein [11]

Since blood and other body fluids are watery so fats need a special transport

system to travel around the body They are carried from one place to another

mixing with protein particles called lipoproteins There are four types of

lipoproteins each having very distinct job These lipoproteins are described as

follows

141 Chylomicrons

Chylomicrons are made by the intestines for carrying new fat to the bodyrsquos cells

These carry mostly triglycerides Chylomicrons carry exogenous lipids to liver

adipose cardiac and skeletal muscle tissue where their triglyceride components are

released by the activity of the enzyme called lipoprotein lipase Consequently

chylomicron remnants are left behind which are taken up by the liver [12] The

density of these particles is less than 095 gml for chylomicrons and 1006 gml for

chylomicron remnants [13]

142 Very-Low-Density Lipoproteins (VLDL)

Very Low Density Lipoproteins are made by the liver and intestine to carry fats

around the body These carry mostly triglycerides

143 Low-Density Lipoproteins (LDL)

Low Density Lipoproteins are made by the liver to transport cholesterol to the

bodyrsquos cells and tissues LDL may form deposits on the walls of arteries and other

blood vessels Therefore they are considered as the lazy or bad cholesterol


4

144 High-Density Lipoproteins (HDL)

High Density Lipoproteins pick up and transport excess cholesterol from the walls

of arteries and bring it back to the liver for processing and removal They are

therefore called the healthy or good cholesterol

15 Classification of hyperlipidemia [14]

Hyperlipidemias are classified according to the Fredrickson classification which is

based on the pattern of lipoproteins on electrophoresis or ultracentrifugation [15] It

was later adopted by the World Health Organization (WHO) It does not directly

account for HDL and it does not distinguish among the different genes that may be

partially responsible for some of these conditions In the past it was a popular

system of classification but is considered out-dated by many experts now

Following are the five types of hyperlipidemia described by Fredrickson

151 Hyperlipoproteinemia Type-I

Hyperlipoproteinemia Type I also called primary hyperlipoproteinaemia or

familial hyperchylomicronemia) is due to deficiency of lipoprotein lipase (LPL) or

altered apo lipoprotein C2 resulting in elevated chylomicrons the particles that

transfer fatty acids from the digestive tract to the liver Its occurrence is 01 of the

population

152 Hyperlipoproteinemia Type-II

Hyperlipoproteinemia Type II the most common form is further classified into

type IIa and type IIb which are as follows


5

1521 Hyperlipoproteinemia Type-IIa

Hyperlipoproteinemia Type-IIa may be sporadic polygenic or truly familial as a

result of mutation either in the LDL receptor gene on chromosome 19 or the Apo B

gene The familial form of this type is characterized by tendon Xanthoma

xanthelasma and premature cardiovascular disease

1522 Hyperlipoproteinemia Type-IIb

Hyperlipoproteinemia Type-IIb is caused by high VLDL levels which are due to

overproduction of substrates including triglycerides acetyl CoA and an increase

in B-100 synthesis They may also be caused by the decreased clearance of LDL

153 Hyperlipoproteinemia Type-III

Hyperlipoproteinemia Type-III is due to high chylomicrons and IDL (intermediate

density lipoprotein) It is also known as broad beta disease or

dysbetalipoproteinemia which is mostly due to the presence of Apo E E2E2

genotype It is due to cholesterol-rich VLDL

154 Hyperlipoproteinemia Type-IV

Hyperlipoproteinemia Type-IV also known as hypertriglyceridemia or pure

hypertriglyceridemia is due to high triglycerides According to the NCEP

(National Cholesterol Education Program) definition of high triglycerides

occurrence is about 16 of adult population [16]

154 Hyperlipoproteinemia Type-V

Hyperlipoproteinemia Type-V is very similar to type I but have high VLDL in

addition to chylomicrons This disease has glucose intolerance and hyperuricemia


6

16 Classification of Antihyperlipidemic Drugs

Several different classes of drugs are used to treat hyperlipidemia These classes

differ not only in their mechanism of action but also in the type of lipid reduction

and the magnitude of the reduction Statins the most common group of

antihyperlipidemic drugs lowers cholesterol by interrupting the cholesterol

biosynthetic pathway [17-18] On the other hand fibrate group decrease fatty acid

and triglyceride levels by stimulating the peroxisomal b-oxidation pathway [19-20]

Apart from these drugs ezetimibe selectively inhibits intestinal cholesterol

absorption [21] cholestyramine colestipol and colesevelam sequester bile acids

[22] torcetrapib inhibits cholesterol ester transfer protein [23] avasimibe inhibits

acyl-CoA cholesterol acyltransferase [24] implitapide inhibits microsomal

triglyceride transfer protein [25] and niacin modifies lipoproteins [21] are several

options to treat hyperlipidemia However statins and fibrates are most popular in

terms of medical use and importance [26] Following are the commonly used group

of drugs to treat dyslipidemia

161 Statins

162 Fibrates

163 Cholesterol absorption inhibitors

161 Statins 3-Hydroxyl-3-methylglutaryl coenzyme A (HMG-CoA) reductase is the enzyme

that catalyzes the conversion of HMG-CoA to mevalonate during cholesterol

synthesis [27] Statins are the drugs that competitively inhibit HMG-CoA

reductase resulting a decrease in serum cholesterol levels [28] Till now there are

seven statins available in pharmaceutical form These are lovastatin simvastatin

pravastatin fluvastatin atorvastatin rosuvastatin and pitavastatin [22 29] Statins

can be classified into naturally derived and chemically synthesized [30-33] The

first statin identified was Mevastatin which is not in use now [34] Cerivastatin


7

was withdrawn from the market by its manufacturers in 2001 after reports of

rhabdomyolysis [35ndash37] Pitavastatin is a new statin available in Japan in

pharmaceutical form and is under trials in Europe and United States [38-39]

Lovastatin and simvastatin are prodrugs that are converted into their active forms in

the liver whereas the other statins are active in their parent forms [31] All statins

show similar function by binding to the active site of 3-hydroxy- 3-methylglutaryl-

coenzyme A reductase (HMGR) and in this way inhibit the enzyme However

structural differences in statins are responsible for differences in potency of enzyme

inhibition [40] Statins are competitive inhibitors of HMGR [41] All statins have a

structural component that is very analogous to the HMG portion of HMG-CoA All

Statins differ from HMG-CoA in being more bulky and more hydrophobic The

naturally derived statins contain a substituted decalin ring structure Fully synthetic

statins with larger flurophenyl groups are linked to the HMG like moiety These

additional groups change the character from very hydrophobic to partly

hydrophobic [42] As all the statins inhibit HMGR at different rates important

structural differences are present in all that distinguish their lipophilicity half-life

and potency [30] As for example lovastatin and simvastatin can cross the blood

brain and placental barriers but pravastatin and fluvastatin can not [43] In addition

rosuvastatin is relatively hydrophilic and has more chances of bonding interactions

with the catalytic site of HMGR compared with mevastatin fluvastatin simvastain

cerivastatin and atorvastatin [28 44ndash47]

The absorption of statins varies from 30 to 98 [48ndash56] All statins are rapidly

absorbed after oral administration and achieve the peak concentrations level within

4 hours Food has no effect on bioavailability of statins except for lovastatin where

it is increased [57] Statins have a slow onset of effect and are therefore insensitive

to temporary changes in unbound plasma drug concentration [58]

Rosuvastatin is glucorinated for excretion while simvastatin lovastatin and

atorvastatin are metabolized by CYP3A4 [59-61] Cerivastatin is metabolized by

CYP3A4 [62] and CYP2C8 [63] and fluvastatin is metabolized by CYP2C9 [64-


8

65] Several reactions are involved during pravastatin metabolism that includes

isomerization sulfonation glutathione conjugation and oxidation [66-68] The

amount of the statin that is excreted in urine as unchanged drug varies from

negligible amounts for atorvastatin [55] to 20 and 30 respectively for

pravastatin and cerivastatin [69-70]

1611 Mechanism of Action of Statins [71]

Statins inhibit HMG-CoA reductase the enzyme that converts HMG-CoA into

mevalonic acid during cholesterol synthesis Statins change the conformation of the

enzyme during binding to its active site In this way HMG-CoA reductase is

prevented from attaining a functional structure Attachment of statins with HMG

CoA reductase is reversible and the affinity of the statins with the enzyme is in the

nanomolar range whereas the attachment of the natural substrate is in micro moles

[72] The reduction of cholesterol in hepatocytes results in increase of hepatic LDL

receptors which measures the reduction of circulating LDL and its precursors

intermediate density and very low density lipoproteins [73] All statins has the

ability to reduce LDL cholesterol non-linearly dose-dependent and after

administration of a single daily dose [74] Efficacy for the reduction of triglycerides

is almost equal to LDL cholesterol reduction [75]

Statins stop hepatic syntesis of apolipoprotein B- 100 which in turn cause a

reduction of the synthesis and secretion of lipoproteins rich in tryglycerides [76]

and increase of receptors producing apolipoproteins BE [77] This can explain why

atorvastatin and simvastatin reduce LDL in patients having homozygous familial

hypercholesterolemia where LDL receptors are not working properly [78-79]

Statins have intermediate effect on HDL increase and therefore has no influence on

lipoprotein(s) concentration [80]

1612 Adverse effects of statin therapy [71]

Statins have generally little side effects The most important adverse effects are

liver and muscle toxicity Myopathy may occur if cytochrom P450 inhibitors or


9

other statins metabolism inhibitors are administered together with statins such as

the azole antifungals [81] Fibrates and niacin increase the risk of myopathy by a

mechanism which does not involve an increase in blood concentration of statins

Other adverse effecfts are hepatic dysfunction renal insufficiency

hypothyroidism advanced age and serious infections Cerivastatin was hence

suspended from the clinical use because of rhabdomyolysis in a number of patients

which confirms that statins cause muscle toxicity

162 Fibrates

Fibrates are another group of antihyperlipidemic agents widely used in the

treatment of different forms of hyperlipidemia and hypercholesterolemia Fibrates

are 2-phenoxy-2-methyl propanoic acid derivatives This group includes

bezafibrate ciprofibrate clofibrate clofibric acid fenofibrate and gemfibrozil

[82] In comparison with statins fibrates does not stop cholesterol biosynthesis

[26] In fact these drugs stimulate b-oxidation of fatty acids mostly in peroxisomes

and partially in mitochondria [19-20 83-84] This group of drugs is therefore

known for decreasing plasma levels of fatty acid and triacylglycerol Clofibrate was

the first fibrate marketed in Japan in the 1960s [85] With this the discovery of

other fibrate drugs such as ciprofibrate bezafibrate fenofibrate and gemfibrozil

begin to start However this period was short because continuous use of some of

these drugs like clofibrate and ciprofibrate causes hepatomegaly and tumor

formation in the rodents liver [86ndash90] Therefore there are objections about

continuous use of these drugs in humans Only gemfibrozil and fenofibrate due to

their milde effect are being used as lipid lowering drugs in humans

1621 Mechanism of Action of Fibrates [26]

One of the functions of fibrate drugs is the activation of peroxisome proliferator

activated receptor (PPAR) PPARs are a collection of three nuclear hormone

receptor isoforms PPAR-g PPAR-a and PPAR-d which are encoded by different


10

genes [91-92] Among the fibrates clofibrate and fenofibrate can activate PPAR-a

with selectivity ten times over PPAR-g [92] Although these drugs activate PPARs

there is no direct binding with PPARs However in response to fibrate drugs

PPAR-a heterodimerizes with retinoid X receptor-a (RXR-a) and the resulting

heterodimer modulates the transcription of genes containing peroxisome

proliferator responsive elements (PPREs) in their promoter sequence [92-93]

B-oxidation of fatty acids occurs mainly in mitochondria In peroxisomes only

very long chain and long-chain fatty acids are b-oxidized [94-95] After chain

shortening in peroxisomes fatty acids are transported into mitochondria for

complete b-oxidation However fibrate drugs can stimulate peroxisomal b-

oxidation mainly [83 84 86] In addition fibrate drugs also stimulate fatty acid w-

oxidation in the liver and they prevent the effects of some fatty acid oxidation

inhibitors such as 4-pentenoate and decanoyl-carnitine Fibrates also increase the

activity of acyl-CoA synthetase and the CoA content of liver while the level of

malonyl-CoA which is the precursor of fatty acid synthesis decreases [96-97] In

addition to stimulating fatty acid oxidation-associated molecules fibrates also

increase lipolysis [98]

Continuous use of fibrates for 40ndash50 weeks in rodents can leads to hepatic tumor

[90 96] Fibrate drugs are believed to cause oxidative stress which ultimately

increases the hepatocyte proliferation and oxidative DNA damage [99]

Fibrates repress cytokine-induced Interleukine-6 (IL-6) production in SMCs iNOS

activity in murine macrophages and VCAM-1 expression in endothelial cells [100-

101] Not only fibrate but PPAR-g ligands also inhibit production of inflammatory

cytokines by monocytes macrophages in vitro [101] Fibrate drugs also show anti-

inflammatory effect in brain cells Although mechanisms of fibrates for the anti-

inflammatory effect is currently unknown it is supposed that these may decrease

inflammation partly by inducing the expression of IkBa which in turn blocks the

activation of NF-kB a transcription factor critical in the activation of pro-

inflammatory molecules [102]


11

1622 Adverse effects of Fibrate therapy [103]

The fibrates are generally well tolerated with very few side-effects The most

common side-effects are gastrointestinal disturbances such as nausea and

diarrhoea Other side-effects include headaches anxiety fatigue vertigo sleep

disorders etc [104-106] The most prominent side-effect is myositis which

commonly occur when renal function is impaired or statins are given

Rhabdomyolysis during statin-fibrate combination therapy is most often observed

Myopathy usually occurs within 2 months of the start of therapy [105107-108]

Fibrates are contraindicated in hepatic or severe liver dysfunction and previous

gallstone disease These drugs should not be used by nursing mothers or during

pregnancy [104 108-109]

163 Cholesterol absorption Inhibitors

Cholesterol absorption inhibitor functions by decreasing the absorption of

cholesterol in the small intestine This cause a decrease in the cholesterol delivery

to the liver which in turn clears more cholesterol from the blood [110] Ezetimibe is

the first of this class of drugs [111-112] In the intestinal mucosa glucoronidation

of ezetimibe to its active metabolite [113] Primarily it is metabolized in the small

intestine and liver through glucuronide conjugation with biliary and renal excretion

[114] Ezetimibe does not affect the absorption of fat-soluble vitamins

triglycerides or bile acids [115] Food administration with this during therapy

cause no effect on the absorption of ezetimibe when used in the 10 mg dose [116-

117]

1631 Mechanism of Action of Ezetimibe [111] Ezetimibe stays at the brush border of the small intestine and selectively inhibits

the absorption of cholesterol from the intestinal lumen into enterocytes [118] After

oral administration ezetimibe is glucuronidated rapidly in the intestines and once

it is glucuronidated undergoes enterohepatic recirculation and hence deliver the


12

drug repeatedly to its site of action The glucuronide of ezetimibe is much more

effective than the parent drug mainly because of its localization at the brush border

of the intestines [119] Both ezetimibe and its glucuronide are recirculated and are

delivered back to their site of action in the intestine resulting in more efficacy

accounting for a half-life of approximately 22 hours [120] The timing of dosing

does not affect its activity [121] In animal models ezetimibe decreased cholesterol

delivery from the intestine to the liver reduce hepatic cholesterol efficiently

regulate LDL cholesterol receptors lying on liver cell membranes and increase

removal of cholesterol from blood [112122-125] In a 2-week clinical study of 18

hypercholesterolemic patients conducted by Sudhop et al ezetimibe 10 mg once

daily causes the inhibition of intestinal cholesterol absorption 54 as compared to

placebo [126]

Monotherapy with ezetimibe can effectively reduce LDL cholesterol in patients

having hypercholesterolemia [127-128]

1632 Adverse Effects of Ezetimibe

The adverse effects of ezetimibe are few and mild In most studies ezetimibe does

not increase myopathy or rhabdomyolysis whether used alone or in combination

with statins although some case reports of myopathy were there due to this agent

In addition ezetimibe can cause mild elevations of liver transaminases when used

in combination with a statin Other side effects are extremely rare [129] The most

commonly reported adverse effects are upper respiratory tract infection diarrhea

arthralgia sinusitis and pain in extremity [130]

17 Combination therapy for Hyperlipidemia Combination therapy for hyperlipidemia especially for combined hyperlipidemia

can have advantages over monotherapy causes better improvement in lipoprotein

risk factors and in turn better prevention of atherothrombotic events [131]


13

Following is the combination therapy that is most commonly used for

hyperlipidemia

171 Statin and ezetimibe combination therapy

172 Statin and fibrate combination therapy

173 Ezetimibe and fibrate combination therapy


Statin and ezetimibe combination therapy is FDA-approved and with this

additional decrease in absolute LDL cholesterol occurs [114132] When used as

monotherapy ezetimibe reduces LDL-C with an average of 17 in patients with

primary hypercholesterolemia [127133] and an additional 9 ndash25 when used in

combination with statins [134-149] The combination therapy of ezetimibe and a

statin is much more effective in reducing LDL-C than either drug alone and it has

been observed in clinical trials comparing simvastatin atorvastatin fluvastatin

pravastatin lovastatin and rosuvastatin alone with each in combination with

ezetimibe Although myalgia was frequently reported in most of these studies (up

to 8) the combination therapy had a safe profile as of statins alone [150] In

another trial 668 subjects with primary hypercholesterolemia were randomly

treated with one of the following 10 regimens for 12 weeks ezetimibe 10 mg

alone simvastatin 10 20 40 or 80 mg alone ezetimibe 10 mg plus simvastatin 10

20 40 or 80 mg or placebo [140] Musculoskeletal pain was observed in nine

patients (3 ) all belonging to simvastatin groups compared to six patients (2 )

in ezetimibe plus simvastatin groups one patient (2 ) in the ezetimibe alone

group and three placebo recipients (4 ) One patient on simvastatin 20 mg had

myopathy The results from the clinical studies suggested that ezetimibe and statin

combination therapy does not induce an increase in myopathy or myalgia compared

with simvastatin monotherapy [143]


14


Statin - Fibrate combination therapy in combined dyslipidemia can decrease LDL

cholesterol more than 40 triglycerides over 50 and raise high-density

lipoprotein (HDL) cholesterol more than 20 [151] Controlled trials showed

regression of atherosclerotic lesions with the combination but also showed increase

risks of myopathy [152-153] In 36 clinical trials in which statin-fibrate

combinations was evaluated 012 of patients developed myopathy but none of

them developed rhabdomyolysis or kidney failure [153] According to experts

myopathy risk is greater with gemfibrozil than with fenofibrate based on

gemfibrozilrsquos inhibition of statin glucuronidation [154] Due to this the maximum

approved daily doses of lovastatin simvastatin and rosuvastatin are lower (20 10

and 10 mg respectively) when used in combination with gemfibrozil [132]

Several trials have studied the safety and efficacy of combination therapy of statins

with fibrates [151] In a trial of 389 patients having familial combined

hyperlipidemia randomized to receive pravastatin 20 mg per day plus gemfibrozil

1200 mg per day simvastatin 20 mg per day plus gemfibrozil 1200 mg per day

or simvastatin 20 mg per day plus ciprofibrate 100 mg per day LDL cholesterol

decreased by 35 39 and 42 and triglycerides level decreased by 48 54

and 57 in the respective groups HDL cholesterol increased by 14 25

and 17 respectively [155] In another study by the same group which was

conducted in 120 type 2 diabetes mellitus patients and combined hyperlipidemia

and without having coronary artery disease the combined of atorvastatin 20 mg

and micronized fenofibrate 200 mg per day decreased LDL cholesterol by 46

and triglyerides by 50 and HDL cholesterol increased by 22 [156] There

were several cases of rhabdomyolysis with renal failure in some cases with this

combination Overall myopathy occurred in approximately 01 to 02 of

patients who received statins in clinical trials and the incidence was dosing related

[36] Of the cases reported to the FDA reporting rate per million prescriptions


15

ranged from a high of 316 with cerivastatin to 019 with lovastatin 012 with

simvastatin 004 with atorvastatin or pravastatin and 0 with fluvastatin [154]

Combination therapy of statins with fibrates requires careful selection and

monitoring of patients Risk factors that can cause myopathy include increased age

female gender renal or liver disease hypothyroidism excessive alcohol intake

trauma surgery and heavy exercise


The ezetimibe and fenofibrate combination was recently approved by the FDA for

treatment of mixed hyperlipidemia This lipid-modifying therapy has the advantage

of the different mechanisms of action of the two individual components Ezetimibe

selectively inhibits intestinal absorption of dietary and biliary cholesterol and

exerts its effect mainly on the low-density lipoprotein cholesterol (LDL-C)

Fenofibrate activates the PPAR-alpha hence increases the tissue lipoprotein lipase

activity and decomposition of triglycerides in VLDL The combination therapy of

ezetimibe and fenofibrate has very good safety profile and represents another

alternative in the clinical treatment of mixed hyperlipidemia [157] McKenney et al

conducted a trial of 587 patients in which they were given ezetimibe 10 mg

fenofibrate 160 mg fenofibrate 160 mg plus ezetimibe 10 mg or placebo

randomly After 12 weeks 576 patients continued into a double-blind 48-week

extension phase in which patients who received ezetimibe or placebo were treated

with fenofibrate plus ezetimibe or fenofibrate alone respectively [158] Fenofibrate

plus ezetimibe produced a 135 greater reduction in LDL-C than fenofibrate

alone as well as significantly greater improvements in triglycerides high-density

lipoprotein total cholesterol nonndashhigh-density lipoprotein cholesterol and apo

lipoprotein B No cases of myopathy were observed in either group over the 48

week of the study Myalgia was not reported [150]

Among all the combination treatments following binary combinations were

selected to be analyzed during this research project The selection of the


16

combinations was based upon the use of combination ease of collecting the

reference standards and drug products etc

1 Atorvastatin 10 mg and Ezetimibe 10 mg

2 Simvastatin 10 mg and ezetimibe 10 mg

3 Lovastatin 20 mg and ezetimibe 10 mg

4 Rosuvastatin 40 mg and ezetimibe 10 mg

5 Atorvastatin 10 mg and gemfibrozil 600 mg

6 Simvastatin 10 mg and gemfibrozil 600 mg

7 Ezetimibe 10 mg and fenofibrate 160 mg

18 Antihyperlipidemic Drugs

The individual details of the drugs mentioned above are given as follows

181 Atorvastatin Calcium

A Origin of substance

Synthetic

B Drug Category

It belongs to the statin family

C Chemical name

It is calcium salt (21) trihydrate of [R-(RR)]-2-(4-f luorophenyl)- b d - d i h y

d r o x y - 5 - (1 -me t h y l e t h y l ) - 3 - p h e n y l - 4[(phenylamino)carbonyl]-

lH-pyrrole-1-heptanoic acid


17

D Structural formula

N

O-

OHOH

O

CH3CH3

O

NH

F

2

Ca+2

3H2O

Figure 11 Chemical structure of atorvastatin calcium

E Molecular Formula

(C33H34 FN2O5)2Cabull3H2O

F Molecular Weight

120942

G Colour

White to off-white crystalline powder

H Solubility

Insoluble in aqueous solutions of pH 4 and below very slightly soluble in

distilled water pH 74 phosphate buffer and acetonitrile slightly soluble in

ethanol and freely soluble in methanol


18

182 Simvastatin


Semi-synthetic

B Drug Category


C Chemical name

[(1S3R7S8S8aR)-8-[2-[(2R4R)-4-hydroxy-6-oxooxan-2-yl]ethyl]-37-

dimethyl-123788a-hexahydronaphthalen-1-yl] 22-dimethylbutanoate


O

CH3

CH3

O

O

CH3

CH3

CH3

OOH

H

Figure 12 Chemical structure of simvastatin

E Molecular Formula

C25H38O5


19

F Molecular Weight

41857

G Colour

White crystalline powder

H Solubility

Practically insoluble in water soluble in methanol ethanol acetonitrile and most

other organic solvents

183 Lovastatin


Semi-synthetic

B Drug Category


C Chemical name


dimethyl-123788a-hexahydronaphthalen-1-yl] (2S)-2-methylbutanoate


20


O

C H 3

CH 3

O

O

C H 3

HCH 3

OH

H

O

Figure 13 Chemical structure of lovastatin

E Molecular Formula

C24H36O5

F Molecular Weight

40454

G Colour

White to off white crystalline powder

H Solubility

Freely soluble in chloroform soluble in acetone in acetonitrile and in methanol

sparingly soluble in alcohol practically insoluble in hexane insoluble in water

184 Rosuvastatin Calcium


Synthetic


21

B Drug Category


C Chemical name

3R5S6E)-7-[4-(4-fluorophenyl)-2-(N-methylmethanesulfonamido)-6-(propan-

2-yl)pyrimidin-5-yl]-35-dihydroxyhept-6-enoic acid


N

N O-

CH3CH3

NS

CH3

O

O

OOHOH

FCH3

Ca+2+2

2

Figure 14 Chemical structure of rosuvastatin calcium

E Molecular Formula

C22H28FN3O6S

F Molecular Weight

100114

G Colour

White to Yellow colured powder


22

H Solubility

Sparingly soluble in water slightly soluble in methanol freely soluble in

acetonitrile and in NN-Dimethyl formamide

185 Gemfibrozil


Synthetic

B Drug Category

It belongs to the fibrate family

C Chemical name

It is 5-(25-dimethylphenoxy)-22-dimethyl-pentanoic acid


O

CH3

CH3OH

OCH3

CH3

Figure 15 Chemical structure of gemfibrozil

E Molecular Formula

C15H22O3


23

F Molecular Weight

25033

G Colour

White waxy crystalline solid

H Solubility

Practically insoluble in water soluble in alcohol in methanol and in chloroform

186 Fenofibrate


Synthetic

B Drug Category


C Chemical name

It is Isopropyl 2-[4-(4-chlorobenzoyl) phenoxy]-2-methylpropionate


24


O

Cl

O

CH3

CH3

O

O CH3

CH3

Figure 16 Chemical structure of fenofibrate

E Molecular Formula

C20H21ClO4

F Molecular Weight

36083

G Colour

A white or almost white crystalline powder

H Solubility

Practically insoluble in water very soluble in methylene chloride slightly soluble

in alcohol

187 Ezetimibe


Synthetic


25

B Drug Category

It belongs to the cholesterol absorption inhibitors family

C Chemical name

It is (3R4S)-1-(4-fluorophenyl)-3-[(3S)-3-(4-fluorophenyl)-3-hydroxypropyl]-4-(4-

hydroxyphenyl)azetidin-2-one


N

O

OH

F

OH F

Figure 17 Chemical structure of ezetimibe

E Molecular Formula

C24H21F2NO3

F Molecular Weight

4094

G Colour

Off white to white crystalline powder


26

H Solubility

Practically insoluble in water Freely soluble in methanol ethanol acetonitrile

and acetone

19 High Performance Liquid Chromatography (HPLC)

High performance liquid chromatography (HPLC) is a form of liquid

chromatography to separate compounds that are dissolved in solution HPLC

apparatus consists of a reservoir for delivering mobile phase a pump an injector a

separation column and a detector The different components in the mixture pass

through the column at different rates due to differences in their partitioning

behavior between the mobile phase and the stationary phase [159]

191 Types of Detectors Used In HPLC

Following types of detectors are generally used during the analysis of

particular components of a mixture depending upon the nature of analytes

Oslash UV-Visible Detector

Oslash Refractive Index Detector

Oslash Fluorescence Detector

Oslash Evaporating Light Scattering Detector

Oslash Electrochemical Detector

Oslash Mass Spectrometric Detector

Among the detectors listed above UV-Visible detector is used for almost 90 of

the compounds


27

192 Chromatographic Terms

1921 Chromatogram

The electronic result of a chromatographic separation which is a plot of detector

signal against elution time It is represented as a series of peaks

1922 Column

A stainless steel tube which contains the stationery phase The stationery phase

interacts differentially with the samplersquos components as they are carried in the

mobile phase

1923 Column Performance

The efficiency of a column is called column performance which is measured as the

number of theoretical plates for a given test compound

1924 Eluent

Sample component carried by the mobile phase and retained on the stationary

phase is called eluent

1925 Flow Rate

The volumetric rate of flow of mobile phase through the column For an analytical

HPLC column typical flow rates are 1 to 2 mlmin

1926 Peak

When the detector registers the presence of a compound the normal baseline signal

it sends to the data system changes resulting in a deflection from the baseline

called a peak

1927 Resolution

The ability of the column to separate chromatographic peaks It is usually

expressed in terms of the separation of two peaks

1928 Retention Factor

Retention factor is how long a compound is retained by the stationary phase

relative to the time it stays in the mobile phase


28

1929 Retention Time

The time between injection and the appearance of the peak maximum is called

retention time

19210 Tailing

The phenomenon in which the normal Gaussian peak has an asymmetry factor

greater than 1 the peak will have tailing edge

193 Method Validation on HPLC [160]

Method validation is the process to confirm that the analytical procedure employed

for a particular test is suitable for its intended purpose Methods need to be

validated or revalidated According to ICH guidelines following are the parameters

for analytical method validation

Linearity

Accuracy

Precision

Specificity

Limits of detection

Limits of quantitation

Robustness

110 Quantitative Analysis

A business or financial analysis technique that is used to understand reaction or

behavior by applying complex mathematical and statistical models measurement

and research is called as quantitative analysis Quantitative analysis is performed

for a number of reasons such as measurement performance evaluation or

evaluation of a financial instrument [161]

In analytical chemistry determination of the absolute or relative concentration of

one several or all substances present in a sample are called quantitative analysis


29

Once it is observed that a certain substance is present in a sample the study of their

concentrations can be helpful in elucidating the certain properties For example

quantitative analysis performed during HPLC of pharmaceutical products can

determine the relative abundance of that particular compound [162]

1101 Quantitative Instrumental Analysis [163]

A number of times during research a researcher want to know the components of a

mixture For this purpose heshe needs analytical instruments such as GC-MS or

HPLC which provides valuable information e g what components are present and

in how much quantity Determining the quantity is called quantitative analysis For

the quantitative analysis of target molecules we must perform an extraction

procedure to separate the analyte in an appropriate solvent All the instruments used

for analysis can detect the analyte to their capacity When analytes enter the

detector an electronic signal is generated which is called response This response

may be named as absorbance intensity abundance etc the computer system

attached with this type of system displays and stores the information

Usually the response is represented in the form of graph having X and Y axis for

retention time and intensity respectively This graph in chromatography is called

chromatogram When no injection is done the response is zero and only a straight

line exists which is called smooth baseline As the analytes are entered in the

detector the response is started to begin The baseline starts moving upward till the

maximum response and then comes down meeting with the baseline This is called

peak which represents the corresponding concentration Size of the peak can be

measured through height width and area However peak area is more reliable and

is used mostly

The concentration of the analyte from the peak area can be calculated by applying

the formula


30

age of Analyte= Peak area of unknown 100 Peak area of standard

It should be noted here that the peak area of unknown and standard should be of the

same concentration The peak area of the standard can be calculated from the

calibration curve that covers the concentration in a wide range

111 Statistics

Statistical methods are necessary part of the development and testing of drug

products Statistics is often thought of as a collection of numbers and averages such

as vital statistics baseball statistics or statistics derived from census Statistical

approaches take the experimental variability into account during analysis [164]

Following statistical tools are used during this study

1111 Average [165]

The average result denoted as X- is calculated by summing the individual results

and dividing this by the number (n) of individual values

X- = X1 + X2 + X3 + X4 + n

1112 Standard Deviation [165]

The standard deviation is a measure of how precise the average is that is how well

the individual numbers agree with each other It is a measure of a type of error

called random error It is calculated as follows

Standard deviation S = radic (X1 ndash X-)2 + (X2 ndash X-)2 + (X3 - X-)2 + n - 1

1113 Relative Standard Deviation [165]

The relative standard deviation (RSD) is often times more convenient It is

expressed in percent and is obtained by multiplying the standard deviation by 100

and dividing this product by the average

Relative standard deviation RSD = 100S X-


31

1114 Linear Regression Analysis

In statistics linear regression refers to any approach that consists of modeling the

relationship between one or more variables denoted by Y and one or more variables

denoted X Such a model is called a linear model Linear regression was the first

type of regression analysis to be studied rigorously and to be used extensively in

practical applications [166] Linear regression determines the relationship between

two variables X and Y For each subject one knows both X and Y and one want to

measure a good straight line through the data In general the purpose of linear

regression is to find the line that best predicts Y from X Linear regression does not

test whether someone s data is linear It assumes that data is linear and finds the

slope and intercept that make a straight line best fit Linear regression analysis can

be represented in the form of linear regression equation which is as follows

Y= mX + C

Where X and Y are two variables m is the slope of the straight line and C is the

intercept The slope quantifies the steepness of the line It equals the change in Y

for each unit change in X It is expressed in the units of the Y-axis divided by the

units of the X-axis If the slope is positive Y increases as X increases If the slope

is negative Y decreases as X increases [167]

1115 Correlation Coefficients [168]

The linear correlation coefficient denoted as ldquorrdquo measures the direction of a linear

relationship between two variables The mathematical formula for computing r is


32

Where n is the number of pairs of data The value of r is -1 to +1 The + and ndash signs

are used for positive linear correlations and negative linear correlations

respectively If x and y have a strong positive linear correlation r is close to +1 If

x and y have a strong negative linear correlation r is close to -1 If there is no linear

correlation or a weak linear correlation r is close to 0 A value near zero means

that there is a random nonlinear relationship between the two variables A perfect

correlation of plusmn 1 occurs only when the data points all lie exactly on a straight line

112 Manufacturing Process of Tablet Dosage form [169]

1121 What is a Tablet

A tablet is a mixture of active substances and excipients usually in powder form

compacted into a solid The excipients may be binders fillers colors etc Some

common excipients include lactose starch avicel and magnesium stearate

1122 Manufacturing Process

In the tablet manufacturing process all ingredients must be dry and free flowing

The main focus should be on the uniform mixing of active ingredient with the

excipients If a homogenous mixing of different components does not occur with

simple mixing the ingredients are granulated before compression

11221 Granulation

Granulation is the process in which bonds are created between the different

components Two types of granulation methods are used for making tablets which

are wet granulation and dry granulation


33

112211 Wet granulation

In wet granulation a liquid binder is used in the powder mixture The amount of

liquid should be kept minimum because over wetting can change the granules to

too hard or soft

112212 Dry granulation

The dry granulation is used for those components which are sensitive to moisture

The dry granulation process may require repeated compaction steps to attain the

proper granules

11222 Tablet Compression

After granulation the granules are compressed into tablet form by tablet presses

also called rotary machines These machines range from very small to very large

and can produce one tablet at a time or many

11223 Tablet coating

Many tablets now a day are coated after compression There are many methods of

coating such as sugar coating and film coating The film coating may be simly film

coating or enteric coating Coating is performed to protect the tablet from

temperature and humidity and also to mask the taste


34

113 AIMS AND OBJECTIVES OF THE RESEARCH WORK

a) To develop simple sensitive rapid and economic methods based upon high

performance liquid chromatography for the determination of statin

ezetimibe and fibrates in binary combinations by searching optimum

chromatographic conditions for these drugs using different stationery

phases and mobile phases

b) Validation of the developed methods according to International Conference

on Harmonization (ICH) and United States Pharmacoepia (USP) guidelines

c) Forced degradation studies on the statins ezetimibe and fibrates and

separation of peaks of interests from degradation products developed due to

forced degradation

d) Application of the newly developed HPLC methods in analysis of

pharmaceutical formulations and synthetic mixtures

CHAPTER 2 LITERATURE REVIEW

35

2 LITERATURE REVIEW

A number of analytical methods have been reported in various journals for the

determination of antihyperlipidemic drugs in pharmaceutical formulations and in

biological samples Some of the work in this area of research is given below for

each drug

21 Analytical Methods for Atorvastatin

Petkovska et al [170] developed and validated a Rapid Resolution Reversed Phase

High-Performance Liquid Chromatography method for the simultaneous

determination of atorvastatin and seven related compounds Experimental design

was used during method optimization and robustness testing Chromatography was

performed with mobile phase containing phosphate buffer pH 35 and a mixture of

10 tetrahydrofuran in acetonitrile as organic modifier A C18 Rapid Resolution

column was used The developed method was able to determine atorvastatin

calcium purity and level of impurities in drug substances

Khedr [171] developed a sensitive selective and validated stability-indicating

high-performance liquid chromatographic assay for atorvastatin in bulk drug and

tablet form Atorvastatin was subjected to different stress conditions including UV

light oxidation acid-base hydrolysis and temperature The analyte and the

degradation products were then analyzed on a C18 column using isocratic elution

with acetonitrile-002 M sodium acetate pH 42 (4555 vv) The samples were

monitored with fluorescence detection at 282 nm (excitation)400 nm (emission)

The method showed good resolution of atorvastatin from its decomposition

products The linear range was 10-1200 nginjection and the limit of quantitation

(LOQ) was 20 nginjection

Sivakumar et al [172] applied statistical experimental design and Derringers

desirability function to develop an improved RP-HPLC (Reverse Phase High


36

Performance Liquid Chromatography) method for the simultaneous analysis of

amlodipine and atorvastatin in pharmaceutical formulations The predicted

optimum for the quality control samples was methanol-acetonitrile-0015 M

dipotassium hydrogen phosphate buffer (pH 533) (1042084792 vvv) as the

mobile phase and 112 mLmin as the flow rate The assay was validated according

to ICH guidelines

Jamshidi et al [173] developed a two-step isocratic chromatography on silica gel

HPTLC layer and densitometric quantitation at λ = 280 nm for the separation of

atorvastatin from plasma constituencies and diclofenac sodium as peak-tracer The

developed HPTLC method was validated in terms of LODLOQ (Limits of

detectionLimits of quantitation) linearity recovery and repeatability The method

was linear in the range 101ndash3535 ngzone The LOD and LOQ were 303 ngzone

and 101 ngzone The recovery and relative standard deviation (RSD) obtained

from between-days analysis were 975ndash1030 and 17ndash34

Ma et al [174] developed a sensitive liquid chromatographicndashelectrospray

ionizationndashmass spectrometric method for direct concentration of atorvastatin in

human plasma Plasma samples were extracted with ethyl acetate and by a simple

reversed-phase chromatography The LOQ was 025 ngmL The assay was linear

from 025ndash20 ngmL Intra-day and inter-day accuracy was better than 15

Stanisz et al [175] developed and validated a rapid HPLC method for determination

of atorvastatin in pharmaceutical dosage forms Separation of atorvastatin was

carried on a C-18 column using water-acetonitrile in the ratio of 4852 adjusted to

pH 20 with 80 ortho-phosphoric acid The wavelength was set as 245 nm The

method was linear in the concentration range of 004 - 04 mgmL The RSD values

for intra and inter day precision were less than 100 and 090 respectively


37

Nirogi et al [176] reported a review paper on published higher performance liquid

chromatographic-mass spectrometric methods for the quantification of presently

available seven statins atorvastatin simvastatin lovastatin pravastatin fluvastatin

rosuvastatin and pitavastatin This review encompass that most of the methods used

for quantification of statins were in plasma and they were suitable for therapeutic

drug monitoring of these drugs

Chaudhari et al [177] described the development and validation of a stability

indicating reverse-phase HPLC method for the simultaneous estimation of

atorvastatin and amlodipine from their combination drug product The developed

RP-HPLC method used a C18 column at ambient temperature The mobile phase

was consisted of acetonitrile and 005 M potassium dihydrogen phosphate buffer

(6040 vv) adjusted to pH 3 plusmn 01 with 10 phosphoric acid at 1 mLmin and

UV detection at 254 nm The described method was linear over the range of 1-90

microgmL and 1-80 microgmL for atorvastatin and amlodipine respectively The mean

recoveries were 9976 and 9812 for atorvastatin and amlodipine respectively

The LOD for atorvastatin and amlodipine were found to be 04 microgmL and 06

microgmL respectively and the LOQ was 10 microgmL for both drugs

Mohammadi et al [178] developed and validated a simple rapid precise and

accurate isocratic stability-indicating RP-HPLC method for the simultaneous

determination of atorvastatin and amlodipine in commercial tablets The method

showed separation of amlodipine and atorvastatin from their associated main

impurities and their degradation products Separation was achieved on an ODS-3

column using a mobile phase consisting of acetonitrile-0025 M sodium dihydrogen

phospahe buffer (pH 45) (5545 vv) at a flow rate of 1 mLmin and UV detection

at 237 nm The linearity of the method was in the range of 2-30 microgmL for

atorvastatin and 1-20 microgmL for amlodipine The LOD were 065 microgmL and 035


38

microgmL for atorvastatin and amlodipine respectively The LOQ were 2 microgmL and 1

microgmL for atorvastatin and amlodipine respectively

Borek-Dohalskyacute et al [179] reported a validated highly sensitive and selective

isocratic HPLC method for quantitative determination of the atorvastatin and its

metabolite 2-hydroxyatorvastatin Detection was performed with a mass

spectrometer equipped with an ESI interface in positive-ionization mode The

method was linear in the concentration range 010-4000 ngmL for both

atorvastatin and 2-hydroxyatorvastatin Inter-day and intra-day precision were less

than 8 for both analytes The LOQ was 002 ngmL for atorvastatin and 007

ngmL for 2-hydroxyatorvastatin

Shen et al [180] developed a specific and accurate reversed-phase HPLC with UV

detection for the assay of atorvastatin in beagle dog plasma After protein

precipitation the extracts were separated on a C8 column with UV wavelength at

270 nm The mobile phase consisted of acetonitrile 01 M ammonium acetate

buffer (pH 40) (6535 vv) at a flow rate of 1 mLmin Linearity was found to be

in the range of 005 microgmL to 25 microgmL The LOQ was 25 ngmL and the LOD

was 8 ngmL The total chromatographic analysis time was less than 9 min

Bahrami et al [181] developed and validated a rapid and sensitive high-

performance liquid chromatographic method for determination of atorvastatin in

human serum After liquid-liquid extraction chromatography was performed using

C18 column with a mobile phase consisting of sodium phosphate buffer (005 M

pH 40) and methanol (3367 vv) at 247 nm The average recovery of the drug was

95 The LOD and LOQ were 1 microgmL and 4 ngmL respectively and the

calibration curves were linear over a concentration range of 4-256 ngmL

Zarghi et al [182] developed a rapid and sensitive high-performance liquid

chromatographic method for the determination of atorvastatin in plasma After


39

protein precipitation by acetonitrile atorvastatin was separated on a C8 column

with mobile phase consisting of sodium dihydrogen phosphate buffer-acetonitrile

(6040 vv) adjusted to pH 55 at a flow rate of 15 mLmin and UV detection at

245 nm The LOD for atorvastatin was 1 ngmL The method was linear over the

concentration range 20-800 ngmL The inter-day and intra-day assay precision was

found to be less than 7

Pasha et al [183] developed and validated a specific accurate precise and

reproducible high-performance liquid chromatographic method for the

simultaneous quantitation of atorvastatin lovastatin pravastatin rosuvastatin and

simvastatin in pharmaceutical formulations and extended it to in vitro metabolism

studies of these drugs Ternary gradient elution at a flow rate of 1 mLmin was

employed on an ODS 3V column at ambient temperature The mobile phase

consisted of 001 M ammonium acetate (pH 50) acetonitrile and methanol at a

wavelength of 237 nm Drugs were found to be 896-1056 of their labels claim

in the pharmaceutical formulations

Hermann et al [184] developed a chromatographic method for the analysis of

atorvastatin o- and p-hydroxyatorvastatin (acid and lactone forms) in human

plasma After solid-phase extraction analytes were separated on an HPLC system

with a linear gradient and a mobile phase consisting of acetonitrile water and

formic acid Detection was done by tandem mass spectrometry in electrospray

positive ion mode Linearity was within the concentration range (02-30 ngmL for

atorvastatin acid and p-hydroxyatorvastatin acid and 05-30 ngmL for o-

hydroxyatorvastatin acid) The LOD was 006 ngmL for atorvastatin and p-

hydroxyatorvastatin and 015 ngmL for o-hydroxyatorvastatin

Ertuumlrk et al [185] developed a simple high-performance liquid chromatographic

method for the analysis of atorvastatin and its impurities in bulk drug and tablets

using gradient RP-HPLC assay with UV detection Best resolution was determined


40

using a C18 column with acetonitrile-ammonium acetate buffer pH 4-

tetrahydrofuran (THF) as mobile phase Samples were eluted gradiently with the

mobile phase at flow rate of 1 mLmin and detected at 248 nm

Jemal et al [186] developed and validated a method for simultaneous quantitation

of both the acid and lactone forms of atorvastatin and both the acid and lactone

forms of its two biotransformation products 2-hydroxyatorvastatin and 4-

hydroxyatorvastatin in human serum by high-performance liquid chromatography

with electrospray tandem mass spectrometry The acid compounds were stable in

human serum at room temperature but the lactone compounds in serum could be

stabilized by lowering the working temperature to 4 0C or lowering the serum pH to

60 The intra-day inter-day precision and the deviations from the nominal

concentrations for all analytes were within 15 The required lower LOQ of 05

ngmL was achieved for each analyte

Bullen et al [187] developed and validated a liquid chromatographicmass

spectrometric method to quantitate atorvastatin and its active metabolites ortho-

hydroxy and para-hydroxy atorvastatin in human dog and rat plasma

Chromatographic separation of analytes was achieved by using a C-18 column with

a mobile phase consisting of acetonitrile-01 acetic acid (7030 vv) Analytes

were detected by tandem mass spectrometry The method proved suitable for

routine quantitation of atorvastatin o-hydroxyatorvastatin and p-

hydroxyatorvastatin over the concentration range of 0250 ngmL to 250 ngmL

Mean recoveries of atorvastatin o-hydroxyatorvastatin and p-hydroxyatorvastatin

from plasma ranged 100 -107 706 -104 and 476 -856

respectively Mean recoveries of the [d5]-AT and [d5]-o-AT internal standards

ranged 980 -999 and 973 respectively Inter assay precision for

atorvastatin o-hydroxyatorvastatin and p-hydroxyatorvastatin was lt or = 719

828 and 127 respectively Inter assay accuracy for atorvastatin o-


41

hydroxyatorvastatin and p-hydroxyatorvastatin was plusmn 106 586 and 158

respectively

22 Analytical Methods for Simvastatin

Apostolou et al [188] developed a fully automated high-throughput liquid

chromatographytandem mass spectrometry method for the simultaneous

quantification of simvastatin and simvastatin acid in human plasma Plasma

samples were treated by acetonitrile for protein precipitation and subsequent two-

step liquid-liquid extraction in 96-deepwell plates using methyl t-butyl ether as the

organic solvent The method was very simple with chromatographic run time of

just 19 min

Basavaiah et al [189] described two sensitive spectrophotometric methods for the

determination of simvastatin in bulk drug and in tablets The methods were based

on the oxidation of simvastatin by cerium (IV) in acid medium followed by

determination of unreacted oxidant by two different reaction schemes In one

procedure (method A) the residual cerium (IV) was reacted with a fixed

concentration of ferroin and the increase in absorbance was measured at 510 nm

The second approach (method B) involved the reduction of the unreacted cerium

(IV) with a fixed quantity of iron (II) and the resulting iron (III) was complexed

with thiocyanate and the absorbance measured at 470 nm In both methods the

amount of cerium (IV) reacted corresponded to simvastatin concentration The

systems obeyed Beers law for 06-75 microgmL and 05-50 microgmL for method A and

method B respectively

Basavaiah et al [190] developed two simple and sensitive spectrophotometric

methods for the determination of simvastatin in pure form and in tablets using in

situ generated bromine and p-phenylenediamine or o-dianisidine as reagents The

methods were based on the bromination of simvastatin by in situ bromine in acid


42

medium followed by the determination of unreacted bromine by reacting with p-

phenylenediamine and measuring the resulting red colour at 510 nm (method A) or

reacting with o-dianisidine and measuring the absorbance at 470 nm (method B)

Beerrsquos law was obeyed over the concentration ranges 20-120 microgmL and 2-12

microgmL for method A and method B respectively The LOD and LOQ for method A

were found to be 296 microgmL and 897 microgmL and the respective values for method

B were 014 microgmL and 042 microgmL The assay precision was less than 5 CV and

the accuracy was 9738-1034

Nigovi et al [191] developed a cathodic square-wave stripping voltammetry method

for the determination of simvastatin at trace levels The voltammetric response was

used to determine drug concentration in the range 1 times 10ndash8 molL to 75 times 10ndash7

molL with LOD of 45 times 10ndash9 molL

Arayne et al [192] developed a simple UV spectrophotometric method for the

determination of simvastatin in methanol and compared this with the existing

pharmacopoeial HPLC method Analytical parameters such as stability selectivity

accuracy and precision were established for the method in tablets and human

serum samples The method was validated according to ICH and USP guidelines

Jitender et al [193] developed and validated a sensitive HPLC assay for simvastatin

and its corresponding simvastatin hydroxyl acid for their simultaneous estimation

in solutions of various studies HPLC separations were achieved on (i) C8 (ii) CN

and (iii) C18 columns The eluents were monitored by diode array detector at 240

nm Retention times were simvastatin 8-9 min and simvastatin hydroxy acid 55-6

min The LOD of both on C-18 column was 005 microgmL and on C8 and CN

columns was 01 microgmL Inter and intra assay precision were less than 6

Malenović et al [194] developed a novel approach for the analysis of simvastatin

and its six impurities applying micro emulsions as mobile phase A micro


43

emulsion eluent containing 09 ww of di-isopropyl ether 17 ww of sodium

dodecyl-sulphate 70 ww of co-surfactant such as n-butanol and 904 ww of

aqueous 0025 M di-sodium phosphate pH 70 was used for the analysis

Separations were performed on a 35 microm X Terra 50 times 46 mm column at 30 0C

Detection was performed at 238 nm and the flow rate of the mobile phase was set

to be 03 mLmin

Coruh et al [195] studied the electrochemical behavior and determination of

simvastatin in aqueous alcohol medium at a stationary glassy carbon electrode

Cyclic voltammetry showed one main oxidation peak between pH 2 and 8

Differential pulse and square wave voltammetric techniques for the determination

of simvastatin in 01 M H2SO4 and a constant amount of methanol (20 ) allowed

quantitation over the 2 x 10-6-1 x 10-4 M range in supporting electrolyte with LOD

of 271 x 10-7 M and 550 x 10-7 M for differential pulse and square wave

voltammetric methods respectively

Abu-Nameh et al [196] proposed a simple and rapid HPLC method for the

determination of simvastatin using a C18 column and acetonitrile-phosphate buffer-

methanol (5 3 1 vvv) as a mobile phase with detection at 230 nm The linear

range for simvastatin was up to 1884 mg and a regression coefficient of 09995

Barrett et al [197] presented a validated highly sensitive and selective isocratic

HPLC method for the quantitative determination of simvastatin and its metabolite

simvastatin hydroxy acid Detection was done on triple quadrupole mass

spectrometer equipped with an ESI interface The linearity was in the concentration

range of 010-1600 ngmL for simvastatin and 010-1600 ngmL for simvastatin

hydroxyl acid Inter and intra-day precisions were lower than 7 for all analytes

The LOQ was 003 ngmL for simvastatin and 002 ngmL for simvastatin hydroxyl

acid


44

Godoy et al [198] developed a simple HPLC method for the determination of

simvastatin in tablet dosage forms The best results were obtained using

acetonitrile-003 M phosphate pH 45 buffer (7030) at a flow rate of 30 mLmin

Separation was achieved at room temperature on a C-18 monolithic column (100 x

46 mm) and the selected detection wavelength was 238 nm The retention time

was 147 minutes

Malenovic et al [199] used a novel and unique approach for retention modeling in

the separation of simvastatin and six impurities by liquid chromatography using a

micro emulsion as mobile phase Optimal conditions for the separation of

simvastatin and its six impurities were obtained using an X Terra 50 x 46 mm

column at 30 0C The mobile phase consisted of 09 ww of diisopropyl ether 22

ww of sodium dodecylsulphate 70 ww of co-surfactant such as n-butanol

and 899 ww of aqueous 0025 M disodium phosphate pH 7

Srinivasu et al [200] developed a micellar electrokinetic chromatographic method

for the quantification of lovastatin and simvastatin Lovastatin and simvastatin were

separated using an electrolyte system consisting of 12 acetonitrile (vv) in 0025

M sodium borate buffer pH 93 containing 0025 M sodium dodecyl sulphate with

an extended light path capillary Calibration curves were linear over the studied

ranges with correlation coefficients greater than 0996 An LOD of 32 microgmL and

LOQ of 106 microgmL were estimated for both the drugs

Tan et al [201] developed and validated a simple and sensitive reversed-phase

liquid chromatographic method for the analysis of simvastatin in human plasma

After extraction with cyclohexane-dichloromethane (351 VV) the drug was

measured by HPLC using a C18 column as stationary phase and an acetonitrile-

water (7030 VV) mixture as mobile phase The flow rate was 12 mLmin and

with UV detection at 237 nm The method was linear in the concentration range of


45

025-500 microgL Intra day and inter-day precision was less than 794 and 858

respectively The recoveries of simvastatin were greater than 933

Wang et al [202] developed a second derivative UV spectroscopic method for the

determination of simvastatin in the tablet dosage form They carefully choose zero-

crossing technique of second derivative UV measurement at 243 nm By using this

the selectivity and sensitivity of simvastatin was comparable to the previously

developed HPLC method

Ochiai et al [203] developed a highly sensitive and selective high performance

liquid chromatographic method for the determination of simvastatin (I) and its

active hydrolyzed metabolite (II) in human plasma Compounds were separately

extracted from plasma into two fractions Compound I in first fraction was

hydrolyzed to II A fluorescent derivative was then prepared by esterification with

1-bromoacetylpyrene in the presence of 18-crown-6 The pyrenacyl ester of II thus

obtained was purified on a phenyl boronic acid solid-phase extraction column and

was measured by column-switching HPLC with fluorescence detection The

calibration curves were linear in the concentration range of 01-10 ngmL The

intra-day precision was less than 110 and the accuracies were between 917

and 117 The LOQ for both analytes were 01 ngmL

Carlucci et al [204] developed and validated a fast simple and accurate method for

determining simvastatin and simvastatin acid concentrations in human plasma This

method involved an extraction procedure using a mixture of acetonitrile-water and

reversed-phase high-performance liquid chromatography with UV detection The

method was linear from 20 ngmL to 1000 ngmL for simvastatin and from 25

ngmL to 1000 ngmL for simvastatin acid respectively Relative standard

deviations less than 23 and relative errors of less than 52 were obtained from

human plasma controls containing simvastatin at identical concentrations


46

23 Analytical Methods for Lovastatin

Wang et al [205] developed a fast and sensitive ultra performance liquid

chromatography tandem mass spectrometry method for the determination of

lovastatin in human plasma Sample pretreatment involved one-step extraction with

n-hexane-methylene dichloride-isopropanol (20101 vvv) of 05 mL plasma

Chromatographic separation was carried out on a C 18 column with mobile phase

consisting of acetonitrile-water (containing 0005 M ammonium acetate 8515

vv) at a flow-rate of 035 mLmin The detection was performed on a triple-

quadrupole tandem mass spectrometer by multiple reactions monitoring via

electrospray ionization source with positive mode The analysis time was shorter

than 17 min per sample The method was linear in the concentration range of

0025-500 ngmL with LOQ of 0025 ngmL The intra and inter-day precision

values were below 11 and the accuracy (relative error) was within 60 at three

quality control levels

Yuan et al [206] developed a selective rapid and sensitive ultra performance liquid

chromatographyndashtandem mass spectrometry method for the quantitative

determination of lovastatin in human plasma Sample pretreatment involved a one-

step extraction with tert-butyl methyl ether The analysis was carried out on a C-18

column with flow rate of 035 mLmin The mobile phase was water and

acetonitrile 80 20 (vv) The detection was performed on a triple-quadrupole

tandem mass spectrometer by multiple reaction monitoring mode via electrospray

ionization (ESI) Method was linear in the concentration range of 008ndash

2450 ngmL with LOQ of 008 ngmL The intra and inter-day precision values

were below 15

Yu et al [207] developed and validated a sensitive and selective liquid

chromatographic tandem mass spectrometric method for analysis of lovastatin in

human plasma Ethyl acetate extraction was used for plasma sample preparation

Chromatographic separation was achieved on a C18 column by isocratic elution


47

with 831701 (vv) methanolndash0002 M aqueous sodium acetatendashformic acid as

mobile phase at a flow rate of 10 mLmin MSndashMS detection was performed using

positive electrospray ionization and multiple-reaction monitoring Method was

linear in the concentration range of 005 ngmL to 20 ngmL with LOQ of 005

ngmL Intra and inter-day precision were ranged from 04 to 114 with the

deviation always less than 15 Extraction recoveries were from 868 to 941

for lovastatin

Zhang et al [208] developed and validated a simple HPLC method for the

determination of lovastatin in rat tissues Samples were prepared by a simple

protein precipitation Separation was carried out on a C-18 column with a mobile

phase of acetonitrile 005 M ammonium acetate at a flow rate of 10 mLmin and

detection at 238 nm The method was linear from 00175 microgmL to 70 microgmL with

LOD of 0006 microgmL

Li et al [209] developed a simple and sensitive method for lovastatin in urine based

on capillary electrophoresis The following optimal conditions were determined for

stacking and separation electrophoretic buffer of 01 M Gly- NaOH (pH 1152)

sample buffer of 002 M Gly-HCl (pH 493) fused-silica capillary of 76 cmtimes75-microm

id (67 cm from detector) and sample injection at 14 mbar for 3 min A 21- to 26-

fold increase in peak height was achieved for detection of lovastatin in urine under

the optimal conditions compared with normal capillary zone electrophoresis The

LOD and LOQ for lovastatin in urine were decreased to 88 ngmL and 292

ngmL respectively The intra day and inter-day precision values were 223ndash361

and 403ndash505 respectively The recoveries of the analyte ranged from 8265

to 10049

Alvarez et al [210] described an HPLC stability-indicating method to study the

hydrolytic behaviour of lovastatin in different simulated fluids The selected

chromatographic conditions were a C-18 column acetonitrilemethanolphosphate


48

buffer solution pH 4 (323335) as mobile phase 45 ordmC temperature column flow

rate of 15 mLmin and UV detection at 238 nm Lovastatin exhibited a pH-

dependent degradation with an instantaneous hydrolysis in alkaline media at room

temperature One or two degradation products were observed when lovastatin was

hydrolyzed in alkaline or acid medium respectively

Orkoula et al [211] developed FT-Raman spectroscopy and HPLC methods for

monitoring the stability of lovastatin in the solid state in the presence of gallic acid

a natural antioxidant A Raman calibration curve was constructed using the area of

the strong but overlapping vibration mode of lovastatin at 1645 cm-1 and of the

gallic acid at 1595 cm-1 Mixtures of the active ingredient with the antioxidant were

heated in the presence of atmospheric air up to 120 0C The molar ratios of

lovastatin and gallic acid in the artificially oxidized mixtures were determined from

their Raman spectra using the calibration curve The HPLC analysis was based on a

reserved-phase C 18 column using a gradient elution program by varying the

proportion of solvent A acetonitrile 100 to solvent B 01 vv phosphoric acid

and a programmable diode array detection at 225 nm

Sharma et al [212] developed a simple validated HPLC method utilizing an

isocratic mobile phase with short retention times for cyclosporine A and lovastatin

Drugs were analysed by a reversed-phase HPLC method using a C18 column An

isocratic mobile phase containing acetonitrile and water in the proportions 7030

and 8020 was used for the HPLC analysis of cyclosporine A and lovastatin

respectively The flow-rate was 1 mLmin and detection was done at 238 nm at 25 0C The LOD were 250 ngmL and 10 ngmL and LOQ were 400 ngmL and 30

ngmL for cyclosporine A and lovastatin respectively The method was linear in

concentration range of 05-6 microgmL for cyclosporine A and 005-04 microgmL for

lovastatin


49

Ye et al [213] developed a simple rapid HPLC assay with ultraviolet detection for

the analytical determination of lovastatin and its acid in human plasma Sample

clean up involved the use of C10 solid-phase extraction cartridges LOQ was 100

ngmL Standard curves were linear from 100 ngmL to 5000 ngmL The assay

was able to measure steady-state lovastatin concentration at the initial dose level in

a phase I trial of lovastatin as a modulator of apoptosis

Strode et al [214] developed a reliable supercritical fluid chromatography method

for the analysis of lovastatin Methanol-modified carbon dioxide was used to elute

the drug and itrsquos dehydro lovastatin and hydroxy acid lovastatin degradation

products from a silica column The hydroxy acid lovastatin was tailed in this

mobile phase This was eliminated by the addition of trifluoroacetic acid to the

mobile phase which permitted the drug and its two main degradation products to

elute from the silica column in under 6 min with symmetrical peak shape

Mazzo et al [215] developed a flow injection method to determine simultaneously

lovastatin and butylated hydroxyanisole in tablets The system involved ultraviolet

absorbance detection for the drug and oxidative amperometric electrochemical

detection for butylated hydroxyanisole The method was found to be reproducible

for routine determinations with accuracy of plusmn 1 for lovastatin and plusmn 4 for

butylated hydroxyanisole Precision for both analytes was approximately plusmn 1

The method with UV detection was specific for the drug in the presence of

potential autoxidation products as well as butylated hydroxyanisole and its

oxidation products

Chaudhari et al [216] developed a simple and reproducible HPTLC method for the

separation and quantitation of simvastatin pravastatin sodium and rosuvastatin

calcium in pharmaceutical dosage forms The stationary phase used was precoated

silica gel The mobile phase was a mixture of chloroform methanol and toluene


50

(622 vvv) All the drugs were extracted from the respective tablets using

methanol The percentage recoveries ranged from 100 to 101 for simvastatin

98 to 101 for pravastatin sodium and 98 to 102 for rosuvastatin calcium

The LOD for simvastatin pravastatin sodium and rosuvastatin calcium were found

to be 15 ngspot 9 ngspot and 8 ngspot respectively and LOQ were 200 ngspot

for simvastatin and 100 ngspot for pravastatin sodium and rosuvastatin calcium

24 Analytical Methods for Rosuvastatin

Suslu et al [217] developed and validated a capillary zone electrophoretic method

with diode array detection for the determination of rosuvastatin calcium in

pharmaceutical formulations Optimum results were obtained with 005 M borate

buffer at pH 95 capillary temperature 30 0C and applied voltage 25 kV The

samples were injected hydrodynamically for 5 s at 50 mbar Detection wavelength

was set at 243 nm The migration times of rosuvastatin calcium and diflunisal were

320 plusmn 001 minutes and 420 plusmn 002 minutes The total time of analysis was less

than 6 minutes

Uyar et al [218] developed a simple rapid and reliable spectrophotometric method

for the determination of rosuvastatin calcium in pharmaceutical preparations The

solutions of standard and pharmaceutical samples were prepared in methanol at 243

nm The developed method was validated with respect to linearity range LOD and

LOQ accuracy precision specificity and ruggedness The linearity range of the

method was 10ndash600 microgmL and LOD was 033 microgmL

Gao et al [219] developed and validated a sensitive liquid chromatographytandem

mass spectrometric method for the determination of rosuvastatin in human plasma

Chromatographic separation was accomplished on a C18 column The mobile

phase consisted of methanol-water (7525 vv adjusted to pH 6 by aqueous

ammonia) Detection was achieved by ESI MSMS in the negative ion mode The


51

LOQ was 002 ngmL The linear range of the method was from 0020 to 600

ngmL The intra and inter-day precisions were lower than 85 and the accuracy

was within -03 to 19 in terms of relative error (RE)

Lan et al [220] developed and validated a simple and sensitive liquid

chromatographytandem mass spectrometry method for the quantification of

rosuvastatin in human plasma The analyte was extracted by simple one-step liquid-

liquid extraction The chromatographic separation was performed on a C18 column

with a mobile phase consisting of 2 formic acidmethanol (2090 vv) at a flow

rate of 100 mLmin The retention time of rosuvastatin was 23 Triple-quadrupole

MSMS detection was operated in positive mode by monitoring the transition of

mz 482--gt258 for rosuvastatin The LOQ was 01ngmL and the assay was linear

from 01-20 ngmL Inaccuracy was less than 84 and imprecision less than 128

at all tested concentration levels

Vittal et al [221] described a simple sensitive and specific high-performance liquid

chromatography method for simultaneous determination of rosuvastatin (RST) and

gemfibrozil (GFZ) in human plasma Following separation the residue was

reconstituted in the mobile phase and injected onto a C18 column The

chromatographic run time was less than 20 min using flow gradient (00-160

mLmin) with a mobile phase consisting of 001 M ammonium acetate acetonitrile

and methanol (504010 vvv) and UV detection at 275 nm Nominal retention

times of RST GFZ and IS were 67 min 139 min and 164 min respectively The

LOQ of RST and GFZ was 003 microgmL and 030 microgmL respectively Linearity

was in the 003-10 microgmL and 03-100 microgmL ranges for RST and GFZ

respectively The inter and intra-day precisions were in the range 237-978 and

092-1008 respectively


52

Kumar et al [222] developed a specific accurate precise and reproducible high-

performance liquid chromatography method for the estimation of rosuvastatin in rat

plasma The assay procedure involved simple liquid-liquid extraction After

separation rosuvastatin was reconstituted in the mobile phase and injected onto a

C18 column Mobile phase consisting of 005 M formic acid and acetonitrile

(5545 vv) was used at a flow rate of 10 mLmin The detection of the analyte

peak was achieved at 240 nm The standard curve for RST was linear in the

concentration range of 002-10 microgmL Absolute recovery of RST was 85-110 The

LOQ was 002 microgmL The inter and intra-day precisions were in the range of 724-

1243 and 228-1023 respectively Accuracy was in the range of 9305-11217

Mehta et al [223] applied a forced degradation study for the development of a

stability-indicating assay for the determination of rosuvastatin in the presence of its

degradation products Degradation of the drug was done at various pH values

Moreover the drug was degraded under oxidative photolytic and thermal stress

conditions The proposed method was able to resolve all of the possible degradation

products formed during the stress study

Hull et al [224] developed a selective accurate and precise assay for the

quantification of the N-desmethyl metabolite of rosuvastatin in human plasma The

method employed automated solid phase extraction followed by HPLC with

positive ion electrospray tandem MS The standard curve range for N-desmethyl

rosuvastatin in human plasma was 05-30 ngmL with 05 ngmL being the value of

LOQ

25 Analytical Methods for Gemfibrozil

Prabu et al [225] developed a simple precise and rapid RP-HPLC method for the

determination of racecadotril in a pharmaceutical formulation using gemfibrozil as


53

internal standard Ratio of the peak area of analyte to internal standard was used for

quantification The chromatographic separation was carried out by using a Reverse

Phase C18 column The mobile phase consisting of a mixture of 002 M phosphate

buffer (pH 35) and acetonitrile in the ratio of (4060) with detection at 230 nm at a

flow rate of 1 mLmin was employed The method was statistically validated for

linearity accuracy and precision

Kim et al [226] developed a sensitive and simple high performance liquid

chromatography for the determination of gemfibrozil in a small plasma sample

The analysis of gemfibrozil in the plasma sample was carried out using a reverse

phase C18 column with fluorescence detection (a maximum excitation at 242 nm

and a minimum emission at 300 nm) A mixture of acetonitrilendash04 phosphoric

acid solution (5347 vv) was used as a mobile phase The detection limit of this

method was 10 ngmL The method was linear over a range of 005 mgmL ndash15

mgmL The inter- and intra-day precision did not exceed 15

Ulu et al [227] developed and validated a simple selective precise and accurate

reversed phase-HPLC assay for analysis of gemfibrozil in tablets Separation and

quantification were achieved on a C-18 column under isocratic conditions using a

mobile phase (methanol water 8020 vv) maintained at 11 mLmin UV-

detection was at 280 nm The method was linear over the range of 05 microgmL ndash30

microgmL The LOD and LOQ were 020 microgmL and 051 microgmL respectively The

intra-day and inter-day precision were below 174 and 183 respectively

Roadcap et al [228] developed and validated a sensitive LCndashMSMS assay for the

quantitative determination of gemfibrozil in dog plasma The assay involved the

extraction of the analyte from dog plasma using Chem Elut cartridges and methyl

tert-butyl ether Chromatography was performed on a Metasil basic column (50times2

mm ID 3 microm) using a mobile phase consisting of 7030 acetonitrilendashammonium


54

acetate (0001 M pH 50) with a flow-rate of 02 mLmin The method showed

inter and intra-assay precision of less than 89 with inter and intra-assay accuracy

between 99 and 101

Gonzaacutelez-Pentildeas et al [229] developed a sensitive high-performance liquid

chromatographic assay for the quantitative determination of gemfibrozil The assay

involved a single cyclohexane extraction and LC analysis with fluorescence

detection Chromatography was performed at 40 0C on an ODS column The

mobile phase was a mixture of a solution of phosphoric acid 04 and acetonitrile

(4555) The detection limit was 0025 microgmL The method was linear from 005 to

05 microgmL Intra and inter-day precision was less than 15 Mean recovery was

9015 for gemfibrozil

Nakagawa et al [230] described sensitive and specific methods for the simultaneous

determination of gemfibrozil and its metabolites in plasma and urine The methods

were based on a fully automated high performance liquid chromatographic system

with fluorescence detection Urine samples diluted with acetonitrile were directly

analysed by HPLC using a flow and eluent programming method In the case of

plasma gemfibrozil and its main metabolites were extracted from acidified samples

and the resulting extracts injected into the chromatographic system The sensitivity

was approximately 100 ngmL for gemfibrozil and its four metabolites

Hengy et al [231] described a sensitive and specific method for the determination

of gemfibrozil at therapeutic concentrations in plasma The method was based on

high performance liquid chromatography Gemfibrozil and the internal standard

ibuprofen were extracted from acidified plasma into cyclohexane and the resulting

residue was analyzed on a commercial reversed phase column with

acetonitrilewater 11 and 02 phosphoric acid as mobile phase The eluted peaks

were detected by UV-absorption at 225 nm The sensitivity was approx 50 ngmL


55

26 Analytical Methods for Fenofibrate

Kadav et al [232] developed and validated a stability indicating UPLC method for

the simultaneous determination of atorvastatin fenofibrate and their impurities in

tablets The chromatographic separation was performed on C18 column (17 microm

21 mm times 100 mm) using gradient elution of acetonitrile and ammonium acetate

buffer (pH 47 001 M) at flow rate of 05 mLmin UV detection was performed at

247 nm Total run time was 3 min within which main compounds and six other

known and major unknown impurities were separated The method was validated

for accuracy repeatability reproducibility and robustness Linearity LOD and

LOQ

Nakarani et al [233] developed two simple and accurate methods to determine

atorvastatin and fenofibrate in combined dosage using second-derivative

spectrophotometry and reversed-phase liquid chromatography Atorvastatin and

fenofibrate in combined preparations were quantitated using the second-derivative

responses at 24564 nm for atorvastatin and 28956 nm for fenofibrate in spectra of

their solution in methanol The method was linear in the concentration range of 3ndash

15 microgmL for atorvastatin and fenofibrate In the HPLC method analysis was

performed on a C-18 column in the isocratic mode using the mobile phase

methanol-water (90 + 10 vv) adjusted to pH 55 with orthophosphoric acid at a

flow rate of 1 mLmin Measurement was made at a wavelength of 24672 nm The

method was linear in the concentration range of 3ndash15 microgmL for atorvastatin and

fenofibrate

Straka et al [234] determined steady-state fenofibric acid serum concentrations

using anion-exchange solid-phase extraction in combination with reverse-phase

HPLC Chromatographic separation under isocratic conditions with use of

ultraviolet detection at 285 nm provided clean baseline and sharp peaks for

clofibric acid 1-napthyl acetic acid (internal standards) and fenofibric acid The


56

assay was employed to quantify fenofibric acid in more than 800 human subject

specimens Fenofibric acid analysis was found to be linear over the range of 05

mgL to 40 mgL Accuracies ranged from 9865 to 1024 whereas the within-

and between-day precisions ranged from 10 to 22 and 20 to 62

respectively

El-Gindy et al [235] presented several spectrophotometric and HPLC methods for

the determination of fenofibrate vinpocetine and their hydrolysis products The

resolution of either fenofibrate or vinpocetine and their hydrolysis products were

accomplished by using numerical spectrophotometric methods as partial least

squares (PLS-1) and principal component regression (PCR) applied to UV spectra

and graphical spectrophotometric methods as first derivative of ratio spectra (1DD)

or first (1D) and second (2D) derivative spectrophotometry for vinpocetine and

fenofibrate respectively In addition HPLC methods were developed using ODS

column with mobile phase consisting of acetonitrile-water (8020 vv pH 4) with

UV detection at 287 nm for fenofibrate and a mobile phase consisting of

acetonitrile-0001 M KH2PO4 containing 01 diethylamine (6040 vv pH 46)

with UV detection at 270 nm for vinpocetine The proposed methods were

successfully applied for the determination of each drug and its hydrolysis product

in laboratory-prepared mixture and pharmaceutical preparation

Yardimci et al [236] investigated the electrochemical reduction of fenofibrate at a

hanging mercury drop electrode by cyclic voltammetry square-wave voltammetry

and chronoamperometry The best analytical signals was found in borate buffer

(pH 90)ndashtetra butyl ammonium iodide mixture containing 125 (vv) methanol at

ndash12 V (versus AgAgCl) According to cyclic voltammetric studies the reduction

was irreversible and diffusion controlled The diffusion coefficient was 238times10ndash

6 cm2 sndash1 as determined by chronoamperometry Under optimized conditions of

square-wave voltammetry a linear relationship was obtained between 0146ndash


57

496 microgmL of fenofibrate with LOD of 0025 microgmL Validation parameters such

as sensitivity accuracy precision and recovery were evaluated

Hernando et al [237] described a multi residue method for the extraction and

determination of two therapeutic groups of pharmaceuticals lipid-regulating agents

(clofibric acid bezafibrate gemfibrocil fenofibrate) and beta-blockers (atenolol

sotalol metoprolol betaxolol) in waters by solid-phase extraction followed by

liquid chromatography-electrospray ionization tandem mass spectrometry

Recoveries obtained from spiked HPLC water as well as from spiked real samples

were all above 60 with the exception of betaxolol with a 52 recovery The

quantitative MS analysis was performed using a multiple reaction monitoring The

LC-MS-MS method gave detection limits ranging from 0017 microgL to 125 microgL in

spiked effluent Precision of the method ranged from 37 to 185

Lossner et al [238] described a sensitive HPLC method for the determination of

fenofibric acid (FA) in serum FA from human serum samples was isolated by an

easy one step extraction procedure with a mixture of n-hexane and ethyl acetate

(9010 vv) The recovery was 848 of the total FA in serum The compound was

separated isocratically on a reversed phase column with acetonitrile and 002 M

phosphoric acid (5545 vv) at a flow-rate of 1 mLmin Absorbance at 287 nm was

recorded for quantification The LOD was 003 microgmL and the LOQ was 01

microgmL

Streel et al [239] developed a new fully automated method for the determination of

fenofibric acid in plasma which involved the solid-phase extraction (SPE) of the

analyte from plasma on disposable extraction cartridges (DECs) and reversed-phase

HPLC with UV detection After extraction 100 microL of the extract was directly

introduced into the HPLC system The liquid chromatographic separation of the

analytes was achieved on a RP-8 stationary phase The mobile phase consisted of a

mixture of methanol and 004 M phosphoric acid (6040 vv) The analyte was

monitored photometrically at 288 nm The absolute recovery was close to 100


58

and a linear calibration curve was obtained in the concentration range from 025

microgmL to 20 microgmL The mean RSD values for repeatability and intermediate

precision were 17 and 39 respectively

Lacroix et al [240] developed HPLC methods for drug content and HPLC and

NMR methods for related compounds in fenofibrate raw materials The HPLC

methods resolved 11 known and six unknown impurities from the drug The HPLC

system was comprised of ODS column a mobile phase consisting of acetonitrile

water trifluoroacetic acid in the ratio of 700300l (vvv) at a flow rate of 1

mLmin and a UV detector set at 280 nm Minimum quantifiable amounts were

about 01 for three of the compounds and less than 005 for the other eight

Individual impurities in 14 raw materials ranged from trace levels to 025 and

total impurities from 004 to 053 (ww) Six unknown impurities were detected

by HPLC all at levels below 010 An NMR method for related compounds was

also developed and it was suitable for 12 known and several unknown impurities

The results for related compounds by the two techniques were consistent The main

differences stem from the low sensitivity of the HPLC method for some of the

related compounds at 280 nm or from the higher limits of quantitation by the NMR

method for several other impurities using the conditions specified Results for the

assay of 15 raw materials by HPLC were within the range 985-1015

Abe et al [241] developed a reliable HPLC method for the determination of

fenofibric acid and reduced fenofibric acid in the biological samples After addition

of the internal standard solution and 05 M HCl to the biological sample fenofibric

acid reduced fenofibric acid and the internal standard were extracted with a mixed

solvent of n-hexane and ethyl acetate (9010) from the mixture The acids were

back-extracted from the organic phase with 01 M Na2HPO4 and then re-extracted

from the aqueous phase with a mixed solution of n-hexane and ethyl acetate (955)

after addition of 05 M HCl The organic phase was evaporated to dryness under


59

the vacuum The residue was dissolved in methanol and diluted with distilled

water An aliquot of the resulting solution was injected on the HPLC

Masnatta et al [242] developed a selective high-performance liquid

chromatographic method to assess either bezafibrate ciprofibrate or fenofibric acid

plasma levels Drugs were extracted with diethyl ether after acidification with

HCL An isocratic acetonitrile-002 M H3PO4 (5545) mobile phase a C18 column

and UV detection were used The LOQ was 025 microgmL for the three fibrates Intra-

and inter-assay accuracy ranged were 90-107 and 82-111 96-115 and 94-

107 94-114 and 94-126 for bezafibrate ciprofibrate and fenofibric acid

respectively Intra- and inter-assay precision were 172-306 and 266-767

188-464 and 062-299 126-469 and 356-717 for the three fibrates

studied

27 Analytical Methods for Ezetimibe

Doshi et al [243] developed and validated a simple precise and accurate HPLC

method for the assay of ezetimibe in tablets and for determination of content

uniformity Reversed-phase liquid chromatographic separation was achieved by use

of phosphoric acid (01 vv)ndashacetonitrile 5050 (vv) as mobile phase The

method was validated for specificity linearity precision accuracy robustness and

solution stability Method was linear in the concentration range of 20ndash80 microgmL

Accuracy was between 1008 and 1027

Dixit et al [244] established a simple selective and stability-indicating HPTLC

method for the analysis of simvastatin and ezetimibe The method used aluminum-

backed silica gel 60F254 TLC plates as stationary phase with n-hexanendashacetone 64

(vv) as mobile phase Densitometric analysis of both drugs was carried out in

absorbance mode at 234 nm Method was linear in the range of 200ndash1600 ngband

The LOD and LOQ were 25 ngband and 150 ngband respectively Simvastatin


60

and ezetimibe were subjected degradation by acid pH 68 phosphate buffer

oxidation dry heat and wet heat The degradation products were well resolved

from the pure drug with significantly different R F values

Sharma et al [245] developed UV first second and third derivative

spectrophotometric methods for the determination of ezetimibe in pharmaceutical

formulation For the first method based on UV spectrophotometry the quantitative

determination of the drug was carried out at 233 nm and the linearity range was

found to be 6-16 microgmL For the first second and third derivative

spectrophotometric methods the drug was determined at 2595 nm 269 nm and 248

nm with the linearity ranges 4-14 microgmL 4-14 microgmL and 4-16 microgmL

Basha et al [246] accomplished simultaneous separation and quantification of

ezetimibe (EZM) and its phase-I metabolite ie ezetimibe ketone (EZM-K) and

phase-II metabolite ie ezetimibe glucuronide (EZM-G) in various matrices by

gradient HPLC with UV detection The assay involved deproteinization of 500 microL

of either incubation or bile sample containing analytes and internal standard (IS

theophylline) with 75 microL acetonitrile containing 25 perchloric acid An aliquot

of 100 microL supernatant was injected onto a C-18 column The chromatographic

separation was achieved by gradient elution consisting of 005 M formic acid

acetonitrile methanol water at a flow rate of 1 mLmin The detection of analyte

peaks were achieved at 250 nm Average extraction efficiencies of EZM EZM-G

and IS was greater than 75-80 and for EZM-K was greater than 50 from all

the matrices tested LOQ for EZM EZM-K and EZM-G was 002 microgmL

Rajput et al [247] developed a simple accurate and precise spectroscopic method

for the simultaneous estimation of ezetimibe and simvastatin in tablets using first

order derivative zero-crossing method Ezetimibe showed zero crossing point at

2454 nm while simvastatin showed zero crossing point at 2652 nm The method

was linear in the range of 5-40 microgmL for ezetimibe at 26520 nm The linear


61

correlation was obtained in the range of 5-80 microgmL for simvastatin at 2454 nm

The limit of detection was 039 microgmL and 012 microgmL for ezetimibe and

simvastatin respectively The LOQ was 110 microgmL and 04 microgmL for ezetimibe

and simvastatin respectively

Singh et al [248] developed a stability-indicating HPLC method for the analysis of

Ezetimibe in the presence of the degradation products Ezetimibe was subjected to

different ICH prescribed stress conditions It involved a C-8 column and a mobile

phase composed of ammonium acetate buffer (002 M pH adjusted to 70 with

ammonium hydroxide) and acetonitrile which was pushed through the column in a

gradient mode The detection was carried out at 250 nm The method was validated

for linearity range precision accuracy specificity selectivity and intermediate

precision

Oliveira et al [249] developed and validated an analytical method based on liquid

chromatography-tandem mass spectrometry for the determination of ezetimibe in

human plasma Ezetimibe and etoricoxib (internal standard) were extracted from

the plasma by liquid-liquid extraction and separated on a C-18 analytical column

with acetonitrile water (8515 vv) as mobile phase Detection was carried out by

positive electrospray ionization (ESI+) in multiple reactions monitoring (MRM)

mode The chromatographic separation was obtained within 20 min and the

method was linear in the concentration range of 025ndash20 ngmL for free ezetimibe

and of 1ndash300 ngmL for total ezetimibe The mean extraction recoveries for free

and total ezetimibe from plasma were 9614 and 6411 respectively

Oswald et al [250] developed a selective assay to measure serum concentrationndash

time profiles renal and fecal elimination of ezetimibe in pharmacokinetic studies

Ezetimibe was measured after extraction with methyl tert-butyl ether using 4-

hydroxychalcone as internal standard and liquid chromatography coupled with

tandem mass spectrometry (LCndashMSMS) for detection The chromatography was


62

done isocratically with acetonitrilewater (6040 vv flow rate 200 microlmin) using

C-18 Column The MSMS analysis was performed in the negative ion mode The

validation ranges for ezetimibe and total ezetimibe were as follows serum 00001ndash

0015 microgmL and 0001ndash02 microgmL urine and fecal homogenate 0025ndash10 microgmL

and 01ndash20 microgmL respectively

Sistla et al [251] developed a rapid specific reversed-phase HPLC method for

assaying ezetimibe in pharmaceutical dosage forms The assay involved an

isocratic elution of ezetimibe on a C18 column using a mobile phase composition

of water (pH 68 005 wv 1-heptane sulfonic acid) and acetonitrile (3070 vv)

The flow rate was 05 mLmin and the analyte monitored at 232 nm The assay was

linear from 05 to 50 microgmL All the validation parameters were within the

acceptance range

CHAPTER 3 EXPERIMENTAL WORK

63

3 EXPERIMENTAL WORK

The experimental requirements used throughout this work are given here including

chemicals reagents and apparatus with detailed description of solvents chemicals

reagents and their source The detailed description of HPLC instruments and other

chromatographic conditions are mentioned against each method

All the chemicals and solvents used in these experiments were of HPLC andor

analytical reagent grade

31 Solvents

The details of solvents and their source are given as

Distilled water (DW) Prepared in our Laboratory

Acetonitrile (ACN) Merck Fluka

Methanol Merck Fluka

32 Chemicals

Chemicals used in these experiments are given as under along with their source

Ammonium acetate Merck Fluka

Acetic acid Merck Fluka

Sodium hydroxide Merck Fluka

Hydrochloric acid Merck Fluka

Hydrogen peroxide Merck Fluka

Starch Schazoo Laboratories Lahore

Magnesium Stearate Schazoo Laboratories Lahore

Lactose Schazoo Laboratories Lahore

Avicel Schazoo Laboratories Lahore

Atorvastatin Schazoo Laboratories Lahore

Simvastatin Schazoo Laboratories Lahore

Lovastatin Xenon Laboratories Lahore


64

Rosuvastatin Schazoo Laboratories Lahore

Gemfibrozil Atco Laboratories Karachi

Fenofibrate Getz Pharma Karachi

Ezetimibe Schazoo Laboratories Lahore

Zetab Plus Tablets Schazoo Laboratories Lahore

Vytorin Tablets Schering-Plough Pharmaceuticals

Whatmann Filter paper No 41 Local Market

33 Analytical equipments

To perform the best procedures for analysis along with its cost effectiveness and

convenient use following analytical instruments were employed

a) Analytical balance Sartorius Gottigen

Model CP324S

Min 00001g

Max 320 g

b) pH meter CHEMCADET

Model 5986-62

c) Vacuum pump Ulvic Sinku Kiko

Model DA-60D

d) Sonicator Notus- Powersonic

Model PS 02000A

e) Nylon Filters (Pore Size 045 microm) Milliopore (USA)

34 Glass Apparatus

To achieve high accuracy and reliability of the results of research work calibrated

glassware was used All glassware was washed thoroughly with distilled water and

then rinsed with methanol and dried before use

a) Beaker (50 mL 100 mL 250 mL 500 mL and 1000 mL capacity)

b) Macro pipettes (10 mL 20 mL 50 mL and 100 mL capacity)


65

c) Micro pipettes (10-100 microL 100-1000 microL)

d) Thermometers (0- 500 OC)

e) Filtration Assembly (Millipore USA)

f) Graduated cylinders (50 mL 100 mL 250 mL and 1000 mL)

g) Measuring flasks (10 mL 50 mL 100 mL 250 mL 500 mL and 1000 mL)

h) Measuring cylinders (50 mL 100 mL 250 mL and 500 mL)

i) Round bottom flasks (500 mL)

j) Glass Funnel


66

35 Atorvastatin calcium and Ezetimibe

351 Preparation of mobile phase

The mobile phase was prepared by mixing 01M ammonium acetate (pH 65) and

acetonitrile in the ratio of 2872 (vv) The pH of the ammonium acetate solution

was adjusted to 65 with 10 glacial acetic acid before mixing with acetonitrile It

was filtered through 045 microm nylon filters and was degassed by sonication before

using in the HPLC system

352 Preparation of standard solution

The standard stock solution of atorvastatin calcium and ezetimibe (02 mgmL

each) was prepared in few mL of methanol by taking 10 mg each of atorvastatin

(base) and ezetimibe in 50 mL volumetric flask and then completing the volume up

to the mark with methanol The solution was prepared in methanol because both

drugs are very much soluble in methanol The working standard solution (32

microgmL for both) was prepared by diluting the stock solution with mobile phase

353 Linearity

The method was linear in the concentration range of 12-52 microgmL for both

atorvastatin and ezetimibe Five different concentrations of solutions in the

mentioned range for both atorvastatin calcium and ezetimibe (12 microgmL 22

microgmL 32 microgmL 42 microgmL and 52 microgmL) were used to verify the linearity Each

concentration was made in triplicate

354 Limits of detection and Limits of quantitation (LOD and LOQ)

Limit of detection (LOD) is the lowest concentration of an analyte that can be

detected by the proposed method It is generally referred to as a concentration when

the signal to noise ratio is usually 31 The limit of quantitation (LOQ) is the lowest

concentration of an analyte that can be determined with acceptable accuracy with a

signal to noise ratio of 101 Two types of solutions ie blank and spiked with

known progressively decreasing concentrations of each analyte were prepared and

analysed The LOD was then calculated by the evaluation of minimum level at

which the analyte can be readily detected The LOQ was calculated by the


67

evaluation of minimum level at which the analyte can be readily quantified with

accuracy

355 Accuracy

The accuracy of the method was evaluated by the addition of known amounts of

atorvastatin calcium and ezetimibe to the sample solution The results obtained

were compared with the theoretical concentration 3 mL sample solution of

atorvastatin calcium and ezetimibe (02 mgmL each) were transferred to four

different 50 mL volumetric flasks already containing 10 20 30 and 40 mL of

standard solution (02 mgmL) The volume was then completed up to the volume

the final concentrations thus obtained was equivalent to 160 microgmL 200 microgmL

240 microgmL and 280 microgmL Each concentration was made in triplicate

356 Precision

Precision of the proposed method was expressed in terms of RSD The within-

day precision was based upon the results of five replicate analysis of three different

concentrations of analytes on a single day The between-day precision was

determined from the same samples analyzed for five consecutive days

357 Selectivity

The selectivity of the proposed method was checked by making a synthetic mixture

of both the analytes with commonly occurring excipients that are found in most

tablet formulations and then measuring the percentage recovery of each component

Also its chromatograms were compared with the chromatograms of reference

solution For synthetic mixture 20 mg each of atorvastatin and ezetimibe and 30 mg

each of starch lactose magnesium stearate and avicel that may be representing as

interfering substances were accurately weighed and transferred into a 100 mL

volumetric flask 70 mL of methanol was added and shaked well The volume was

then completed with methanol and the mixture was filtered 4 mL of this filtrate

was transferred into a 25 mL volumetric flask and the mobile phase was added up

to volume to give a final concentration of 32 microgmL each


68

358 Robustness

Robustness of the proposed method was evaluated by intentionally modifying the

chromatographic conditions such as composition and flow rate of the mobile phase

and pH of the buffer solution The percentage recovery along with the classical

chromatographic parameters of each analyte such as retention time tailing factor

and number of theoretical plates were measured at each changed conditions

359 Forced Degradation study

Forced degradation study was carried out using different ICH prescribed stress

conditions such as acidic basic oxidative and thermal stresses to assess the

specificity of the method For acidic stress 4 mL of the standard stock solution was

refluxed for 1 hour with 1 mL of 1M hydrochloric acid cooled neutralized with

1M NaOH and diluted up to 25 mL with mobile phase For basic stress 4 mL of

standard stock solution was treated with 1 mL of 1M NaOH stayed it at room

temperature for 3 hours neutralized with 1M HCl and then diluted to 25 mL with

mobile phase For oxidative stress 1 mL of 5 H2O2 and 4 mL of standard stock

solution were refluxed for 30 minutes cooled to room temperature and then diluted

up to 25 mL with mobile phase For thermal stress 4 mL of the standard stock

solution was refluxed for 3 hours cooled and then diluted to 25 mL with mobile

phase The stressed samples after completion of stress conditions were analyzed by

the proposed method and the percentage degradation of each analyte was calculated

under each condition

3510 Stability of Solutions

The stability of each component in the presence of other in solution was assessed

by analyzing the samples after 24 48 and 72 hrs and then determining their

RSD

3511 Application of the Method

Twenty tablets were accurately weighed to get their average weight and then they

were ground manually using pestle and mortar An amount of powder equivalent to

20 mg each of atorvastatin and ezetimibe was accurately weighed and transferred to


69

a 100 mL volumetric flask About 70 mL of methanol was then added and it was

shaked for 5 minutes to extract all the active analytes After that the volume was

made up to volume with methanol The concentration thus achieved was 02

mgmL atorvastatin and 02 mgmL ezetimibe The solution was filtered manually

using Whatmann No 41 filter paper and a glass funnel After filtration the

solutions were diluted with mobile phase to get a final concentration of 32 microgmL

each

3512 HPLC Set Up

1 HPLC System Varian Prostar

2 HPLC Pump Prostar 210

3 Detector UV

4 Wavelength 242 nm

5 Injector Rheodyne

6 Mobile Phase 01M ammonium acetate (pH 65) and

acetonitrile in the ratio of 2872 (vv)

7 Flow rate 05 mLmin

8 Temperature Room temperature (25 plusmn 2 0C)

9 Column Phenyl-2 column (25046 mm)

10 Particle size 5 microm


70

36 Ezetimibe and Simvastatin


A mobile phase was prepared by mixing 01M ammonium acetate buffer pH 50

and acetonitrile in the ratio of 3070 vv The mobile phase was filtered using 045

microm nylon filters and was degassed by sonication before use


A stock standard solution containing 04 mgmL each of ezetimibe and simvastatin

was prepared by dissolving 20 mg each of ezetimibe and simvastatin in mobile

phase in 50 mL volumetric flask and raising the volume up to the mark To prepare

the working standard solution (40 microgmL for both ezetimibe and simvastatin) the

stock standard solution was diluted with mobile phase

363 Linearity


ezetimibe and simvastatin Five solutions in the range of 20-60 microgmL for both

ezetimibe and simvastatin (20 microgmL 30 microgmL 40 microgmL 50 microgmL and 60

microgmL) were used to evaluate the linearity Each concentration was used in

triplicate

364 Limit of detection and Limits of quantitation

Two types of solutions ie blank and spiked with known progressively decreasing

concentrations of each analyte were prepared and analysed The limit of detection

(LOD) and limit of quantification (LOQ) was then established by evaluating the

minimum level at which the analyte can be readily detected and quantified with

accuracy

365 Accuracy

The accuracy of the method was performed by adding known amounts of ezetimibe

and simvastatin to placebo solution and then comparing the added amount with the

observed amount Three levels of solutions were made which correspond to 50

100 and 150 of the nominal analytical concentration ie 40 microgmL each Each

level was made in triplicate


71

366 Precision




determined from the same samples analyzed for three consecutive days

367 Selectivity



tablet formulations and then measuring the percentage recovery of both ezetimibe

and simvastatin along with chromatographic parameters Also its chromatograms

were compared with the chromatograms of reference solution For synthetic

mixture 20 mg each of ezetimibe and simvastatin and 30 mg each of starch

lactose magnesium stearate and avicel were transferred to a 50 mL volumetric

flask sonicated with 30 mL of mobile phase for 15 minutes and then diluted up to

the mark with mobile phase The solution was filtered using Whatmann filter paper

no 41 and the filtrate was diluted with mobile phase to get a final concentration of

40 microgmL for both ezetimibe and simvastatin

368 Robustness

Robustness of the proposed method was evaluated by intentionally but slightly

modifying the chromatographic conditions such as composition and flow rate of the

mobile phase and pH of the buffer solution The percentage recovery along with the

classical chromatographic parameters of each analyte such as retention time tailing

factor and number of theoretical plates were measured at each changed conditions

369 Forced degradation study


conditions such as acidic basic oxidative and thermal stresses For acidic stress

25 mL of the standard stock solution was refluxed for 1 hour with 2 mL of 1M

hydrochloric acid cooled neutralized with 1M NaOH and diluted up to 25 mL

with mobile phase For basic stress 25 mL of standard stock solution was treated


72

with 1mL of 1M NaOH stayed it at room temperature for 2 hours neutralized with

1M HCl and then diluted to 25 mL with mobile phase For oxidative stress 2 mL of

5 H2O2 and 25 mL of standard stock solution were refluxed for 3 hours cooled

to room temperature and then diluted up to 25 mL For thermal stress 25 mL of

the standard stock solution was refluxed for 3 hours cooled and then diluted to 25

mL with mobile phase The stressed samples after completion of stress conditions

were analyzed by the proposed method and the percentage degradation of each

analyte was calculated under each condition

3610 Stability of solutions

The stability of each component in the presence of other was assessed by analyzing

the samples after 24 48 and 72 hrs and then determining their RSD


Twenty tablets were accurately weighed to get the average weight and then they

were homogenized by grinding manually using pestle and mortar An accurately

weighed quantity of homogenized powder equivalent to 20 mg each of ezetimibe

and simvastatin was placed in 50 mL volumetric flask 30 mL mobile phase was

added and the flask was shaken for 5 minutes so as to completely extract all the

drugs The volume was then made up to the mark with mobile phase to get a

solution containing 04 mgmL ezetimibe and 04 mgmL simvastatin Solution was

then filtered using Whatmann filter paper No 41 manually using a glass funnel and

diluted with mobile phase to obtain a final concentration of 40 microgmL ezetimibe

and 40 microgml simvastatin


73

3612 HPLC Set Up

1 HPLC System Shimadzu LC-10A

2 HPLC Pump LC-10AT pump

3 Detector UV

4 Wavelength 240 nm

5 Injector Rheodyne





9 Column C-18 column (25046 mm)



74

37 Gemfibrozil and Simvastatin






A Stock solution of gemfibrozil and simvastatin was prepared at about 60 mgmL

and 01 mgmL respectively in mobile phase The working standard solution 240

microgmL for gemfibrozil and 4 microgmL for simvastatin were prepared by diluting the

stock solution with mobile phase

373 Linearity

Linearity of the proposed method was checked by analyzing seven solutions in the

range of 60-420 microgmL for gemfibrozil (60 microgmL 120 microgmL 180 microgmL 240

microgmL 300 microgmL 360 microgmL 420 microgmL) and 1-7 microgmL for simvastatin (1

microgmL 2 microgmL 3 microgmL 4 microgmL 5 microgmL 6 microgmL 7 microgmL) Each level was

made in triplicate


For calculating the LOD and LOQ values solutions with known decreased

concentrations of analytes were injected into the HPLC system The limit of

detection (LOD) and quantification (LOQ) were then measured by calculating the

minimum level at which the analytes can be readily detected and quantified with

accuracy respectively

375 Accuracy

Method accuracy was performed by adding known amounts of gemfibrozil and

simvastatin to the pre-analysed synthetic mixture solution and then comparing the

added concentration with the found concentration Three levels of solutions were

made which correspond to 50 100 and 150 of the nominal analytical

concentration (240 microgmL for gemfibrozil and 4 microgmL for simvastatin) Each level

was made in triplicate


75

376 Precision

Precision of the proposed method was expressed in terms of RSD For

evaluating the within-day precision results of five replicate analysis of three

different concentrations of samples were calculated on a single day The between-

day precision was calculated from the same samples analyzed on five different

days

377 Selectivity



tablet formulations and then calculating its percentage recovery in the presence of

excipients Also the chromatograms of synthetic mixture were compared with the

chromatogram of the reference standard to check any kind of interference

Synthetic mixture containing 600 mg gemfibrozil 10 mg simvastatin and 20 mg

each of starch lactose magnesium stearate and avicel which are present as

excipients in the pharmaceutical formulation were accurately weighed and

transferred into 100 mL volumetric flask The mixture was shaked well with 70 mL

mobile phase and then the volume was completed with mobile phase and filtered 1

mL of this filtrate was transferred into 25 mL volumetric flask and mobile phase

was then added to volume to obtain a final solution containing 240 microgmL

gemfibrozil and 4 microgmL simvastatin

378 Robustness

Robustness of the method was performed by intentionally but slightly changing the


and pH of the buffer solution The percentage recovery along with chromatographic

parameters of each analyte such as retention time tailing factor and number of

theoretical plates were measured at each changed conditions



conditions such as acidic basic oxidative and thermal stresses


76

For acidic stress 2 mL of the standard stock solution was refluxed for 1 hour with

1 mL of 1M hydrochloric acid cooled after reflux neutralized with 1M NaOH and

diluted up to 50 mL with mobile phase For basic stress 2 mL of standard stock

solution was refluxed with 1mL of 1M NaOH for 2 hours cooled after the

completion of reflux neutralized with 1M HCl and then diluted to 50 mL with



up to 50 mL For thermal stress 2 mL of the standard stock solution was refluxed

for 3 hours cooled and then diluted to 25 mL with mobile phase The stressed

samples after completion of stress conditions were analyzed by the proposed

method and the percentage degradation of each analyte was calculated under each

condition




3711 HPLC Set Up



3 Detector UV

4 Wavelength 237 nm

5 Injector Rheodyne








77

38 Ezetimibe and Fenofibrate





382 Preparation of standard solutions

To prepare the standard stock solution of ezetimibe and fenofibrate (02 mgmL and

32 mgmL respectively) 20 mg of ezetimibe and 320 mg of fenofibrate reference

standards were accurately weighed in 100 mL of volumetric flask 70 mL of mobile

phase was added sonicated for 15 minutes to dissolve completely and then volume

was completed up to the mark with mobile phase The working standard solution

(16 microgmL ezetimibe and 256 microgmL fenofibrate) was prepared by diluting 2 mL of

the standard stock solution to 25 mL with mobile phase

383 Linearity

To prepare the calibration curve and to evaluate the linearity five different

concentrations were made and analyzed in the range of 08 to 40 microgmL for

ezetimibe (08 microgmL 16 microgmL 16 microgmL 28 microgmL and 40 microgmL) and 128

to 640 microgmL for fenofibrate (128 microgmL 256 microgmL 256 microgmL 448 microgmL

and 640 microgmL) Each concentration was made and analyzed in triplicate

384 Limit of detection and limit of quantitation

To calculate the LOD and LOQ values serials of dilutions were made and analysed

by the proposed method The limit of detection and quantification were then

established by evaluating the level at which the analyte can be readily detected and

quantified with accuracy respectively

385 Accuracy

To determine the accuracy known amounts of the ezetimibe and fenofibrate were

added to pre-quantified sample solution and then experimental and theoretical

results were compared Three levels of solutions were made which corresponds to


78

50 100 and 150 of the nominal analytical concentration ie 16 microgmL

ezetimibe and 256 microgmL fenofibrate

386 Precision




day precision was calculated from the same samples analyzed on three different

days

387 Selectivity





chromatogram of the reference standard to check any kind of interference For

synthetic mixture 20 mg of ezetimibe 320 mg of fenofibrate and 30 mg each of

starch lactose magnesium stearate and avicel were transferred to a 100 mL

volumetric flask sonicated with 70 mL of mobile phase for 15 minutes and then

diluted up to the mark with mobile phase The solution was filtered using

Whatmann filter paper no 41 and the filtrate was diluted with mobile phase to get a

final concentration of 16 microgmL ezetimibe and 256 microgmL fenofibrate

388 Robustness

Deliberate modifications were made in the operating conditions of the method to

assess the robustness of the method For this purpose slight changes were made in

the composition of the mobile phase flow rate and pH of the ammonium acetate

solution and then percentage recovery of each analyte along with chromatographic

parameters such as retention time tailing factor and number of theoretical plates

were calculated




79

conditions such as acidic basic oxidative and thermal stresses For acidic stress 2

mL of the standard stock solution was refluxed for 2 hours with 1 mL of 1M

hydrochloric acid cooled neutralized with 1 M NaOH and diluted up to 25 mL


with 1 mL of 1 M NaOH stayed it at room temperature for 3 hours neutralized

with 1 M HCl and then diluted to 25 mL with mobile phase For oxidative stress

1mL of 5 H2O2 and 2 mL of standard stock solution were refluxed for 3 hours

cooled to room temperature and then diluted up to 25 mL For thermal stress 2 mL

of the standard stock solution was refluxed for 3 hours cooled and then diluted to

25 mL with mobile phase The stressed samples after completion of stress

conditions were analyzed by the proposed method and the percentage degradation

of each analyte was calculated under each stress




3811 Application of the method



weighed quantity of homogenized powder equivalent to 10 mg of ezetimibe and

160 mg fenofibrate was placed in 50 mL volumetric flask 30 mL mobile phase was



solution containing 02 mgmL ezetimibe and 32 mgmL fenofibrate Solution was



and 256 microgmL fenofibrate


80

3812 HPLC Set Up


2 HPLC Pump LC-20AT

3 Detector photodiode array (PDA) detector

4 Wavelength 240 nm

5 Injector Rheodyne








81

39 Ezetimibe and Lovastatin


The mobile phase was prepared by mixing 01M ammonium acetate buffer (pH

50) and acetonitrile in the ratio of 2872 (vv) The mobile phase was then filtered

through 045 microm nylon filters and degassed before use


The standard stock solution of lovastatin and ezetimibe was prepared by dissolving

20 mg lovastatin and 10 mg ezetimibe to a small amount of mobile phase in a 50

mL volumetric flask and then raising the volume up to the mark with mobile phase

The concentration thus achieved was equivalent to 400 microgmL and 200 microgmL for

lovastatin and ezetimibe respectively To prepare the working solution a volume

equal to 25 mL of the standard solution was taken to 50 mL measuring flask and

raised its level up to the mark with mobile phase This furnishes a concentration of

20 microgmL and 10 microgmL lovastatin and ezetimibe respectively

393 Linearity

To prepare the calibration curve and to evaluate the linearity seven different

concentrations were made and analyzed in the range of 02-100 microgmL for

ezetimibe (02 microgmL 08 microgmL 25 microgmL 10 microgmL 25 microgmL 50 microgmL and

100 microgmL) and 04-200 microgmL for lovastatin (04 microgmL 16 microgmL 5 microgmL 20

microgmL 50 microgmL 100 microgmL and 200 microgmL) Each concentration was made and

analyzed in triplicate

394 Limits of detection and Limits of quantitation





395 Accuracy

To determine the accuracy known amounts of the ezetimibe and lovastatin were

added to pre-quantified synthetic mixture solution and then experimental and


82

theoretical results were compared Three levels of solutions were made which

corresponds to 50 100 and 150 of the nominal analytical concentration ie

10 microgmL for ezetimibe and 20 microgmL for lovastatin

396 Precision





days

397 Selectivity




excipients and also comparing its chromatogram with the chromatograms of

standard solution to check any kind of interference Synthetic mixture containing

10 mg ezetimibe 20 mg lovastatin and 30 mg each of starch lactose magnesium

stearate and avicel which are present as excipients in the pharmaceutical

formulation were accurately weighed and transferred into 100 mL volumetric flask

The mixture was shaked well with about 70 mL of mobile phase and then the

volume was completed with mobile phase and filtered 25 mL of this filtrate was

transferred into 25 mL volumetric flask and mobile phase was then added to

volume to obtain a final solution containing 10 microgmL for ezetimibe and 20 microgmL

for lovastatin

398 Robustness


assess the robustness of the method For this purpose slight change were made in


solution and then percentage recovery each analyte along with chromatographic


83


were calculated

399 Forced Degradation Study

Degradation studies were performed to evaluate the specificity of the method Four

types of degradation studies were performed to both lovastatin and ezetimibe in

combination This includes acidic basic oxidative and thermal stress

For acidic stress 1 mL of 1M HCl was added to 1 mL of lovastatin and ezetimibe

standard solution and was refluxed for 1 hour After completion of stress the

solution was neutralized with 1 M NaOH solution (as required) and was then

finally diluted up to 25 mL with mobile phase For basic stress 1 mL of 1 M NaOH

was added to 1 mL of lovastatin and ezetimibe standard solution This solution was

kept at room temperature for 30 minutes Afterwards the solution was neutralized

with 1M HCl solution and was diluted up to 25 mL with mobile phase For

oxidative stress 1 mL of 5 H2O2 was added to 1mL of lovastatin and ezetimibe

standard solution and was refluxed for 15 minutes Finally it was diluted to 25 mL

with mobile phase For thermal stress 1 mL of lovastatin and ezetimibe stock

solution was refluxed for 2 hours and then diluted up to 25 mL with mobile phase

The stressed samples after completion of stress conditions were analyzed by the

proposed method and the percentage degradation of each analyte was calculated

under each stress





84

3911 HPLC Set Up


2 HPLC Pump LC-20AT


4 Wavelength 240 nm

5 Injector Rheodyne








85

310 Atorvastatin and Gemfibrozil



50) and acetonitrile in the ratio of 4555 (vv) It was then filtered through 045 microm

nylon filters and degassed prior to use


The standard stock solution of atorvastatin and gemfibrozil (02 mgmL and 12

mgmL respectively) was prepared by dissolving 10 mg atorvastatin and 600 mg

gemfibrozil to a small amount of mobile phase in a 50 mL volumetric flask and

then raising the volume upto the mark with mobile phase To prepare the working

solution a volume equal to 1 mL of the standard solution was taken to 25 mL

measuring flask and raised its level upto the mark with mobile phase This

furnishes a concentration of 8 microgmL and 480 microgmL atorvastatin and gemfibrozil

respectively

3103 Linearity

The method was linear in the concentration range of 01-20 microgmL for atorvastatin

and 6-1200 microgmL for gemfibrozil Seven solutions in the range of 01-20 microgmL

for atorvastatin (01 microgmL 05 microgmL 1 microgmL 25 microgmL 8 microgmL 15 microgmL

and 20 microgmL) for atorvastatin and 6-1200 microgmL (6 microgmL 30 microgmL 60 microgmL

150 microgmL 480 microgmL 900 microgmL and 1200 microgmL) for gemfibrozil were used to

evaluate the linearity Each concentration was made and analyzed in triplicate






accuracy


86

3105 Accuracy

The accuracy of the method was performed by adding known amounts of

atorvastatin and gemfibrozil to pre-quantified standard solution and then comparing

the added amount with the observed amount Three levels of solutions were made

which correspond to 50 100 and 150 of the nominal analytical

concentration Each level was made in triplicate

3106 Precision

The precision was expressed in terms of RSD The within-day precision was

based upon the results of five replicate analysis of three different concentrations of

analytes on a single day The between-day precision was determined from the same

samples analyzed for three consecutive days

3107 Selectivity






10 mg atorvastatin 600 mg gemfibrozil and 30 mg each of starch lactose

magnesium stearate and avicel which are present as excipients in the

pharmaceutical formulation were accurately weighed and transferred into 100 mL

volumetric flask The mixture was shaked well with about 70 mL of mobile phase

and then the volume was completed with mobile phase and filtered 2 mL of this

filtrate was transferred into 25 mL volumetric flask and mobile phase was then

added to volume to obtain a final solution containing 8 microgmL for atorvastatin and

480 microgmL for gemfibrozil

3108 Robustness



and pH of the buffer solution The classical chromatographic parameters of each


87

analyte such as retention time tailing factor and number of theoretical plates were

measured at each changed conditions



type of degradation was performed that is acidic basic oxidative and thermal 1

mL of 1M HCl was added to 1 mL of atorvastatin and gemfibrozil standard

solution and was refluxed for 1hour Afterwards the solution was neutralized with

1M NaOH solution and was finally diluted upto 25 mL with mobile phase 1 mL of

1M NaOH was added to 1 mL of atorvastatin and gemfibrozil standard solution and

was refluxed for 45 minutes Afterwards the solution was neutralized with 1M HCl

solution and was finally diluted upto 25 mL with mobile phase 1 mL of 5 H2O2

was added to 1mL of atorvastatin and gemfibrozil and standard solution and was

refluxed for 30 minutes Finally it was diluted to 25 mL with mobile phase 1 mL

of gemfibrozil stock solution was refluxed for 3 hours and then diluted up to 25 mL

with mobile phase The stressed samples after completion of stress conditions were

analyzed by the proposed method and the percentage degradation of each analyte

was calculated under each condition



the samples after 24 48 and 72 hrs


88

31011 HPLC Set Up


2 HPLC Pump LC-20AT


4 Wavelength 240 nm

5 Injector Rheodyne








89

311 Rosuvastatin and Ezetimibe


A mobile phase was prepared by mixing 1 phosphoric acid and acetonitrile in the

ratio of 4060 vv The mobile phase was filtered using 045 microm nylon filters and

was degassed by sonication before use


The standard stock solution of rosuvastatin and ezetimibe was prepared by taking

40 mg rosuvastatin and 10 mg ezetimibe in 50 mL volumetric flask About 30 mL

of mobile phase was added and the mixture was shaken for 15 minutes to dissolve

all the components This provided a concentration of rosuvastatin and ezetimibe

equivalent to 800 microgmL and 200 microgmL respectively The working standard

solution (80 microgmL rosuvastatin and 20 microgmL ezetimibe) was prepared by diluting

5 mL of the standard stock solution to 50 mL with mobile phase

3113 Preparation of sample solution



weighed quantity of homogenized powder equivalent to 40 mg of rosuvastatin and

10 mg ezetimibe was placed in 50 mL volumetric flask 30 mL mobile phase was



solution containing 08 mgmL rosuvastatin and 02 mgmL ezetimibe Solution

was then filtered using Whatmann filter paper No 41 manually using a glass funnel

and diluted with mobile phase to obtain a final concentration of 80 microgmL

rosuvastatin and 20 microgmL ezetimibe

3114 Linearity



rosuvastatin (08 microgmL 5 microgmL 20 microgmL 80 microgmL 120 microgmL 140 microgmL

and 160 microgmL) and 02 to 40 microgmL for ezetimibe (02 microgmL 125 microgmL 5


90

microgmL 20 microgmL 30 microgmL 35 microgmL and 40 microgmL) Each concentration was

made and analyzed in triplicate



by the proposed method The limit of detection (LOD) and quantification (LOQ)

were then established by evaluating the level at which the analyte can be readily

detected and quantified with accuracy respectively

3116 Accuracy

To determine the accuracy known amounts of the rosuvastatin and ezetimibe were


results were compared Three levels of concentrations were made which


80 microgmL rosuvastatin and 20 microgmL ezetimibe

3117 Precision

The precision of the proposed method was expressed in terms of RSD For



day precision was calculated from the same samples analyzed in three different

days

3118 Selectivity

For checking selectivity a synthetic mixture of rosuvastatin and ezetimibe with

commonly occurring tablet excipients was prepared and analyzed by the proposed

method and then calculating its percentage recovery in the presence of excipients

and also comparing its chromatogram with the chromatograms of standard solution

to check any kind of interference For synthetic mixture 80 mg of rosuvastatin 20

mg of ezetimibe and 30 mg each of starch lactose magnesium stearate and avicel

were transferred to a 100 mL volumetric flask sonicated with 60 mL of mobile

phase for 15 minutes and then diluted up to the mark with mobile phase The

solution was filtered using Whatmann filter paper no 41 and the filtrate was


91

diluted with mobile phase to get a final concentration of 80 microgmL rosuvastatin and

20 microgmL ezetimibe

3119 Robustness



the composition of the mobile phase flow rate and concentration of phosphoric

acid in the solution and the percentage recovery of the analytes along with

chromatographic parameters such as retention time tailing factor and number of

theoretical plates were calculated




For acidic stress 25 mL of the standard stock solution was refluxed for 2 hours

with 1 mL of 1M hydrochloric acid cooled neutralized with 1M NaOH and


solution was treated with 1 mL of 1M NaOH stayed it at room temperature for 3

hours neutralized with 1M HCl and then diluted to 25 mL with mobile phase For

oxidative stress 1 mL of 5 H2O2 and 25 mL of standard stock solution were

refluxed for 3 hours cooled to room temperature and then diluted up to 25 mL For

thermal stress 25 mL of the standard stock solution was refluxed for 3 hours

cooled and then diluted to 25 mL with mobile phase The stressed samples after

completion of stress conditions were analyzed by the proposed method and the

percentage degradation of each analyte was calculated under each stress





92

31112 HPLC Set Up


2 HPLC Pump LC-20AT


4 Wavelength 240 nm

5 Injector Rheodyne

6 Mobile Phase 1 phosphoric acid and acetonitrile in the

ratio of 4060 (vv)





CHAPTER 4 RESULTS AND DISCUSSIONS

93

4 RESULTS AND DISCUSSIONS 41 Atorvastatin calcium and Ezetimibe 411 Method Development and Optimization

In this work the aim was to develop a simple isocratic accurate and sensitive

HPLC method for the simultaneous determination of atorvastatin and ezetimibe in

their fixed dose combination Initially various mobile phases and stationery phases

were tested to obtain the best separation and resolution between atorvastatin and

ezetimibe The mobile phase of 01M ammonium acetate (pH 65) and acetonitrile

in the ratio of 2872 (vv) and Hypersil Phenyl-2 column were found to be the most

appropriate for the separation of both the components at a the flow rate of 05 mL

min Using the mentioned chromatographic conditions well resolved sharp peaks

can be obtained at retention time of 306 and 446 minutes for atorvastatin and

ezetimibe respectively The chromatograms of standard and tablet solutions of

atorvastatin and ezetimibe are shown in Fig 41 and 42

Method development was started with less polar mobile phase (50 acetonitrile)

however no peak could be obtained The polarity of the mobile phase was then

increased by the addition of 01M ammonium acetate A ratio of 2872 (vv) for

ammonium acetate and acetonitrile resulted in good separation and sharp peaks

The optimum mobile phase composition was found to be 01M ammonium acetate

(pH 65) and acetonitrile in the ratio of 2872 (vv)

412 Method validation

The developed chromatographic method for the simultaneous determination of

atorvastatin calcium and ezetimibe was validated using ICH guidelines [252-253]

Validation parameters performed include linearity limit of detectionquantitation

selectivity specificity accuracy precision robustness and stability of solutions

4121 Linearity

Linearity of the proposed method was verified by analyzing five solutions in the

range of 12-52 microgmL for both atorvastatin and ezetimibe (12 microgmL 22 microgmL

32 microgmL 42 microgmL and 52 microgmL) Each concentration was used in triplicate


94

Good linearity was observed over the above range for both atorvastatin and

ezetimibe The calibration curve was made using concentration of the analytes

versus peak area The coefficient of determination from the linear regression

analysis was calculated and found to be greater than 09966 in case of both the

analytes This indicates that there exists a good linear relationship between

concentration of drugs and the peak area The linear regression equation for

atorvastatin was Y= 00154 x + 00238 with value of coefficient of determination

equal to 09966 whereas the linear regression equation for ezetimibe was Y=

00448 x + 00665 with 09993 as the value of coefficient of determination


Two types of solutions ie blank and spiked with known concentrations of each

analyte were prepared and analysed The limit of detection (LOD) and

quantification (LOQ) were then established by evaluating the signal to noise ratio

of 31 and 101 respectively The LOD was found to be 011 microgmL and 007

microgmL for atorvastatin and ezetimibe respectively The LOQ was found to be 025

microgmL and 018 microgmL for atorvastatin and ezetimibe

4123 Accuracy

The accuracy of the method was performed by making synthetic mixtures

containing various amounts of atorvastatin and ezetimibe (160 200 240 and 280

microgmL each) and then analyzed by the proposed method The mean percentage

recovery and the RSD were calculated from recovery experiments The data is

shown in Table 41 The recovery range and the relative standard deviation for each

of the analytes were found to be 9825-10175 and 011-124 respectively

4124 Precision

The precision of the proposed method was determined by the analysis of three

different concentrations in terms of RSD The within-day precision was based

upon the results of five replicate analysis of three different concentrations of



95

samples analyzed for five consecutive days The results of within-day and between-

day precision are given in Table 42

4125 Selectivity



tablet formulations such as starch lactose magnesium stearate and avicel The

percentage recovery of each component was then calculated in the presence of

excipients Also its chromatograms were compared with the chromatograms of

standard solution to check any kind of interference The results showed no

interference as evident from recovery results and no co-eluting peaks The data is

given in Table 43


The stability of each component in the presence of other in solution was checked

by determining the percentage RSD of replicate injections of the same solution

over a period of 72 hours The analytes were stable for the mentioned period as

given in Table 44

4127 Robustness

Robustness of the method was performed by intentionally but slightly modifying

the chromatographic conditions The results showed that the slight change in the

chromatographic conditions had no pronounced effects on the chromatographic

parameters The results of the robustness study are given in Table 45 and 46


96

Figure 41 Chromatograms of atorvastatin calcium and ezetimibe reference substance

Figure 42 Chromatograms of atorvastatin calcium and ezetimibe Tablets


97

Table41 Recovery experiments of the proposed HPLC method

Drug Concentration Amount recovered Recovery RSD

(microgmL) (microgmL) ()

Atorvastatin calcium 160 1616 10100 105

200 2028 10140 029

240 2368 9867 042

280 2812 10043 124

Ezetimibe 160 1588 9925 057

200 1965 9825 086

240 2442 10175 168

280 2782 9936 011

Table42 Within-day and Between-day precision of the proposed HPLC method

Compound Conc n Within-day precision Between-day precision

(microgmL) Mean RSD () Mean RSD ()

Atorvastatin calcium 160 5 1628 111 1636 159

320 5 3215 103 3248 151

480 5 4772 086 4861 125

Ezetimibe 160 5 1570 070 1633 135

320 5 3252 083 3158 089

480 5 4882 039 4802 110


98

Table43 Selectivity of the proposed HPLC method

Atorvastatin calcium

Added Recovered recovery

(microgmL) (microgmL)

Ezetimibe



32 3218 10056

32 3162 9881

32 3178 9931

32 3252 10162

Mean recovery = 10008

RSD = 126

32 3251 10159

32 3186 9956

32 3158 9869

32 3224 10075


RSD = 128


99

Table44 Stability study of atorvastatin calcium and ezetimibe in solution

Concentration Recovered concentration (microgmL)

(microgmL) After 24 hrs After 48 hrs After 72 hrs RSD ()


160 1573 1582 1615 138

320 3148 3168 3150 035

480 4818 4798 4880 089

Ezetimibe

160 1632 1611 1630 074

320 3281 3242 3218 094

480 4772 4848 4820 114


100

Table 45 Robustness study of Atorvastatin

Conditions Assay RT1 (min) Theoretical plates Tailing

Acetonitrile buffer (7228) 10029 306 3425 122

Acetonitrilebuffer (7030) 10105 345 3640 118


Flow rate (04mLmin) 10184 383 3507 125

Flow rate (06 mLmin) 9858 255 3310 141

Buffer (pH 63) 10089 303 3401 120

Buffer (pH 67) 10154 302 3467 121

1RT Retention Time

Table 46 Robustness study of Ezetimibe





Flow rate (04mLmin) 9802 558 5221 115

Flow rate (06 mLmin) 9915 372 5019 118

Buffer (pH 63) 10022 441 5186 110

Buffer (pH 67) 10005 443 5125 111

1RT Retention Time


101


To evaluate the specificity of the proposed method different stress conditions were

applied to both atorvastatin and ezetimibe in combination form The stress

conditions applied were acid base oxidation and thermal stress Under acidic

conditions atorvastatin was degraded up to 40 whereas the degradation of

ezetimibe was only 52 Under basic conditions no degradation occurred for

atorvastatin whereas ezetimibe was degraded up to 45 Oxidative stress

conditions degraded atorvastatin to 88 and to ezetimibe to only 6 Thermal

stress had no effect on the degradation of ezetimibe whereas atorvastatin was

degraded to only 2 In all the stress conditions the degradation products peaks

were separated from the peaks of both the analytes which shows that the method is

specific in the presence of degradation products

413 Application of the method in tablets

The application of the proposed HPLC method was checked by analyzing the

atorvastatin calcium and ezetimibe in their combined tablet formulations The

results obtained showed high percentage recoveries (9900-10203) and low RSD

(048-146) values These results confirm the suitability of the proposed method for

the routine determination of atorvastatin and ezetimibe in their combined tablet

formulations The results are given in Table 47


102

Table47 Analysis of atorvastatin calcium and ezetimibe in tablets




Ezetimibe



32 3262 10194

32 3215 10047

32 3168 9900

Mean recovery =10047

RSD = 146

32 3256 10175

32 3256 10056

32 3248 10203


RSD = 048


103


421 Method Development and Optimization

Simvastatin is an official drug in United States Pharmacoepia [254] while

ezetimibe is not found in any Pharmacoepial convention The HPLC method for

simvastatin tablets described by USP used phosphate buffer pH 45 and acetonitrile

in the ratio of 3565 (vv) as a mobile phase and C-18 column as stationary phase

The column temperature is maintained at 45 oC The USP method therefore offers

stringent chromatographic conditions that can also have a negative impact on the

column life

The aim of the present study was to develop a simple isocratic accurate and

sensitive HPLC method for the simultaneous determination of ezetimibe and

simvastatin in their fixed dose combination Initially various mobile phases and

stationery phases were tested to obtain the best separation and resolution between

ezetimibe and simvastatin The mobile phase consisting of 01M ammonium

acetate buffer pH 50 and acetonitrile in the ratio of (3070 vv) was found

appropriate for separation of both the components using a Merck C-18 column The

chromatographic conditions were optimized to get good resolution between the two

analytes The mobile phase composition was varied from 4060 (vv) buffer-

acetonitrile to 2080 (vv) buffer-acetonitrile in order to assess the impact of the

acetonitrile content on the separation and chromatographic parameters like

resolution tailing factor and number of theoretical plates Although increase of

acetonitrile contents to 80 reduced the retention time of simvastatin to 6 minutes

and resolution between ezetimibe and simvastatin to about 7 but tailing was greater

than 13 with fewer theoretical plates as compared to the plates obtained using

optimum mobile phase composition (3070 vv buffer-acetonitrile) The decrease

of acetonitrile contents to 60 resulted in the elution of simvastatin after 18

minutes with almost the same tailing factor So by applying the optimum

chromatographic conditions resolved sharp peaks that belong to ezetimibe and


104

simvastatin were obtained at retention times of 295 and 980 minutes respectively

[Figure 43 and 44]



ezetimibe and simvastatin was validated using ICH guidelines Assessed validation

parameters include linearity limit of detectionquantitation selectivity specificity

accuracy precision robustness and stability of solutions

4221 Linearity

Linearity of the proposed method was done by analyzing five solutions in the range

of 20-60 microgmL for both ezetimibe and simvastatin (20 microgmL 30 microgmL 40

microgmL 50 microgmL and 60 microgmL) Each concentration was used in triplicate Good

linearity was observed over the above range for both ezetimibe and simvastatin

The calibration curve was made using concentration of the analytes versus peak

area The correlation coefficient from the linear regression analysis was calculated

and found to be greater than 09996 in case of both the analytes This indicates that

there exists a good linear relationship between concentration of drugs and the peak

area The linear regression equation for ezetimibe was Y= 001868 x -000302 with

value of correlation coefficient equal to 09996 whereas the regression equation for

simvastatin was Y= 002284 x -000548 with 09992 as the value of correlation

coefficient

4222 Limit of detection and Limit of quantitation



and quantification was then established by evaluating the minimum level at which

the analyte can be readily detected and quantified with accuracy The LOD was

found to be 006 microgmL and 005 microgmL for ezetimibe and simvastatin respectively

(signal to noise ratio of 31) The LOQ was found to be 019 microgmL and 017

microgmL for ezetimibe and simvastatin (signal to noise ratio of 101)


105

Figure 43 Chromatograms of ezetimibe and simvastatin reference substance

Figure 44 Chromatograms of ezetimibe and simvastatin Tablets


106

4223 Accuracy




100 and 150 of the nominal analytical concentration Each level was made in

triplicate The recovery range and the relative standard deviation for each of the

analytes were found to be 9912-10150 and 038-138 respectively [Table

48]

4224 Precision




determined from the same samples analyzed for three consecutive days The results

of within-day and between-day precision are given in Table 49

4225 Selectivity




in the presence of excipients along with chromatographic parameters Also its

chromatograms were compared with the chromatograms of reference substance

The results show no interference from the excipients [Table 410]



by analyzing the samples after 24 48 and 72 hrs The relative standard deviation of

peak area was less than 044 The results are presented in Table 411 which

indicates good stability for each drug


107

Table 48 Results of recovery experiments of the proposed HPLC method

Drug Level n Concentration Amount recovered Recovery RSD

() (microgmL) (microgmL) () ()

Ezetimibe 50 3 200 2005 10025 138

100 3 400 3965 9912 068

150 3 600 6020 10033 086

Simvastatin 50 3 200 2030 10150 038

100 3 400 4025 10062 115

150 3 600 6060 10100 102

Table 49 Within and Between-day precision of the proposed HPLC method



Ezetimibe 200 5 1986 110 1995 078

400 5 4012 105 3990 115

600 5 5996 028 6012 120

Simvastatin 200 5 2024 145 2010 056

400 5 4056 068 3975 132

600 5 5942 075 6025 088


108

Table 410 Selectivity of the proposed HPLC method

Drugs age recovery n RT1 Resolution Tailing Factor TP2

Ezetimibe 10062 5 296 - 123 6781

Simvastatin 9943 5 980 1964 106 13752

1 Retention time 2 Theoretical Plates

Table 411 Stability study of ezetimibe and simvastatin in solution

Concentration Recovered concentration


After 24hrs After 48hrs After 72hrs RSD ()

Ezetimibe

200 2032 2009 1985 024

400 4076 3990 4040 043

600 5970 6025 6056 044

Simvastatin

200 2012 1995 2005 008

400 4035 4025 4020 008

600 6015 6025 5975 026


109

4227 Robustness


the chromatographic conditions The results showed that the change of the

conditions had no pronounced effects on the chromatographic parameters The

results of the robustness study are given in Table 412 amp 413



applied to both ezetimibe and simvastatin in combination form The percentage

degradation of each analyte was then calculated under each stress condition The

stress conditions applied were acid base oxidation and thermal stress Under

acidic conditions ezetimibe was degraded up to 5 whereas the degradation of

simvastatin was 43 Under basic conditions ezetimibe was degraded up to 45

whereas simvastatin to only 13 Oxidative conditions degraded ezetimibe to

about 20 and to simvastatin to only 3 Thermal stress had no effect on the

degradation and the drugs remain almost intact during this treatment In all the

stress conditions the degradation products peaks were separated from the peaks of

both the analytes which shows that the method is specific in the presence of

degradation products


The proposed HPLC method was applied for the determination of ezetimibe and

simvastatin in their pharmaceutical formulations [Table 414] The recovery of the

data and the agreement between the label claim and the amount found were

excellent This confirms the suitability of the proposed method for the routine

quality control determination of ezetimibe and simvastatin in pharmaceutical

formulations


110


Conditions Assay RT (min) Theoretical plates Tailing




Flow rate (14 mLmin) 9842 318 6810 125

Flow rate (16 mLmin) 9905 278 6566 127

Buffer (pH 48) 10022 299 6685 125

Buffer (pH 52) 10089 301 6628 124

Table 413 Robustness study of Simvastatin





Flow rate (14 mLmin) 10033 1052 13927 105

Flow rate (16 mLmin) 9915 919 13425 107

Buffer (pH 48) 10129 985 13564 108

Buffer (pH 52) 10086 984 13416 108


111

Table 414 Results of analysis of ezetimibe and simvastatin in tablets

Drug n Amount claimed Amount found Mean Recovery RSD

(mg per tablet) (mg per tablet) () ()

Ezetimibe 5 10 1012 10125 075

Simvastatin 5 10 1005 10050 115


112



Gemfibrozil is a cholesterol lowering drug belonging to the fibrate class In

addition to cholesterol lowering it also has the ability to lower the incidence of

coronary heart disease in human beings [255-256] Simvastatin is an HMG CoA

reductase inhibtor lowering cholesterol with the same mechanism as other statins

Many patients with coronary artery disease do not respond well with single agent

therapy The combination of gemfibrozil and an HMG CoA reductase are ideal and

recent reports confirm the efficacy of combination of gemfibrozil and an HMG

CoA reductase [257-264] The combination is also FDA approved and in view of

the efficacy of this combination many pharmaceutical companies are going to

launch the combination of gemfibrozil with simvastatin and atorvastatin In the

present work therefore the conditions were optimized for the development and

validation of a simple and accurate HPLC method for the simultaneous

determination of gemfibrozil and simvastatin in synthetic mixture form for future

possible use in the combined form Method development was started with 01 M

ammonium acetate pH 50 and acetonitrile in the ratio of 3070 (vv) based on our

previous results obtained during method development for ezetimibe and simvastatin

combination At this composition although both components were eluted but

resolution was greater than 20 and retention time of simvastatin was about 16

minutes The acetonitrile contents of the mobile phase were then increased to

decrease resolution and retention time At the composition of 1585 (01 M

ammonium acetate pH 50 and acetonitrile) both components were eluted with a

good resolution The most appropriate mobile phase composition was thus found to

be 01M ammonium acetate pH 50 and acetonitrile in the ratio of 1585 (vv)

Under the described experimental conditions sharp peaks that belong to

gemfibrozil and simvastatin were obtained at retention times of 465 and 768

minutes respectively as shown in Figure 45


113


The developed chromatographic method was validated using ICH guidelines

Validation parameters performed include linearity limit of detection and

quantitation selectivity specificity robustness accuracy precision and stability of

solutions

4321 Linearity

The calibration curve was linear over the concentration range of 60-420 microgmL for

gemfibrozil and 1-7 microgmL for simvastatin Good linearity was observed over the

above range for both gemfibrozil and simvastatin The calibration curve was made

using concentration of the analytes versus peak area The correlation coefficient in

both cases was found to be greater than 09999 which manifests a linear

relationship between concentration and the peak area The linear regression

equation for gemfibrozil was found to be Y= 5112 x + 226 with correlation

coefficient equal to 099995 The linear regression equation for simvastatin was

found to be Y= 35679 x ndash 0365 with value of correlation coefficient equal to

099997

4322 Limits of detection and Quantitation




minimum level at which the analytes can be readily detected (signal to noise ratio

of 31) and quantified (signal to noise ratio of 101) with accuracy respectively In

this study the LOD was found to be 013 microgmL and 002 microgmL for gemfibrozil

and simvastatin respectively The LOQ was found to be 039 microgmL and 006

microgmL for gemfibrozil and simvastatin respectively

4323 Accuracy





114



was made in triplicate The recovery and the relative standard deviation for each of

the analytes are given in Table 415

4324 Precision





days The results of within-day and between-day precision are presented in Table

416

4325 Selectivity





chromatogram of the reference standard to check any kind of interference The

percentage recovery is presented in Table 417 The chromatogram of gemfibrozil

and simvastatin in synthetic mixtures is given in Figure 46 showing selectivity of

the proposed method



the samples after 24 48 and 72 hrs The relative standard deviation of peak area

was less than 130 The results are presented in Table 418 which indicates good

stability for each drug


115

Figure 45 Chromatograms of Gemfibrozil and simvastatin reference substance


116

Table 415 Accuracy of the proposed HPLC method

Drug level n Added Conc Found Conc recovery RSD

() (microgmL) (microgmL)

Gemfibrozil 50 5 1200 12022 10018 095

100 5 2400 23734 9889 043

150 5 3600 35421 9839 042

Simvastatin 50 5 20 202 10100 133

100 5 40 406 10150 119

150 5 60 593 9883 074

Table 416 Precision of the proposed HPLC method



Gemfibrozil 1200 5 12125 078 11958 125

2400 5 24456 095 24258 102

3600 5 36521 124 36321 085

Simvastatin 20 5 202 144 201 106

40 5 396 111 395 058

60 5 607 036 602 131


117

Figure 46 Chromatograms of Gemfibrozil and simvastatin in a synthetic mixture


118


Gemfibrozil



Simvastatin



240 23645 9852

240 24142 10059

240 24356 10148

240 23988 9995


RSD = 125

4 405 10125

4 396 9900

4 398 9950

4 393 9825


RSD = 128


119

Table 418 Stability study of gemfibrozil and simvastatin in solution




Gemfibrozil

1200 11808 11788 11756 022

2400 24262 23943 23640 130

3600 35828 35641 35494 047

Simvastatin

20 201 199 197 101

40 398 395 391 089

60 602 595 591 093


120

4327 Robustness


the chromatographic conditions The results showed that the variance of the

conditions had no pronounced effects to that of actual The results of the robustness

study are given in Table 419 amp 420



applied to both gemfibrozil and simvastatin in combination form The stress


conditions gemfibrozil was degraded up to 14 whereas the degradation of

simvastatin was 27 Under basic conditions gemfibrozil was degraded to about

31 whereas simvastatin to only 8 Oxidative conditions degraded gemfibrozil

to about 45 whereas no degradation occurred for simvastatin under these

conditions Thermal stress had no effect on the degradation of gemfibrozil whereas

degradation of simvastatin was only 3 In all the stress conditions the

degradation products peaks were separated from the peaks of both the analytes

which shows that the method is specific in the presence of degradation products


121

Table 419 Robustness study of Gemfibrozil

Conditions Assay () RT1 (min) TP2 Tailing




Flow rate (11mLmin) 9869 424 7118 128

Flow rate (09 mLmin) 10041 518 8002 122

Buffer (pH 52) 9889 465 7719 123

Buffer (pH 48) 10115 465 7662 123

1Retention Time

2Theoretical Plates






Flow rate (11mLmin) 9921 698 10220 129

Flow rate (09 mLmin) 9903 853 12515 122

Buffer (pH 52) 10069 765 11308 122

Buffer (pH 48) 10098 766 11015 122

1Retention Time

2Theoretical Plates


122


441 Method development and Optimization

In this work chromatographic conditions were developed and optimized for the

development and validation of an isocratic and simple HPLC method for the

simultaneous determination of ezetimibe and fenofibrate The main aim during this

method development was to apply the simple mobile phase with short retention

time tailing factor less than 15 and good resolution between the ezetimibe and

fenofibrate and also the degradation products produced through forced degradation

study To achieve this different composition of acetonitrile and 01M ammonium

acetate pH 50 were tested The optimum mobile phase composition was then found

to be acetonitrile and 01M ammonium acetate pH 50 in the ratio of 7525 vv

Upon application of these chromatographic conditions well-resolved sharp peaks

for both ezetimibe and fenofibrate were achieved at retention times of 244 and

878 minutes respectively The represented chromatograms of ezetimibe and

fenofibrate are given in Fig 47 and 48

442 Method Validation


Validation parameters which were performed include linearity accuracy precision

robustness specificity selectivity limit of detectionquantitation and stability of

solutions

4421 Linearity

To observe the linearity and to prepare the calibration curve five different

concentrations for both ezetimibe and fenofibrate were prepared and analyzed in

the concentration range of 08-40 microgmL for ezetimibe and 1256-640 microgmL for

fenofibrate The peak areas of the drugs against the concentration were used to

prepare a linear regression equation and to calculate the value of correlation

coefficient The correlation coefficient for both the drugs was greater than 09999

which clearly manifests an excellent linear curve between the concentration and

detectors response The linear regression equation for ezetimibe was Y= 3463 x +


123

1248 with value of correlation coefficient equal to 099998 and linear regression

equation for fenofibrate was Y= 3419 x + 2986 with value of correlation

coefficient equal to 099999


The limit of detection and quantification were determined by making serials of

dilutions The LOD and LOQ were then measured by calculating the minimum

level at which the analytes can be readily detected and quantified with accuracy

respectively The LOD was found to be 006 microgmL and 048 microgmL for ezetimibe

and fenofibrate respectively with a signal to noise ratio of 31 The LOQ was found

to be 019 microgmL and 16 microgmL for ezetimibe and fenofibrate respectively with a

signal to noise ratio of 101

4423 Accuracy




corresponds to 50 100 and 150 of the nominal analytical concentrations From

these levels the percentage recovery and relative standard deviation were

calculated The results of accuracy are given in Table 421

4424 Precision

The within-day precision was evaluated by analyzing three different concentrations

of ezetimibe and fenofibrate five times in a day The between-day precision was

evaluated by analyzing the same solutions kept in dark in three different days

From the results RSD values were calculated which were less than 2 as given in

Table 422

4425 Selectivity

The selectivity of ezetimibe and fenofibrate was checked by making a synthetic

mixture of both the analytes with commonly occurring tablet excipients The

percentage recovery of each analyte was calculated in the presence of excipients

Also the chromatograms of synthetic mixture were compared with the


124


results are given in Table 423 which shows no interference of excipients with

analytes and an excellent recovery


The stability of each component in the presence of other in solution form was

assessed by analyzing the samples after 24 48 and 72 hrs The relative standard

deviation of peak area was less than 134 The results are presented in Table 424

which indicates good stability for each drug

4427 Robustness

Robustness of the method was evaluated by slight by deliberate modifications in

the operating conditions of the method and then percentage recovery retention

time tailing factor and theoretical plates were calculated at each modified

condition The results are given in Table 425 and 426 It is evident from the tables

that slight modifications in the chromatographic conditions have no effect on the

recovery of the analytes and chromatographic parameters remains acceptable

4428 Forced degradation Study


applied to both ezetimibe and fenofibrate in combination form The stress


conditions ezetimibe was degraded up to 95 whereas the degradation of

fenofibrate was only 19 The major degradation occurred under basic conditions

where ezetimibe was degraded to 44 whereas fenofibrate to only 4 Oxidative

conditions degraded ezetimibe to 18 and to fenofibrate to only 22 Thermal

stress had no effect on the degradation and the drugs remain almost intact during

this treatment From the stress studies it is evident that fenofibrate is more stable

under applied stress conditions whereas ezetimibe is more vulnerable and degraded

easily especially under basic conditions In all the stress conditions the degradation

products were well separated from the analyte peaks


125

Figure 47 Chromatogram of ezetimibe and fenofibrate reference standard

Figure 48 Chromatogram of ezetimibe and fenofibrate Tablets


126


Drug n level Conc Amount recovered recovery RSD


Ezetimibe 5 50 80 788 9850 095

5 100 160 1581 9881 031

5 150 240 2405 10021 033

Fenofibrate 5 50 1280 12924 10097 018

5 100 2560 25492 9958 051

5 150 3840 38850 10117 075

Table 422 Within-day and between day precision of the proposed HPLC method

Compound Conc n Within-day Precision Between-day precision


Ezetimibe 160 5 158 138 157 151

160 5 1608 095 1611 107

400 5 3995 055 3991 085

Fenofibrate 256 5 2550 096 2553 063

256 5 2548 033 2545 051

640 5 6373 022 6355 039


127


Ezetimibe



Fenofibrate



160 1611 10089

160 1593 9956

160 1588 9925

160 1590 9938


RSD = 076

2560 2538 9914

2560 2543 9934

2560 2581 10082

2560 2546 9945


RSD = 077


128

Table 424 Stability study of Ezetimibe and Fenofibrate in solution




Ezetimibe

160 157 156 155 064

160 1618 1590 1576 134

400 3988 3942 3912 041

Fenofibrate

256 2484 2465 2456 058

2560 25512 25349 25215 059

6400 63841 63555 63373 037


129


Conditions Assay RT (min) Theoretical plate Tailing




Flow rate (14mLmin) 10098 261 6372 136

Flow rate (16 mLmin) 9962 234 4803 116

Buffer (pH 52) 10126 243 6005 122

Buffer (pH 48) 10085 244 6078 123

Table 426 Robustness study of Fenofibrate





Flow rate (14mLmin) 10049 950 14337 121

Flow rate (16 mLmin) 9979 808 9991 107

Buffer (pH 52) 10021 876 12885 115

Buffer (pH 48) 9905 877 12687 116


130



fenofibrate in their pharmaceutical formulations The results are given in Table

427 The results show an excellent agreement with the claimed value This

confirms the suitability of the proposed method for the routine quality control

determination of ezetimibe and fenofibrate in pharmaceutical formulations


131

Table 427 Analysis of Ezetimibe and Fenofibrate in tablets

Ezetimibe



Fenofibrate



16 1624 10150

16 1605 10031

16 1591 9944


RSD = 103

256 25894 10115

256 25536 9975

256 25748 10058


RSD = 070


132



Ezetimibe is a selective inhibitor of intestinal cholesterol and related phytosterol

absorption whereas lovastatin is a cholesterol-reducing drug belonging to the

family of statins and is widely used in the treatment of hypercholesterolemia [254]

The combination therapy of ezetimibe with any statin is FDA approved and with

this therapy additional 12 to 21 absolute LDL cholesterol is reduced [114] In a

study conducted by Kerzner et al [144] the coadministration of ezetimibe with

lovastatin was shown to be more effective in decreasing plasma concentrations of

LDL cholesterol than either lovastatin or ezetimibe alone In addition the co

administration of ezetimibe with lovastatin was well tolerated with no reports of

myopathy or rhabdomyolysis [144] Ezetimibe (10mg) is therefore prescribed for

reducing hyperlipidemia along with lovastatin (20mg) In this work therefore a

stability indicating reverse phase HPLC method was developed and validated for

the simultaneous determination of lovastatin and ezetimibe in binary combination

for its future use in the combination form as many companies have passion to

launch this combination in near future Method development was started using 01

M ammonium acetate buffer pH 50 and acetonitrile in the ratio of 30 70 vv

based on our previous method development for ezetimibe and simvastatin At this

composition ezetimibe and lovastatin were eluted with good sharp peaks but the

retention time of lovastatin was greater than 12 minutes The mobile phase

composition was then changed by increasing the organic phase to reduce the

retention time At the composition of 2872 (buffer acetonitrile) both components

were eluted with total run time less than ten minutes This composition was

suitable for use in the synthetic mixture and all the degradation products were

separated from the main peaks of analytes Further increase of acetonitrile resulted

in the co-elution of degradation products peaks with the main peaks of analytes So

the final composition thus used was 2872 (buffer acetonitrile) Upon application

of the proposed method well separated sharp peaks were obtained for both


133

ezetimibe and lovastatin within 10 minutes The represented chromatograms of

ezetimibe and lovastatin are given in Figure 49

Later the method was also applied for the determination of these two drugs in

spiked human plasma under the same chromatographic conditions There was no

interference from the plasma peaks showing that it can also be applied for in vivo

studies Extraction recovery precision accuracy specificity and stability of

analytical solutions were determined and were found within range (data not

shown)


The developed method was validated according to ICH guidelines The validation

parameters that were performed include linearity precision accuracy selectivity

specificity robustness LODLOQ and stability of solutions

4521 Linearity of the method

The developed analytical method was linear in the concentration range of 02-100

microgmL for ezetimibe and 04-200 microgmL for lovastatin Seven solutions were made

for linearity for both ezetimibe and lovastatin in the range of 02-100 microgmL for



microgmL 50 microgmL 100 microgmL and 200microgmL) The peak area of drugs was plotted

against the corresponding concentrations and a linear regression equation was made

and the value of correlation coefficient was calculated The method was linear in

the mentioned ranges with linear regression equation Y= 00568 x ndash 006892 for

ezetimibe and Y= 0026355 x ndash 011561 for lovastatin The correlation coefficient

value was 09957 and 09956 for ezetimibe and lovastatin respectively

4522 Limit of detection and quantitation

The LOD and LOQ were calculated by analyzing a series of solutions with

progressively decreasing concentration of each analyte The limit of detection was

then estimated at approximately about the concentration where there was a signal to

noise ratio of 31 The limit of quantitation was calculated from the limit of


134

detection by multiplying LOD with 33 The LOD values were found to be 006

microgmL for ezetimibe and 012 microgmL for lovastatin The LOQ values were 02

microgmL and 04microg mL for ezetimibe and lovastatin respectively

4323 Accuracy

The accuracy of the method in was performed by adding known amounts of

ezetimibe and lovastatin to already analyzed synthetic mixture solutions and then

comparing the added amount with the observed amount Three levels of solutions

were made which correspond to 50 100 and 150 of the nominal analytical

concentration (10 microgmL for ezetimibe and 20 microgmL for lovastatin) Each level



4524 Precision


day precision was evaluated by analyzing the three different concentrations of

analytes each in triplicate within the same day and calculating their RSD The

between-day precision was evaluated by analyzing the same solutions for five

different days stored at 4 0C and calculating their RSD values The results of

within-day and between-day precision are presented in Table 429

4525 Selectivity






percentage recovery is presented in Table 430 The chromatogram of ezetimibe

and lovastatin in synthetic mixtures is given in Figure 410 showing selectivity of

the proposed method


135

Figure 49 Chromatogram of ezetimibe and lovastatin reference substance


136




Ezetimibe 50 3 50 508 10160 102

100 3 100 988 9880 146

150 3 150 1541 10273 039

Lovastatin 50 3 100 1022 10220 063

100 3 200 1944 9720 119

150 3 300 2928 9760 093




Ezetimibe 080 5 082 122 081 163

250 5 2458 086 2443 138

1000 5 9869 074 9805 108

Lovastatin 16 5 156 111 155 151

500 5 5059 055 5046 149

2000 5 20241 032 19968 098


137

Figure 410 Chromatogram of ezetimibe and lovastatin in synthetic mixture form


138


Ezetimibe



Lovastatin



100 1023 10230

100 992 9920

100 986 9860

100 1018 10180


RSD = 184

200 2054 10270

200 2036 10180

200 1978 9890

200 1986 9930


RSD = 185


139






4527 Robustness


the chromatographic conditions The results showed that the slight change of the

chromatographic conditions had no appreciable effects on the chromatographic

parameters The results of the robustness study are given in Table 432 amp 433


Specificity of the method was evaluated by performing degradation studies on both

the analytes in their mixture form For this purpose the analytes were treated with

acidic basic oxidative and thermal conditions Ezetimibe degraded up to 8 12

3 and 4 with acidic basic oxidative and thermal stresses respectively

whereas lovastatin showed 85 100 90 and 36 degradation for acidic

basic oxidative and thermal stresses respectively In all the stress conditions the

degradation products were well separated from the analyte peaks showing

specificity of the method in the presence of degradation products


140

Table 431 Stability study of Ezetimibe and Lovastatin in solution




Ezetimibe

080 081 078 079 193

250 2484 2466 2448 073

1000 10098 9922 9805 148

Lovastatin

160 163 161 159 124

500 4963 4921 4893 071

2000 20098 19852 19646 114


141






Flow rate (09 mLmin) 9946 453 5100 134

Flow rate (11 mLmin) 9905 370 4886 140

Buffer (pH 52) 10048 405 5454 139

Buffer (pH 48) 10215 404 5404 139

Table 433 Robustness study of Lovastatin





Flow rate (09 mLmin) 9932 1080 8004 126

Flow rate (11 mLmin) 10068 883 7575 136

Buffer (pH 52) 10046 967 7785 130

Buffer (pH 48) 9885 966 7715 130


142



Atorvastatin is the member of statins and reduces the LDL whereas the gemfibrozil

is a member of fibrates that not only increases the HDL but also decreases the LDL

level In addition to cholesterol lowering gemfibrozil also has the ability to lower

the incidence of coronary heart disease in human beings [255-256] Many patients

with coronary artery disease do not respond well with single agent therapy The

combination of an HMG CoA reductase and gemfibrozil are ideal and recent

reports confirm the efficacy of combination of an HMG CoA reductase and

gemfibrozil [257-264] The combination is also FDA approved and in view of the

efficacy of this combination many pharmaceutical companies are going to launch

the combination of gemfibrozil with simvastatin and atorvastatin In this work the

stability indicating reverse phase HPLC method for atorvastatin and gemfibrozil in

binary combination was developed and validated for future possible use in the

combined form Method development was started using 01 M ammonium acetate

buffer pH 50 and acetonitrile in the ratio of 7030 (vv) based on our previous

experiments At this composition both atorvastatin and gemfibrozil were eluted

with total run time of just 7 minutes This composition was suitable for the elution

of both components in the synthetic mixture but when applied forced degradation

samples degradation product peaks strongly interfered with both atorvastatin and

gemfibrozil The composition of mobile phase was then changed by increasing the

polarity of the mobile phase At the composition of 4555 vv (ammonium acetate

buffer pH 50 acetonitrile) both the components were eluted without any

interference from each other and from degradation products Upon application of

the proposed method well separated sharp peaks were obtained for both

atorvastatin and gemfibrozil within 12 minutes The represented chromatograms of

atorvastatin and gemfibrozil are given in Figure 411


143






shown)


The developed chromatographic method was validated in accordance with ICH

guidelines Validation parameters performed include linearity precision accuracy

selectivity specificity robustness limit of detection and quantitation and stability

of solutions

4621 Linearity






evaluate the linearity Each concentration was made and analyzed in triplicate The

peak areas obtained against each concentration of the analytes were used to build a

linear regression equation and to determine value of correlation coefficient Good

linearity was observed over the above mentioned range with linear regression

equation y = 4873 x + 298 for atorvastatin and y = 3063 x ndash 227 for gemfibrozil

The value of correlation coefficient was found to be 09997 for atorvastatin and



To calculate the limit of detection and limit of quantitation a blank solution and a

solution spiked with known progressively decreasing concentrations of each

analyte were prepared and analyzed by the developed method The LOD and LOQ

was the minimum concentration at which the analyte can be detected and quantified


144

with accuracy respectively The LOD values were found to be 003 microgmL for

atorvastatin and 013 microgmL for gemfibrozil The LOQ values were 01microgmL and

040 microgmL for atorvastatin and gemfibrozil respectively

4623 Accuracy





concentration (8 microgmL for atorvastatin and 480 microgmL for gemfibrozil) Each level



4624 Precision






435

4625 Selectivity








the proposed method


145

Figure 411 Chromatogram of Atorvastatin and gemfibrozil reference substance


146




Atorvastatin 50 3 40 406 10150 163

100 3 80 789 9863 126

150 3 120 1212 10100 069

Gemfibrozil 50 3 2400 23658 9858 101

100 3 4800 48863 10180 065

150 3 7200 73356 10188 053




Atorvastatin 05 5 052 198 051 223

80 5 795 086 786 155

200 5 1984 063 1982 141

Gemfibrozil 300 5 2963 101 2951 129

4800 5 48212 073 47871 122

12000 5 118648 088 118002 125


147

Figure 412 Chromatograms of Atorvastatin and gemfibrozil in synthetic mixture form


148


Atorvastatin



Gemfibrozil



80 808 10100

80 796 9950

80 805 10063

80 793 9912


RSD = 090

4800 47222 9838

4800 47805 9959

4800 48215 10045

4800 47329 9860


RSD = 097


149






4627 Robustness



conditions had no pronounced effects to the chromatographic parameters The



Specificity of the method was performed by performing degradation studies of both


acidic basic oxidative and thermal conditions Atorvastatin degraded 4058 2

8754 and 74 with acidic basic oxidative and thermal stresses

respectively similarly gemfibrozil showed 1411 294 4487 and 23

degradation for acidic basic oxidative and thermal stresses respectively In all the

stress conditions the degradation products were well separated from the analyte

peaks which showed the specificity of the method in the presence of degradation

products

The stress condition under oxidative condition was prolonged for two months and

after that a novel degradation product was isolated in crystalline form The scheme

of degradation of atorvastatin under oxidative conditions is given in Figure 413

whereas the X-ray structure of atorvastatin degradation product is given in Figure

414


150

Table 437 Stability study of Atorvastatin and Gemfibrozil in solution




Atorvastatin

05 052 051 052 112

80 794 797 790 044

200 2022 1995 1990 086

Gemfibrozil

300 3046 2983 2955 156

4800 48258 47626 47298 069

12000 119239 118658 118022 051


151






Flow rate (14 mLmin) 10169 380 3310 136

Flow rate (16 mLmin) 10043 334 2866 141

Buffer (pH 48) 9932 354 3164 141

Buffer (pH 52) 9978 355 3214 140






Flow rate (14 mLmin) 9911 1255 4002 131

Flow rate (16 mLmin) 10009 1098 3687 135

Buffer (pH 48) 9969 1169 3998 134

Buffer (pH 52) 9955 1169 4008 135


152

Figure 413 Scheme showing degradation of atorvastatin in the presence of hydrogen peroxide

Figure 414 X-Ray structure of atorvastatin degradation product produced under oxidative stress

Ca2+

3H2O

N

O

NH

CH3

CH3

F

OHOH

O-

O CH3OH H2O2

Room TempO

O

NH

O

OH

OCH3

OHCH3


153



Both rosuvastatin and ezetimibe are not found yet in any pharmacopeia in

combined dosage form Therefore the aim behind this work was to develop a

simple isocratic accurate and sensitive HPLC method for the simultaneous

determination of rosuvastatin and ezetimibe in their fixed dose combination

Method development was started with 01M ammonium acetate buffer pH 50 and

acetonitrile in various ratios with Merck C-18 column but in the entire conditions

peak tailing of rosuvastatin was greater than 15 and also the peak shape was not

good The column was then replaced with new Hypersil C-18 column Discovery

monolithic column and phenyl column but peak shape and tailing of rosuvastatin

was not improved The mobile phase was then switched from ammonium acetate to

phosphoric acid solution Phosphoric acid solution along with acetonitrile was good

enough to separate both the analytes with good peak shape with tailing less than

15 The chromatographic conditions were then optimized to get good resolution

between the two analytes The best results were obtained using mobile phase of 1

phosphoric acid and acetonitrile in the ratio of (4060 vv) on a Merck C-18

column So by applying the optimum chromatographic conditions resolved sharp

peaks that belong to rosuvastatin and ezetimibe were obtained at retention times of

430 and 633 minutes respectively [Figure 415 and 416]



rosuvastatin and ezetimibe was validated using ICH guidelines Assessed validation


accuracy robustness precision and stability of solutions

4721 Linearity

Linearity of the proposed method was done by analyzing seven solutions in the

range of 08 to 160 microgmL for rosuvastatin (08 microgmL 5 microgmL 20 microgmL 80

microgmL 120 microgmL 140 microgmL and 160 microgmL) and 02 to 40 microgmL for ezetimibe


154

(02 microgmL 125 microgmL 5 microgmL 20 microgmL 30 microgmL 35 microgmL and 40

microgmL) Each concentration was made and analyzed in triplicate Good linearity

was observed over the above range for both rosuvastatin and ezetimibe The

calibration curve was made using concentration of the analytes versus peak area

The correlation coefficient from the linear regression analysis was calculated and

found to be greater than 0999 in case of both the analytes This indicates that there

exists a good linear relationship between concentration of drugs and the peak area

The linear regression equation for rosuvastatin was Y= 2321 x + 222 with value of

correlation coefficient equal to 09993 whereas the regression equation for

ezetimibe was Y= 872 x + 183 with 09996 as the value of correlation coefficient



by the proposed method The limit of detection and quantification was then

established by evaluating the minimum level at which the analyte can be readily

detected and quantified with accuracy The LOD was found to be 026 microgmL and

006 microgmL for rosuvastatin and ezetimibe respectively (signal to noise ratio of

31) The LOQ was found to be 08 microgmL and 02 microgmL for rosuvastatin and

ezetimibe (signal to noise ratio of 101)


155

Figure 415 Chromatograms of rosuvastatin and ezetimibe reference substance

Figure 416 Chromatograms of rosuvastatin and ezetimibe Tablets


156

4723 Accuracy


rosuvastatin and ezetimibe to pre-quantified sample solution and then comparing



concentration Each level was made in triplicate The recovery range and the

relative standard deviation for each of the analytes were found to be 9760-10240

and 096-145 respectively [Table 440]

4724 Precision




determined from the same samples analyzed in three different days The results of

within-day and between-day precision are given in Table 441

4725 Selectivity






results show no interference from the excipients [Table 442]







157




Rosuvastatin 50 3 400 4069 10172 145

100 3 800 7888 9860 115

150 3 1200 11821 9851 096

Ezetimibe 50 3 100 981 9810 139

100 3 200 2048 10240 121

150 3 300 2928 9760 111




Rosuvastatin 50 5 505 144 503 189

800 5 7925 119 7805 169

1600 5 16228 095 16059 128

Ezetimibe 125 5 123 151 122 205

200 5 2051 076 2028 128

400 5 3965 105 3921 156


158


Rosuvastatin



Ezetimibe



800 8089 10111

800 7866 9832

800 8129 10161

800 8052 10065


RSD = 145

200 2048 10240

200 1963 9815

200 1983 9915

200 1972 9860


RSD = 193


159

Table 443 Stability study of Rosuvastatin and ezetimibe in solution over 72 hours




Rosuvastatin

50 497 498 495 031

800 7942 7881 7885 043

1600 16152 15922 15905 086

Ezetimibe

125 123 121 122 082

200 1982 1975 1955 071

400 4008 3928 3911 131


160

4727 Robustness

Robustness of the method was performed by intentionally modifying the

chromatographic conditions The results showed that the change of the conditions

had no pronounced effects on the chromatographic parameters The results of the

robustness study are given in Table 444 amp 445



applied to both Rosuvastatin and ezetimibe in combination form The stress


conditions Rosuvastatin was degraded up to 20 whereas the degradation of

ezetimibe was about 10 The major degradation occurred under basic conditions

where ezetimibe was degraded to 45 whereas no degradation was occurred for

rosuvastatin Oxidative conditions degraded rosuvastatin to 12 and to ezetimibe

to 18 Thermal stress had no effect on the degradation and the drugs remain

almost intact during this treatment In all the stress conditions the degradation

products were well separated from the analyte peaks which showed the specificity

of the method in the presence of degradation products


The proposed HPLC method was applied for the determination of rosuvastatin and

ezetimibe in their pharmaceutical formulations The results are given in Table 446

The results show an excellent agreement with the claimed value This confirms the

suitability of the proposed method for the routine quality control determination of

Rosuvastatin and ezetimibe in pharmaceutical formulations


161

Table 444 Robustness study of Rosuvastatin


ACN 1 H3PO4 (6040) 10025 430 3126 135

ACN 1 H3PO4 (5842) 10011 476 3316 133

ACN 1 H3PO4 (6238) 9865 408 3040 141

Flow rate (11mLmin) 9985 391 2866 140

Flow rate (09 mLmin) 10141 478 3264 135

H3PO4 Conc (09 ) 9955 428 3167 139

H3PO4 Conc (11 ) 9941 427 3114 133



ACN 1 H3PO4 (6040) 10069 633 4139 142

ACN 1 H3PO4 (5842) 9965 715 4267 141

ACN 1 H3PO4 (6238) 10025 595 3964 148

Flow rate (11mLmin) 10095 575 4040 145

Flow rate (09 mLmin) 9926 703 4220 142

H3PO4 Conc (09 ) 10068 635 3998 144

H3PO4 Conc (11 ) 10029 636 4002 139


162

Table446 Results of analysis of Rosuvastatin and ezetimibe in tablets



Rosuvastatin 5 40 4052 10130 103

Ezetimibe 5 10 1021 10210 131


163

48 Conclusion

In this study simple sensitive and economic HPLC methods were developed for

seven binary combinations widely used for hyperlipidemia

For the first combination containing atorvastatin and Ezetimibe a simple and

economic HPLC method was developed and validated in solid dosage forms The

method is highly selective and specific for the two components and is not interfered

by the tablet excipients and degradation products The total run time for the two

components is less than 5 min The method is accurate and precise so it can be used

for the simultaneous determination of these two components in pharmaceutical

formulations

In the second method simultaneous determination of ezetimibe and simvastatin in

their pharmaceutical formulation has been successfully achieved by the use of a

validated analytical method The method is accurate and precise for reliable quality

control evaluation of drugs with good accuracy and precision From these values it

is concluded that the new HPLC method is suitable for the simultaneous

determination of ezetimibe and simvastatin in their pharmaceutical formulations

For the binary combination of gemfibrozil and simvastatin a simple and accurate

reverse phase HPLC method was developed for the simultaneous determination of

gemfibrozil and simvastatin The method was validated by testing its linearity

accuracy precision limits of detection and quantitation selectivity specificity and

robustness The run time of less than ten minutes allows its application for the

routine determination of gemfibrozil and simvastatin

The binary combination of ezetimibe and fenofibrate was successfully analyzed

after developing a simple and accurate HPLC method The method was validated

by testing its linearity accuracy precision recovery robustness limits of

detectionquantitation and specificity The method is specific in the presence of the

degradation products as evident from the forced degradation studies The total run

time of less than ten minutes not only allows its suitability for the routine


164

determination of ezetimibe and fenofibrate but also for stability studies

In the fifth method a simple and accurate HPLC method for the simultaneous

determination of ezetimibe and lovastatin was developed The method was

validated by testing its linearity accuracy precision recovery robustness limits of


degradation products as evident from the forced degradation studies The method

was also applied to spiked human plasma and showed good results The total run


determination of lovastatin and ezetimibe but also for stability studies and in

human plasma

For the sixth binary combination comprising of atorvastatin and gemfibrozil a

simple and accurate reverse phase HPLC method was developed The method was

validated by testing its linearity accuracy precision limits of detection and

quantitation selectivity specificity and robustness The method was also applied to

spiked human plasma and showed good results As the method can separate the

degradation products from the main peaks of analytes so it can be used not only for

routine analysis but also for stability studies and in human plasma

In the seventh binary combination analysis a simple and economic HPLC method

was developed and validated for the simultaneous determination of rosuvastatin

and ezetimibe in their pharmaceutical formulation The method is accurate and

precise for the determination of these drugs with good accuracy and precision

From these values it is concluded that the new HPLC method is suitable for the

simultaneous determination of these two components in their pharmaceutical

formulations

CHAPTER 5 REFERENCES

165

5 REFERENCES

1 Reynolds JEF Martindale the extra pharmacopoeia 30th edition 1993

Page 979 Published by Info access and distribution Pte Ltd Singapore

2 Murchison L E Br Med J 1985 290 535-538

3 Joel GH amp Lee EL Goodman and Gilmanrsquos The Pharmacological basis

of therapeutics International edition 10th edition Mc Grow Hill 2001

Page 971

4 Sharma SB amp Dwivedi S Indian Drugs 1997 34 (5) 242-251

5 Elnasri HA amp Ahmed AM Eastern Mediterranean Health Journal

2008 14(2) 314-324

6 httpwwwnetdoctorcoukatediabetes202338html Accessed on

141108

7 httpwwwvascularweborgpatientsNorthPointHyperlipidemiahtml

Accessed on 141108

8 httpwwwhealthcentralcomencyclopedia408366html Accessed on

141108

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141108

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151108

12 httpwwwanswerscomtopicchylomicron Accessed on 151108

13 Gotto A amp Pownall H The Manual of Lipid Disorders Reducing the

Risk for Coronary Heart Disease 3rd ed Lippincott Williams amp Wilkins

New York 2003

14 httpenwikipediaorgwikiHyperlipidemia Accessed on 161108

15 Frederickson DS amp Lee RS Circulation 1965 31 321-7

16 Third Report of the National Cholesterol Education Program (NCEP)

Expert Panel on Detection Evaluation and Treatment of High Blood


166

Cholesterol in Adults (Adult Treatment Panel III) Final Report Circulation

2002 106 3240

17 Krukemyer J J amp Talbert R L Pharmacotherapy 1987 7 198ndash210

18 Hebert PR Gaziano JM Chan KS amp Hennekens CH JAMA 1997

278 313ndash321

19 Watts G F amp Dimmitt S B Curr Opin Lipidol 1999 10 561ndash574

20 Ozasa H Miyazawa S Furuta S Osumi T amp Hashimoto T J

Biochem (Tokyo) 1985 97 1273ndash1278

21 Vasudevan AR amp Jones PH Curr Cardiol Rep 2005 7 471ndash479

22 Steinmetz KL Am J Health Syst Pharm 2002 59 932ndash939

23 Gauthier A Lau P Zha X Milne R amp McPherson R Arterioscler

Thromb Vasc Biol 2005 25 2177ndash2184

24 Kharbanda RK Wallace S Walton B Donald A Cross JM amp

Deanfield J Circulation 2005 111 804ndash807

25 Ueshima K Akihisa-Umeno H Nagayoshi A Takakura S Matsuo M

amp Mutoh S Biol Pharm Bull 2005 28 247ndash252

26 Pahan K Cell Mol Life Sci 2006 63 1165ndash1178

27 Goldstein JL amp Brown MS Nature 1990 343 425-430

28 Istvan ES amp Deisenhofer J Science 2001 292 1160-1164

29 Asztalos BF Horvath KV McNamara JR Roheim PS Rubinstein

JJ amp Schaefer EJ Atherosclerosis 2002 164 361ndash369

30 Illignworth DR amp Tobert JA Adv Protein Chem 2001 56 77ndash114

31 Corsini A Maggi FM Catapano AL Pharmacol Res 1995 34 9ndash27

32 Thompson GR amp Naoumova RP Expert Opin Invest Drugs 2000 9

2619ndash2628

33 Dujovne CA amp Moriarty PM Clin Ther 1996 18 392ndash410

34 Endo A Tsujita Y Kuroda M amp Tanzawa K Eur J Biochem 1977

77 31ndash36

35 Farmer JA Lancet 2001 358 1383ndash1385


167

36 Staffa JA Chang J amp Green L N Engl J Med 2002 346 539ndash540

37 Pogson GW Kindred LH amp Carper BG Am J Cardiol 1999 83

1146

38 Kajinami K Mabuchi H amp Saito Y Expert Opin Investig Drugs

2000 9 2653ndash2661

39 Mukhtar RYA Reid J amp Reckless JPD Int J Clin Pract 2005 59

239ndash252

40 Istvan ES Atheroscler Suppl 2003 4 3ndash8

41 Endo A Kuroda M amp Tanzawa K FEBS Lett 1976 72 323ndash326

42 Nirogi R Mudigonda K amp Kandikere V J Pharm Biomed Anal 2007

44 379ndash387

43 Drug Evaluations by American Medical Association 1995 2486

44 McTaggart F Buckett L Davidson R Holdgate G McCormick A

Schneck D Smith G amp Warwick M Am J Cardiol 2001 87 28Bndash

32B

45 Martin PD Warwick MJ Dane AL Hill SJ Giles PB Phillips

PJ amp Lenz E Clin Ther 2003 25 2822ndash2835

46 Blasetto JW Stein EA Brown WV Chitra R amp Raza A Am J

Cardiol 2003 91 3Cndash10C

47 Jones PH Davidson MH Stein EA Bays HE McKenney JM

Miller E Cain VA amp Blasetto JW Am J Cardiol 2003 93 152ndash160

48 Appel S amp Dingemanse J Drugs Today 1996 32 39ndash55

49 Christians U Jacobsen W amp Floren LC Pharmacol Ther 1998 80

1ndash34

50 Dain JG Fu E Gorski J Nicoletti J amp Scallen TJ Drug Metab

Dispos 1993 21 567ndash572

51 Muck W Ritter W Dietrich H Frey R amp Kuhlmann J Int J Clin

Pharmacol Ther 1997 35 261ndash264


168

52 Muck W Ritter W Ochmann K Unger S Ahr G Wingender W amp

Kuhlmann J Int J Clin Pharmacol Ther 1997 35 255ndash260

53 Muck W Drugs 1998 56 (Suppl 1) 15ndash23

54 Muck W Ochmann K Mazzu A amp Lettieri J Int J Med Res 1999

27 107ndash114

55 Posvar EL Radulovic LL Cilla DD Whitfield LR amp Sedman AJ

JClin Pharmacol 1996 36 728ndash731

56 Tse FLS Jaffe JM amp Troendle A J Clin Pharmacol 1992 32630ndash

638

57 Lennernas H amp Fager G Clin Pharmacokinet 1997 32 403ndash425

58 Tobert JA Am J Cardiol 1988 62 28Jndash34J

59 Prueksaritanont T Gorham LM Ma B Liu L Yu X Zhao JJ

Slaughter DE Arison BH amp Vyas KP Drug Metab Dispos 1997

25 1191ndash1199

60 Zhou LX Finley DK Hassell AE amp Holtzman JL J Pharmacol

Exp Ther 1995 273 121ndash127

61 Chong PH amp Seeger JD Pharmacotherapy 1997 17 1157ndash1177

62 Plosker GL Dunn CJ amp Figgit DP Drugs 2000 60 1179ndash1206

63 Wolfgang M Drugs 1998 56 (Suppl 1) 15ndash23

64 Fischer V Johanson L Heitz F Tullman R Graham E Baldeck JP

amp Robinson WT Drug Metab Dispos 1999 27 410ndash416

65 Transon C Leemann T Vogt N amp Dayer P Clin Pharmacol Ther

1995 58 412ndash417

66 Haria M amp McTavish D Drugs 1997 53 299ndash336

67 Everett DW Chando TJ Didonato GC Singhvi SM Pan HY amp

Weinstein SH Drug Metab Dispos 1991 19 740ndash748

68 Kitazawa E Tamura N Iwabuchi H Uchiyama M Muramatsu S

Takahagi H amp Tanaka M Biochem Biophys Res Commun 1993 192

597ndash602


169

69 McClellan KJ Wiseman LR amp McTavish D Drugs 1998 55 415ndash

420

70 Singhvi SM Pan HY Morrison RA amp Willard DA Br J Clin

Pharmacol 1990 29 239ndash243

71 Stancu C amp Sima A JCellMolMed 2001 5(4) 378-387

72 Corsini A Bellosta S Baetta R Fumagalli R amp Bernini F

Pharmacol Ther 1999 84 413-28

73 Sehayek E Butbul E amp Avner R Eur J Clin Invest 1994 24 173-8

74 Blum CB Am J Cardiol 1994 73 3D-11D

75 Stein EA Lane M amp Laskarzewski P Am J Cardiol 1998 81 66B-

69B

76 Ramakrishnan R amp Desnick RJ J Clin Invest 1987 80 1692-1697

77 Gaw A Packard CJ amp Murray EF Arterioscler Thromb 1993 13

170-89

78 Marais AD Naumova RP Firth JC Penny C amp Neuwirth CK J

Lipid Res 1997 38 2071-2078

79 Raal FJ Pilcher GJ Illingworth DR Pappu AS Stein EA

Laskarzewski P Mitchel YB amp Melino MR Atherosclerosis 1997

135 249- 256

80 Kostner GM Gavish D Leopold B Bolzano K Weintraub MS amp

Breslow JL Circulation 1989 80 1313-1319

81 Maron DJ Fazio S amp Linton MF Circulation 2000 101 207-213

82 Komsta L Misztal G Majchrzak E amp Hauzer A J Pharm Biomed

Anal 2006 41(2) 408-414

83 Moody D E amp Reddy J K Am J Pathol 1978 90 435ndash450

84 Reddy JK Goel SK Nemali MR Carrino JJ Laffler TG Reddy

MK Sperbeck SJ Osumi T Hashimoto T amp Lalwani ND Proc

Natl Acad Sci USA 1986 83 1747ndash 1751

85 Ozawa H amp Ozawa T Yakushigaku Zasshi 2002 37 84ndash94


170

86 Lazarow PB Shio H amp Leroy-Houyet MA J Lipid Res 1982 23

317ndash 326

87 Gray TJ Beamand JA Lake BG Foster JR amp Gangolli SD

Toxicol Lett 1982 10 273ndash279

88 Reddy JK amp Krishnakantha TP Science 1975 190 787ndash789

89 Leighton F Coloma L amp Koenig C J Cell Biol 1975 67 281ndash309

90 Rao MS Subbarao V amp Reddy JK J Natl Cancer Inst 1986 77

951ndash956

91 Kliewer SA Xu HE Lambert MH amp Willson TM Recent Prog

Horm Res 2001 56 239ndash263

92 Willson TM amp Wahli W Curr Opin Chem Biol 1997 1 235ndash 241

93 Chu R Lin Y Rao MS amp Reddy JK J Biol Chem 1995 270

29636ndash29639

94 Lazarow PB J Inherit Metab Dis 1987 10 (suppl 1) 11ndash 22

95 Singh I Moser AE Goldfischer S amp Moser HW Proc Natl Acad

Sci USA 1984 81 4203ndash 4207

96 Yu S Rao S amp Reddy JK Curr Mol Med 2003 3 561ndash572

97 Reddy J K amp Hashimoto T Annu Rev Nutr 2001 21 193ndash230

98 Staels B Schoonjans K Fruchart JC amp Auwerx J Biochimie 1997

79 95ndash99

99 Yeldandi AV Rao MS amp Reddy JK Mutat Res 2000 448 159ndash177

100 Delerive P De Bosscher K Besnard S Vanden Berghe W Peters

JM Gonzalez FJ Fruchart J Tedgui A Haegeman G amp Staels B J

Biol Chem 1999 274 32048ndash32054

101 Daynes RA amp Jones DC Nat Rev Immunol 2002 2 748ndash759

102 Delerive P Gervois P Fruchart JC amp Staels B J Biol Chem 2000

275 36703ndash 36707

103 Elisaf M Curr Med Res Opin 2002 18(5) 269-276

104 Adkins JC amp Faulds D Drugs 1997 54 615-33


171

105 Davignon P Can J Cardiol 1994 10(Suppl B) 61B-71B

106 Shepherd J Eur Heart J 1995 16 5-13

107 Munoz A Guichard JP amp Reginault PH Atherosclerosis 1999 110

S45-S48

108 Alexandridis G Pappas G amp Elisaf M Am J Med 2000 109 261-2

109 Kiortsis DN Milionis H Bairaktari E amp Elisaf M Eur J Clin

Pharmacol 2000 56 631-5

110 httpwwwlipidnursecapdf_filesezetimibepdf Accessed on 251108

111 Davidson MH amp Toth PP Progress in Cardiovascular Diseases 2004

47(2) 73-104

112 Catapano AL Eur Heart J 2001 Suppl 3 E6ndashE10

113 Salisbury BG Davis HR Burrier RE Burnett DA Bowkow G

Caplen MA Clemmons AL Compton DS Hoos LM amp McGregor

DG Atherosclerosis 1995 115 45-63

114 Jeu L amp Cheng JW Clin Ther 2003 25 2352-87

115 Nutescu EA amp Shapiro NL Pharmacotherapy 2003 23 1463-1474

116 Courtney RD Kosoglou T amp Statkevich P Clin Pharmacol Ther

2002 71 80

117 Al-Shaer MH Choueiri NE amp Suleiman ES Lipids in Health and

Disease 2004 3 22

118 Davis HR Compton DS Hoos L Tetzloff G Caplen MA amp

Burnett DA Eur Heart J 2000 21 636(Suppl)

119 Van Heek M Farley C Compton DS Hoos L Alton KB Sybertz

EJ amp Davis Jr HR Br J Pharmacol 2000 129 1748- 1754

120 Zetia [prescribing information] North Wales PA MerckSchering-Plough

Pharmaceuticals 2002

121 Bays HE Moore PB Drehobl Rosenblatt S Toth PD Dujovne

CA Knopp RA Lipka LJ LeBeaut AP Yang B Mellars LE

Cuffie-Jackson C amp Veltri EP Clin Ther 2001 23 1209-1230


172

122 Van Heek M France CF Compton DS Mcleod RL Yumibe NP

Alton KB Sybertz EJ amp Davis Jr HR J Pharmacol Exp Ther 1997

283 157-163

123 Rosenblum SB Huynh T Afonso A Davis Jr HR Yumibe N

Clader JW amp Burnett DA J Med Chem 1998 41 973- 980

124 Van Heek M Farley C Compton DS Hoos L amp Davis HR Br J

Pharmacol 2001 134 409-417

125 Van Heek M Compton DS amp Davis HR Eur J Pharmacol 2001 415

79-84

126 Sudhop T Lutjohann D Kodal A Igel M Tribble DL Shah S

Perevozskaya I amp Von Bergmann K Circulation 2002 106 1943-1948

127 Dujovne CA Ettinger MP McNeer JF Lipka LJ LeBeaut AP

Suresh R Yang B amp Veltri EP Am J Cardiol 2002 90 1092- 1097

128 Knopp RH Gitter H Truitt T Bays H Manion CV Lipka LJ

LeBeaut AP Suresh R Yang B amp Veltri EP Eur Heart J 2003 24

729-741

129 Florentin M Liberopoulos EN amp Elisaf MS International Journal of

Clinical Practice 2007 62(1) 88 ndash 96

130 httpwwwrxlistcomzetia-drughtm Accessed on 01012009

131 Guyton JR Current Cardiology Reports 1999 1 244ndash250

132 Saseen J amp Tweed E J Fam Practic 2006 55(1) 70-72

133 Knopp RH Dujovne CA Le Beaut A Lipka LJ Suresh R amp Veltri

EP Int J Clin Pract 2003 57 363ndash368

134 Ballantyne CM Abate N Yuan Z King TR amp Palmisano J Am

Heart J 2005 149 464ndash473

135 Ballantyne CM Blazing MA King TR Brady WE amp Palmisano J

Am J Cardiol 2004 93 1487ndash1494


173

136 Ballantyne CM Houri J Notarbartolo A Melani L Lipka LJ

Suresh R Sun S LeBeaut Ap Sager PT amp Veltri EP Circulation

2003 107 2409ndash 2415

137 Ballantyne CM Lipka LJ Sager PT Strony J Alizadeh J Suresh

R amp Veltri EP Int J Clin Pract 2004 58 653ndash 658

138 Ballantyne CM Weiss R Moccetti T Vogt A Eber B Sosef F amp

Duffield E Am J Cardiol 2007 99 673ndash 680

139 Bays HE Ose L Fraser N Tribble DL Quinto K Reyes R

Johnson-Levonas AO Sapre A amp Donahue SR Clin Ther 2004 26

1758 ndash1773

140 Davidson MH McGarry T Bettis R Melani L Lipka LJ LeBeaut

AP Suresh R Sun S amp Veltri EP J Am Coll Cardiol 2002 40

2125ndash2134

141 Feldman T Davidson M Shah A Maccubbin D Meehan A Zakson

M Tribble D Veltri E amp Mitchel Y Clin Ther 2006 28 849ndash859

142 Gagneacute C Bays HE Weiss SR Mata P Quinto K Melino M Cho

M Musliner TA amp Gumbiner B Am J Cardiol 2002 901084 ndash1091

143 Goldberg AC Sapre A Liu J Capece R amp Mitchel YB Mayo Clin

Proc 2004 79 620ndash 629

144 Kerzner B Corbelli J Sharp S Lipka LJ Melani L LeBeaut A

Suresh R Mukhopadhyay P amp Veltri EP Am J Cardiol 2003 91

418ndash424

145 Landray M Baigent C Leaper C Adu D Altmann P Armitage J

Ball S Baxter A Blackwell L Cairns HS Carr S Collins R

Kourellias K Rogerson M Scoble JE Tomson CRV Warwick G

amp Wheeler DC Am J Kidney Dis 2006 47 385ndash395

146 Melani L Mills R Hassman D Lipetz R Lipka L LeBeaut A

Suresh R Mukhopadhyay P amp Veltri E Eur Heart J 2003 24 717ndash

728


174

147 Stein E Stender S Mata P Sager P Ponsonnet D Melani L Lipka

L Suresh R MacCubbin D amp Veltri E Am Heart J 2004 148 447ndash

455

148 Kastelein JJP Akdim F Stroes ES Zwinderman AH Bots ML

Stalenhoef AFH Visseren FLJ Sijbrands EJG Trip MD Stein

EA Gaudet D Duivenvoorden R Veltri EP Marais AD amp de Groot

E N Engl J Med 2008 3581431ndash1443

149 Alvarez-Sala LA Cachofeiro V Masana L Suarez C Pinilla B

Plana N Trias F Moreno MA Gambus G Lahera V amp Pintoacute X

Clin Ther 2008 30 84 ndash97

150 Slim H amp Thompson PD Journal of Clinical Lipidology 2008 2 328ndash

334

151 Xydakis AM Ballantyne CM Am J Cardiol 2002 90(10B) 21Kndash9K

152 Hunninghake D Jr Insull W Toth P Davidson D Donovan JM amp

Burke SK Atherosclerosis 2001 158 407ndash416

153 Shek A amp Ferrill MJ Ann Pharmacother 2001 35 908ndash917

154 Pasternak RC Smith SC Jr Bairey-Merz CN Grundy SM

Cleeman JI amp Lenfant C J Am Coll Cardiol 2002 40 567ndash572

155 Athyros VG Papageorgiou AA Hatzikonstandinou HA Didangelos

TP Carina MV Kranitsas DF amp Kontopoulos AG Am J Cardiol

1997 80 608ndash613

156 Athyros VG Papageorgiou AA Athyrou VV Demitriadis DS amp

Kontopoulos AG Diabetes Care 2002 25 1198ndash 1202

157 Moon YSK Chun P amp Chung S Drugs Today 2007 43(1) 35

158 McKenney JM Farnier M Lo K Bays HE Perevozkaya I Carlson

G Davies MJ Mitchel YB amp Gumbiner B J Am Coll Cardiol 2006

47 1584 ndash1587

159 Christian G D Analytical Chemistry John Wiley amp Sons Inc New York

5th Edition 1994 23-25 51-53


175

160 ldquoThe United States Pharmacoepiardquo 26th ed US Pharmacoepial

Convention Rockville MD 2003 p 1151

161 httpwwwinvestopediacomtermsqquantitativeanalysisasp Accessed on

01082009

162 httpenwikipediaorgwikiQuantitative_analysis_(chemistry) Accessed

on 01082009

163 mhttpwwwgmuedudepartmentsSRIFtutorialgcdquanthtmethod

Accessed on 03082009

164 David B T R The science and practice of pharmacy 21st edition

Liipincott Williams and Wilkins Maryland USA 2006 p 128

165 httpwwwchemtamueduclassfypmathrevstd-devpdf Accessed on

03082009

166 httpenwikipediaorgwikiLinear_regression Accessed on 03082009

167 httpwwwcurvefitcomlinear_regressionhtm Accessed on 04082009

168 httpmathbitscomMathbitsTISectionStatistics2correlationhtm


169 httpenwikipediaorgwikiTablet Accessed on 04082009

170 Petkovska R Cornett C amp Dimitrovska A Analytical Letters 2008 41

992ndash1009

171 Khedr A J AOAC Int 2007 90(6) 1547-53

172 Sivakumar T Manavalan R Muralidharan C amp Valliappan K J Sep

Sci 2007 30(18) 3143-53

173 Jamshidi A amp Nateghi AR Chromatographia 2007 65 (11-12) 763-

766

174 Ma L Dong J Chen XJ amp Wang GJ Chromatographia 2007 65

(11-12) 737-741

175 Stanisz B amp Kania L Acta Pol Pharm 2006 63(6) 471-6


44(2) 379-87


176

177 Chaudhari BG Patel NM amp Shah PB Chem Pharm Bull 2007 55(2)

241-6

178 Mohammadi A Rezanour N Ansari Dogaheh M Ghorbani Bidkorbeh

F Hashem M amp Walker RB J Chromatogr B Analyt Technol Biomed

Life Sci 2007 846(1-2) 215-21

179 Borek-Dohalskyacute V Huclovaacute J Barrett B Nemec B Ulc I amp Jeliacutenek

I Anal Bioanal Chem 2006 386(2) 275-85

180 Shen HR Li ZD amp Zhong MK Pharmazie 2006 61(1) 18-20

181 Bahrami G Mohammadi B Mirzaeei S amp Kiani A J Chromatogr B

Analyt Technol Biomed Life Sci 2005 826(1-2) 41-5

182 Zarghi A Shafaati A Foroutan SM amp Khoddam A

Arzneimittelforschung 2005 55(8) 451-4

183 Pasha MK Muzeeb S Basha SJ Shashikumar D Mullangi R amp

Srinivas NR Biomed Chromatogr 2006 20(3) 282-93

184 Hermann M Christensen H amp Reubsaet JL Anal Bioanal Chem 2005

382(5) 1242-9

185 Ertuumlrk S Sevinccedil Aktaş E Ersoy L amp Ficcedilicioğlu S J Pharm Biomed

Anal 2003 33(5) 1017-23

186 Jemal M Ouyang Z Chen BC amp Teitz D Rapid Commun Mass

Spectrom 1999 13(11) 1003-15

187 Bullen WW Miller RA amp Hayes RN J Am Soc Mass Spectrom

1999 10(1) 55-66

188 Apostolou C Kousoulos C Dotsikas Y Soumelas GS Kolocouri F

Ziaka A amp Loukas YL J Pharm Biomed Anal 2008 46(4) 771-9

189 Basavaiah K amp Devi OZ Eclet Quiacutem 2008 33 (2 ) 1-6

190 Basavaiah K amp Tharpa K Chemical Industry amp Chemical Engineering

Quarterly 2008 14(3) 205minus210

191 Nigovic B Komorsky-Lovric S amp Devcic D Crotica Chemica Acta

2008 81(3) 453-459


177

192 Arayne MS Sultana N Hussain F amp Ali SA Journal of Analytical

Chemistry 2007 62(6 ) 536-541

193 Jitender M Vikrant T Dwivedi AK amp Satyawan S Journal of

scientific amp industrial research 2007 66 (5) 371-376

194 Malenović A Medenica A Ivanović D amp Jančic B

Chromatographia 2006 63 S95-S100

195 Coruh O amp Ozkan SA Pharmazie 2006 61(4) 285-90

196 Abu-Nameh ESM Shawabkeh RA amp Ali A Journal of Analytical

Chemistry 2006 61 (1 ) 63-66

197 Barrett B Huclovaacute J Borek-Dohalskyacute V Nemec B amp Jeliacutenek I J

Pharm Biomed Anal 2006 41(2) 517-26

198 Godoy R Godoy CG De Diego M amp Gomez C J Chil Chem Soc

2004 49 (4) 289-289

199 Malenovic A Ivanovic D Medenica M Jancic B amp Markovic S J

Sep Sci 2004 27(13) 1087-92

200 Srinivasu MK Narasa Raju A amp Om Reddy G J Pharm Biomed Anal

2002 29 (4) 715-721

201 Tan L Yang LL Zhang X Yuan YS amp Ling SS Se Pu 2000

18(3) 232-4

202 Wang L amp Asgharnejad M J Pharm Biomed Anal 2000 21(6) 1243-8

203 Ochiai H Uchiyama N Imagaki K Hata S amp Kamei T J

Chromatogr B Biomed Sci Appl 1997 694(1) 211-7

204 Carlucci G Mazzeo P Biordi L amp Bologna M J Pharm Biomed Anal

1992 10(9) 693-7

205 Wang D Wang D Qin F Chen L amp Li F Biomed Chromatogr

2008 22(5) 511-8

206 Yuana H Wanga F Tua J Penga W amp Huande Li J Pharm Biomed

Anal 2008 46(4) 808-813


178

207 Yu XR Sondi M Hangi TJ amp Wen AD Acta Chromatographica

2008 20 399ndash410

208 Zhang Z amp Yang Z Chromatographia 2007 66 487ndash491

209 Min Li Fan L Zhang W amp Cao C Anal Bioanal Chem 2007 387

2719ndash2725

210 Aacutelvarez-Lueje A Pastine J Squella JA amp Nunez-Vergara LJ J Chil

Chem Soc 2005 50(4) 639-646

211 Orkoula MG Kontoyannis CG Markopoulou CK amp Koundourellis

JE J Pharm Biomed Anal 2004 35(5)1011-6

212 Sharma P Chawla H amp Panchagnula R J Chromatogr B Analyt Technol

Biomed Life Sci 2002 768(2) 349-59

213 Ye LY Firby PS amp Moore MJ Ther Drug Monit 2000 22(6) 737-

41

214 Strode JT Taylor LT Howard AL amp Ip D J Pharm Biomed Anal

1999 20(1-2) 137-43

215 Mazzo DJ Biffar SE Forbes KA Bell C amp Brooks MA J Pharm

Biomed Anal 1988 6(3) 271-6

216 Chaudhari BG Patel NM amp Shah PB Indian Journal of

Pharmaceutical Sciences 2007 69 (1) 130-132

217 Suslu I Celebier M amp Altınoz S Chromatographia 2007 66 S65ndashS72

218 Uyar B Celebier M amp Altinoz S Pharmazie 2007 62(6) 411-413

219 Gao J Zhong D Duan X amp Chen X J Chromatogr B Analyt Technol

Biomed Life Sci 2007 856(1-2) 35-40

220 Lan K Jiang X Li Y Wang L Zhou J Jiang Q amp Ye L J Pharm

Biomed Anal 2007 44(2) 540-6

221 Vittal S Shitut NR Kumar TR Vinu MC Mullangi R amp Srinivas

NR Biomed Chromatogr 2006 20(11) 1252-9

222 Kumar TR Shitut NR Kumar PK Vinu MC Kumar VV

Mullangi R amp Srinivas NR Biomed Chromatogr 2006 20(9) 881-7


179

223 Mehta TN Patel AK Kulkarni GM amp Suubbaiah G J AOAC

International 2005 88 (4) 1142-1147

224 Hull CK Martin PD Warwick MJ amp Thomas E J Pharm Biomed

Anal 2004 35(3) 609-14

225 Prabu S Singh T Joseph A Kumar C amp Shirwaikar A Indian J

Pharm Sci 2007 69 819-21

226 Kim C Jae J Hwang H Ban E Maeng J Kim M amp Piao X J Liq

Chromat Relat Technol 2006 29 403ndash414

227 Ulu ST Chromatographia 2006 64 447ndash451

228 Roadcap BA Musson DG Rogers JD amp Zhao JJ J Chromatogra

B 2003 791 161ndash170

229 Gonzaacutelez-Pentildeas E Agarraberes S Loacutepez-Ocariz A Garciacutea-Quetglas

E Campanero MA Carballal JJ amp Honorato J J Pharm Biomed

Anal 2001 26(1) 7-14

230 Nakagawa A Shigeta A Iwabuchi H Horiguchi M Nakamura K amp

Takahagi H Biomed Chromatogr 1991 5(2) 68-73

231 Hengy H amp Koumllle EU Arzneimittelforschung 1985 35(11) 1637-9

232 Kadav AA amp Vora DN J Pharm Biomed Anal 2008 48(1) 120-126

233 Nakarani NV Bhatt KK Patel RD amp Bhatt HS J AOAC

International 2007 90(3) 700-705

234 Straka RJ Burkhardt RT amp Fisher JE Ther Drug Monit 2007 29(2)

197-202

235 El-Gindy A Emara S Mesbah MK amp Hadad GM Farmaco 2005

60(5) 425-38

236 Yardmici C amp Oumlzaltin N Anal Bioanal Chem 2004 378(2) 495-498

237 Hernando MD Petrovic M Fernaacutendez-Alba AR amp Barceloacute D

J Chromatogr A 2004 1046(1-2) 133-40

238 Lossner A Banditt P amp Troger U Pharmazie 2001 56(1) 50-1


180

239 Streel B Hubert P amp Ceccato A J Chromatogr B Biomed Sci Appl

2000 742(2) 391-400

240 Lacroix PM Dawson BA Sears RW Black DB Cyr TD amp

Ethier JC J Pharm Biomed Anal 1998 18(3) 383-402

241 Abe S Ono K Mogi M amp Hayashi T Yakugaku Zasshi 1998

118(10) 447-55

242 Masnatta LD Cuniberti LA Rey RH amp Werba JP

J Chromatogr B Biomed Appl 1996 687(2) 437-42

243 Doshi AS Kachhadia PK amp Joshi HS Chromatographia 2008 67(1-

2) 137-142

244 Dixit RP Barhate CR amp Nagarsenker MS Chromatographia 2008

67(1-2) 101-107

245 Sharma M Mhaske DV Mahadik M Kadam SS amp Dhaneshwar

SR Ind J Pharm Sci 2008 70(2) 258-260

246 Basha SJ Naveed SA Tiwari NK Shashikumar D Muzeeb S

Kumar TR Kumar NV Rao NP Srinivas N Mullangi R amp

Srinivas NR J Chromatogr B Analyt Technol Biomed Life Sci 2007

853(1-2) 88-96

247 Rajput SJ amp Raj HA Ind J Pharm sci 2007 69(6) 759-762

248 Singh S Singh B Bahuguna R Wadhwa L amp Saxena R J Pharm

Biomed Anal 2006 41(3) 1037-40

249 Oliveira PR Brum Junior L Fronza M Bernardi LS Masiero

SMK amp Dalmora SL Chromatographia 2006 63(7-8) 315-320

250 Oswald S Scheuch E Cascorbi I amp Siegmund W J Chromatography

B 2006 830(1)143-150

251 Sistla R Tata VS Kashyap YV Chandrasekar D amp Diwan PV J

Pharm Biomed Anal 2005 39(3-4) 517-22


181

252 ICH (Q2A) Note for guidance on validation of analytical methods

definition and terminology International conference on Harmonisation

IFPMA Geneva 1994

253 ICH (Q2B) Note for guidance on validation of analytical procedures

methodology International conference on Harmonisation IFPMA Geneva

1996

254 USP 29-NF 24 The United States Pharmacoepial Convention 12601

Twinbrook Parkway Rockville MD 20852 2006 1965-1966

255 Craig CR amp Stitzel RE Modern Pharmacology fourth ed Little Brown

and Company Boston 1994 p 207

256 Tadd PA amp Ward A Drugs 1988 36 32-35

257 Vanhanen HT amp Miettinen T A Atherosclerosis 1995 115 135-146

258 Smit JW Jansen GH de Bruin TW amp Erkelens DW Am J Cardiol

1995 76(2) 126A-128A

259 Pasternak RC Brown LF Stone PH Silverman DI Gibson M amp

Sacks FM Ann Intern Med 1996 125 529-540

260 Rosenson RS amp Frauenheim WA Am J Cardiol 1994 74 499-509

261 Illingworth DR amp Bacon S Circulation 1989 79 590-596

262 Athyros V Papageorgiou A Hagikonstantinou H Papadopoulos G

Zamboulis C amp Kontoponlos A Drug Invest 1994 7 134-142

263 Da Col PG Fonda M amp Fisicaro M Curr Ther Res 1993 53 473-483

264 Wirebaugh SR Shapiro ML McIntyre TH amp Whitney EJ

Pharmacotherapy 1992 12 445-450

265 OrsquoConnor P Feely J amp Shepherd J BMJ 1990 300 667-672

Title_pages_PhDpdf

GC UNIVERSITY LAHORE PAKISTAN

Muhammad Ashfaq


RESEARCH COMPLETION CERTIFICATE


Supervisor

Prof Dr M Saeed Iqbal

To

Abbreviationspdf

ICH= International Conference on Harmonization

List_of_Tablespdf

List of Tables

TAB DESCRIPTIONPAGE

List_of_Figurespdf

FIG DESCRIPTIONPAGE

List_of_Publicationspdf

List of Publications

Table_of_Contentspdf

Table of Contents

DESCRIPTION PAGE

CHAPTER 1INTRODUCTION1-34

110 Quantitative Analysis28

1122 Manufacturing Process32 11221 Granulation32

112211 Wet granulation33

112212 Dry granulation33

11222 Tablet Compression33

11223 Tablet coating33

113 Aims and objective of the research work34

3512 HPLC Set Up69

3611 Application of the Method72

3612 HPLC Set Up73

3711 HPLC Set Up76

3811 Application of the method79

3812 HPLC Set Up80

3911 HPLC Set Up84

31011 HPLC Set Up88

31112 HPLC Set Up92

CHAPTER 4RESULTS AND DISCUSSION93-164

48S-1pdf









48S-2pdf

Kumar et al [222] developed a specific accurate precise and reproducible high-performance liquid chromatography method for t

Mehta et al [223] applied a forced degradation study for the development of a stability-indicating assay for the determination

48S-3pdf

3 EXPERIMENTAL WORK




3512 HPLC Set Up



3612 HPLC Set Up

3711 HPLC Set Up


3812 HPLC Set Up

3911 HPLC Set Up

31011 HPLC Set Up


31112 HPLC Set Up

48S-4pdf

4 RESULTS AND DISCUSSIONS



























ACN 1 H3PO4 (6040) 10025 430 3126 135


ACN 1 H3PO4 (6040) 10069 633 4139 142



Submitted to GC University Lahore in partial fulfillment of the requirements

for the award of degree of

DOCTOR OF PHILOSOPHY

in

CHEMISTRY

by

Muhammad Ashfaq Session 2004-2008

13-GCU-PhD-Chem-04


DECLARATION














______

Dated Supervisor


Submitted Through









Dedicated

To



ACKNOWLEDGEMENTS








work


























forgetfulness

Muhammad Ashfaq

Abbreviations

Abbreviations




LIST OF TABLES

xiv

List of Tables





















LIST OF TABLES

xv
























LIST OF TABLES

xvi




LIST OF FIGURES

xvii

List of Figures


reference substance



LIST OF FIGURES

xviii






xiii











ABSTRACT

i

ABSTRACT






formulations







5 min








138









ABSTRACT

ii






fenofibrate

























ABSTRACT

iii












in each study

TABLE OF CONTENTS

iv

Table of Contents

DESCRIPTION PAGE

Abstract i-iii









141 Chylomicrons 03













161 Statins 06



162 Fibrates 09


TABLE OF CONTENTS

v











182 Simvastatin 18

183 Lovastatin 19


185 Gemfibrozil 22

186 Fenofibrate 23

187 Ezetimibe 24





1922 Column 27


1924 Eluent 27

1925 Flow Rate 27

1926 Peak 27

1927 Resolution 27



19210 Tailing 28


TABLE OF CONTENTS

vi



111 Statistics 30

1111 Average 30























31 Solvents 63

32 Chemicals 63



TABLE OF CONTENTS

vii




353 Linearity 66


355 Accuracy 67

356 Precision 67

357 Selectivity 67

358 Robustness 68




3512 HPLC Set Up 69




363 Linearity 70


365 Accuracy 70

366 Precision 71

367 Selectivity 71

368 Robustness 71




3612 HPLC Set Up 73




373 Linearity 74


TABLE OF CONTENTS

viii

375 Accuracy 74

376 Precision 75

377 Selectivity 75

378 Robustness 75



3711 HPLC Set Up 76




383 Linearity 77


385 Accuracy 77

386 Precision 78

387 Selectivity 78

388 Robustness 78




3812 HPLC Set Up 80




393 Linearity 81


395 Accuracy 81

396 Precision 82

397 Selectivity 82

398 Robustness 82



TABLE OF CONTENTS

ix

3911 HPLC Set Up 84




3103 Linearity 85


3105 Accuracy 86

3106 Precision 86

3107 Selectivity 86

3108 Robustness 86








3114 Linearity 89


3116 Accuracy 90

3117 Precision 90

3118 Selectivity 90

3119 Robustness 91








4121 Linearity 93

TABLE OF CONTENTS

x


4123 Accuracy 94

4124 Precision 94

4125 Selectivity 95


4127 Robustness 95






4221 Linearity 104


4223 Accuracy 106

4224 Precision 106



4227 Robustness 109






4321 Linearity 113


4323 Accuracy 113

4324 Precision 114



4327 Robustness 120


TABLE OF CONTENTS

xi




4421 Linearity 122


4423 Accuracy 123

4424 Precision 123



4427 Robustness 124








4323 Accuracy 134

4524 Precision 134



4527 Robustness 139





4621Linearity 143


4623 Accuracy 144

4624 Precision 144

TABLE OF CONTENTS

xii



4627 Robustness 149





4721 Linearity 153


4723 Accuracy 156

4724 Precision 156



4727 Robustness 160



48 Conclusion 163



1

1 INTRODUCTION




























2


























3






follows

141 Chylomicrons
















4


















population





5
























6
















161 Statins

162 Fibrates











7































8






























9








162 Fibrates





















10































11











pregnancy [104 108-109]











117]






12












placebo [126]















13


hyperlipidemia


























14






























15






























16














Synthetic

B Drug Category


C Chemical name





17


N

O-

OHOH

O

CH3CH3

O

NH

F

2

Ca+2

3H2O


E Molecular Formula


F Molecular Weight

120942

G Colour


H Solubility





18

182 Simvastatin


Semi-synthetic

B Drug Category


C Chemical name




O

CH3

CH3

O

O

CH3

CH3

CH3

OOH

H


E Molecular Formula

C25H38O5


19

F Molecular Weight

41857

G Colour


H Solubility



183 Lovastatin


Semi-synthetic

B Drug Category


C Chemical name




20


O

C H 3

CH 3

O

O

C H 3

HCH 3

OH

H

O


E Molecular Formula

C24H36O5

F Molecular Weight

40454

G Colour


H Solubility





Synthetic


21

B Drug Category


C Chemical name




N

N O-

CH3CH3

NS

CH3

O

O

OOHOH

FCH3

Ca+2+2

2


E Molecular Formula

C22H28FN3O6S

F Molecular Weight

100114

G Colour



22

H Solubility



185 Gemfibrozil


Synthetic

B Drug Category


C Chemical name



O

CH3

CH3OH

OCH3

CH3


E Molecular Formula

C15H22O3


23

F Molecular Weight

25033

G Colour


H Solubility


186 Fenofibrate


Synthetic

B Drug Category


C Chemical name



24


O

Cl

O

CH3

CH3

O

O CH3

CH3


E Molecular Formula

C20H21ClO4

F Molecular Weight

36083

G Colour


H Solubility


in alcohol

187 Ezetimibe


Synthetic


25

B Drug Category


C Chemical name




N

O

OH

F

OH F


E Molecular Formula

C24H21F2NO3

F Molecular Weight

4094

G Colour



26

H Solubility


and acetone


















the compounds


27


1921 Chromatogram



1922 Column



mobile phase




1924 Eluent



1925 Flow Rate



1926 Peak



called a peak

1927 Resolution







28

1929 Retention Time


retention time

19210 Tailing








Linearity

Accuracy

Precision

Specificity

Limits of detection


Robustness










29
























is used mostly


the formula


30





111 Statistics






1111 Average [165]



X- = X1 + X2 + X3 + X4 + n












31













Y= mX + C










32


















11221 Granulation





33




too hard or soft




proper granules











34











forced degradation




35

2 LITERATURE REVIEW




each drug























36






to ICH guidelines





















37




























38




























39





























40





























41


respectively








just 19 min


















42




























43






to be 03 mLmin




















acid


44






was 147 minutes






















45




























46
























were below 15






47







for lovastatin

















to 10049





48

























lovastatin


49






















oxidation products






50















than 6 minutes













51



























52






















LOQ





53




























54



between 99 and 101

























55










LOQ












fenofibrate







56





respectively
























57




















microgmL










58





























59












studied
















60





























61












precision

















62












acceptance range


63

3 EXPERIMENTAL WORK







31 Solvents





32 Chemicals















64












Model CP324S

Min 00001g

Max 320 g


Model 5986-62


Model DA-60D


Model PS 02000A


34 Glass Apparatus







65








j) Glass Funnel


66















353 Linearity
















67


accuracy

355 Accuracy









356 Precision





357 Selectivity













68

358 Robustness
























RSD






69







each

3512 HPLC Set Up



3 Detector UV

4 Wavelength 242 nm

5 Injector Rheodyne








70












363 Linearity





triplicate






accuracy

365 Accuracy







71

366 Precision





367 Selectivity












368 Robustness













72
























73

3612 HPLC Set Up



3 Detector UV

4 Wavelength 240 nm

5 Injector Rheodyne








74











373 Linearity





made in triplicate







375 Accuracy








75

376 Precision





days

377 Selectivity














378 Robustness










76












condition




3711 HPLC Set Up



3 Detector UV

4 Wavelength 237 nm

5 Injector Rheodyne








77














383 Linearity











385 Accuracy





78



386 Precision





days

387 Selectivity












388 Robustness






were calculated




79




























80

3812 HPLC Set Up


2 HPLC Pump LC-20AT


4 Wavelength 240 nm

5 Injector Rheodyne








81















393 Linearity












395 Accuracy




82




396 Precision





days

397 Selectivity













for lovastatin

398 Robustness






83


were calculated


















under each stress





84

3911 HPLC Set Up


2 HPLC Pump LC-20AT


4 Wavelength 240 nm

5 Injector Rheodyne








85














respectively

3103 Linearity












accuracy


86

3105 Accuracy






3106 Precision





3107 Selectivity














3108 Robustness





87






















88

31011 HPLC Set Up


2 HPLC Pump LC-20AT


4 Wavelength 240 nm

5 Injector Rheodyne








89

























3114 Linearity






90








3116 Accuracy






3117 Precision





days

3118 Selectivity











91



3119 Robustness

























92

31112 HPLC Set Up


2 HPLC Pump LC-20AT


4 Wavelength 240 nm

5 Injector Rheodyne


ratio of 4060 (vv)






93
























4121 Linearity





94

















4123 Accuracy







4124 Precision






95



4125 Selectivity








given in Table 43





given in Table 44

4127 Robustness






96




97





200 2028 10140 029

240 2368 9867 042

280 2812 10043 124

Ezetimibe 160 1588 9925 057

200 1965 9825 086

240 2442 10175 168

280 2782 9936 011





320 5 3215 103 3248 151

480 5 4772 086 4861 125

Ezetimibe 160 5 1570 070 1633 135

320 5 3252 083 3158 089

480 5 4882 039 4802 110


98





Ezetimibe



32 3218 10056

32 3162 9881

32 3178 9931

32 3252 10162


RSD = 126

32 3251 10159

32 3186 9956

32 3158 9869

32 3224 10075


RSD = 128


99





160 1573 1582 1615 138

320 3148 3168 3150 035

480 4818 4798 4880 089

Ezetimibe

160 1632 1611 1630 074

320 3281 3242 3218 094

480 4772 4848 4820 114


100






Flow rate (04mLmin) 10184 383 3507 125

Flow rate (06 mLmin) 9858 255 3310 141

Buffer (pH 63) 10089 303 3401 120

Buffer (pH 67) 10154 302 3467 121

1RT Retention Time






Flow rate (04mLmin) 9802 558 5221 115

Flow rate (06 mLmin) 9915 372 5019 118

Buffer (pH 63) 10022 441 5186 110

Buffer (pH 67) 10005 443 5125 111

1RT Retention Time


101





















102





Ezetimibe



32 3262 10194

32 3215 10047

32 3168 9900


RSD = 146

32 3256 10175

32 3256 10056

32 3248 10203


RSD = 048


103









column life





















104


[Figure 43 and 44]






4221 Linearity












coefficient










105




106

4223 Accuracy







48]

4224 Precision






4225 Selectivity













107




Ezetimibe 50 3 200 2005 10025 138

100 3 400 3965 9912 068

150 3 600 6020 10033 086

Simvastatin 50 3 200 2030 10150 038

100 3 400 4025 10062 115

150 3 600 6060 10100 102




Ezetimibe 200 5 1986 110 1995 078

400 5 4012 105 3990 115

600 5 5996 028 6012 120

Simvastatin 200 5 2024 145 2010 056

400 5 4056 068 3975 132

600 5 5942 075 6025 088


108



Ezetimibe 10062 5 296 - 123 6781

Simvastatin 9943 5 980 1964 106 13752






Ezetimibe

200 2032 2009 1985 024

400 4076 3990 4040 043

600 5970 6025 6056 044

Simvastatin

200 2012 1995 2005 008

400 4035 4025 4020 008

600 6015 6025 5975 026


109

4227 Robustness
























formulations


110






Flow rate (14 mLmin) 9842 318 6810 125

Flow rate (16 mLmin) 9905 278 6566 127

Buffer (pH 48) 10022 299 6685 125

Buffer (pH 52) 10089 301 6628 124






Flow rate (14 mLmin) 10033 1052 13927 105

Flow rate (16 mLmin) 9915 919 13425 107

Buffer (pH 48) 10129 985 13564 108

Buffer (pH 52) 10086 984 13416 108


111




Ezetimibe 5 10 1012 10125 075

Simvastatin 5 10 1005 10050 115


112






























113





solutions

4321 Linearity










099997










4323 Accuracy





114





4324 Precision






416

4325 Selectivity








the proposed method







115



116




Gemfibrozil 50 5 1200 12022 10018 095

100 5 2400 23734 9889 043

150 5 3600 35421 9839 042

Simvastatin 50 5 20 202 10100 133

100 5 40 406 10150 119

150 5 60 593 9883 074




Gemfibrozil 1200 5 12125 078 11958 125

2400 5 24456 095 24258 102

3600 5 36521 124 36321 085

Simvastatin 20 5 202 144 201 106

40 5 396 111 395 058

60 5 607 036 602 131


117



118


Gemfibrozil



Simvastatin



240 23645 9852

240 24142 10059

240 24356 10148

240 23988 9995


RSD = 125

4 405 10125

4 396 9900

4 398 9950

4 393 9825


RSD = 128


119





Gemfibrozil

1200 11808 11788 11756 022

2400 24262 23943 23640 130

3600 35828 35641 35494 047

Simvastatin

20 201 199 197 101

40 398 395 391 089

60 602 595 591 093


120

4327 Robustness


















121






Flow rate (11mLmin) 9869 424 7118 128

Flow rate (09 mLmin) 10041 518 8002 122

Buffer (pH 52) 9889 465 7719 123

Buffer (pH 48) 10115 465 7662 123

1Retention Time

2Theoretical Plates






Flow rate (11mLmin) 9921 698 10220 129

Flow rate (09 mLmin) 9903 853 12515 122

Buffer (pH 52) 10069 765 11308 122

Buffer (pH 48) 10098 766 11015 122

1Retention Time

2Theoretical Plates


122




















solutions

4421 Linearity










123












4423 Accuracy







4424 Precision





Table 422

4425 Selectivity






124









4427 Robustness





















125




126




Ezetimibe 5 50 80 788 9850 095

5 100 160 1581 9881 031

5 150 240 2405 10021 033

Fenofibrate 5 50 1280 12924 10097 018

5 100 2560 25492 9958 051

5 150 3840 38850 10117 075




Ezetimibe 160 5 158 138 157 151

160 5 1608 095 1611 107

400 5 3995 055 3991 085

Fenofibrate 256 5 2550 096 2553 063

256 5 2548 033 2545 051

640 5 6373 022 6355 039


127


Ezetimibe



Fenofibrate



160 1611 10089

160 1593 9956

160 1588 9925

160 1590 9938


RSD = 076

2560 2538 9914

2560 2543 9934

2560 2581 10082

2560 2546 9945


RSD = 077


128





Ezetimibe

160 157 156 155 064

160 1618 1590 1576 134

400 3988 3942 3912 041

Fenofibrate

256 2484 2465 2456 058

2560 25512 25349 25215 059

6400 63841 63555 63373 037


129






Flow rate (14mLmin) 10098 261 6372 136

Flow rate (16 mLmin) 9962 234 4803 116

Buffer (pH 52) 10126 243 6005 122

Buffer (pH 48) 10085 244 6078 123






Flow rate (14mLmin) 10049 950 14337 121

Flow rate (16 mLmin) 9979 808 9991 107

Buffer (pH 52) 10021 876 12885 115

Buffer (pH 48) 9905 877 12687 116


130








131


Ezetimibe



Fenofibrate



16 1624 10150

16 1605 10031

16 1591 9944


RSD = 103

256 25894 10115

256 25536 9975

256 25748 10058


RSD = 070


132































133








shown)























134




4323 Accuracy








4524 Precision







4525 Selectivity








the proposed method


135



136




Ezetimibe 50 3 50 508 10160 102

100 3 100 988 9880 146

150 3 150 1541 10273 039

Lovastatin 50 3 100 1022 10220 063

100 3 200 1944 9720 119

150 3 300 2928 9760 093




Ezetimibe 080 5 082 122 081 163

250 5 2458 086 2443 138

1000 5 9869 074 9805 108

Lovastatin 16 5 156 111 155 151

500 5 5059 055 5046 149

2000 5 20241 032 19968 098


137



138


Ezetimibe



Lovastatin



100 1023 10230

100 992 9920

100 986 9860

100 1018 10180


RSD = 184

200 2054 10270

200 2036 10180

200 1978 9890

200 1986 9930


RSD = 185


139






4527 Robustness















140





Ezetimibe

080 081 078 079 193

250 2484 2466 2448 073

1000 10098 9922 9805 148

Lovastatin

160 163 161 159 124

500 4963 4921 4893 071

2000 20098 19852 19646 114


141






Flow rate (09 mLmin) 9946 453 5100 134

Flow rate (11 mLmin) 9905 370 4886 140

Buffer (pH 52) 10048 405 5454 139

Buffer (pH 48) 10215 404 5404 139






Flow rate (09 mLmin) 9932 1080 8004 126

Flow rate (11 mLmin) 10068 883 7575 136

Buffer (pH 52) 10046 967 7785 130

Buffer (pH 48) 9885 966 7715 130


142





























143






shown)





of solutions

4621 Linearity



















144




4623 Accuracy








4624 Precision






435

4625 Selectivity








the proposed method


145



146




Atorvastatin 50 3 40 406 10150 163

100 3 80 789 9863 126

150 3 120 1212 10100 069

Gemfibrozil 50 3 2400 23658 9858 101

100 3 4800 48863 10180 065

150 3 7200 73356 10188 053




Atorvastatin 05 5 052 198 051 223

80 5 795 086 786 155

200 5 1984 063 1982 141

Gemfibrozil 300 5 2963 101 2951 129

4800 5 48212 073 47871 122

12000 5 118648 088 118002 125


147



148


Atorvastatin



Gemfibrozil



80 808 10100

80 796 9950

80 805 10063

80 793 9912


RSD = 090

4800 47222 9838

4800 47805 9959

4800 48215 10045

4800 47329 9860


RSD = 097


149






4627 Robustness














products





414


150





Atorvastatin

05 052 051 052 112

80 794 797 790 044

200 2022 1995 1990 086

Gemfibrozil

300 3046 2983 2955 156

4800 48258 47626 47298 069

12000 119239 118658 118022 051


151






Flow rate (14 mLmin) 10169 380 3310 136

Flow rate (16 mLmin) 10043 334 2866 141

Buffer (pH 48) 9932 354 3164 141

Buffer (pH 52) 9978 355 3214 140






Flow rate (14 mLmin) 9911 1255 4002 131

Flow rate (16 mLmin) 10009 1098 3687 135

Buffer (pH 48) 9969 1169 3998 134

Buffer (pH 52) 9955 1169 4008 135


152



Ca2+

3H2O

N

O

NH

CH3

CH3

F

OHOH

O-

O CH3OH H2O2

Room TempO

O

NH

O

OH

OCH3

OHCH3


153


























4721 Linearity





154




















155




156

4723 Accuracy








4724 Precision






4725 Selectivity













157




Rosuvastatin 50 3 400 4069 10172 145

100 3 800 7888 9860 115

150 3 1200 11821 9851 096

Ezetimibe 50 3 100 981 9810 139

100 3 200 2048 10240 121

150 3 300 2928 9760 111




Rosuvastatin 50 5 505 144 503 189

800 5 7925 119 7805 169

1600 5 16228 095 16059 128

Ezetimibe 125 5 123 151 122 205

200 5 2051 076 2028 128

400 5 3965 105 3921 156


158


Rosuvastatin



Ezetimibe



800 8089 10111

800 7866 9832

800 8129 10161

800 8052 10065


RSD = 145

200 2048 10240

200 1963 9815

200 1983 9915

200 1972 9860


RSD = 193


159





Rosuvastatin

50 497 498 495 031

800 7942 7881 7885 043

1600 16152 15922 15905 086

Ezetimibe

125 123 121 122 082

200 1982 1975 1955 071

400 4008 3928 3911 131


160

4727 Robustness
























161



ACN 1 H3PO4 (6040) 10025 430 3126 135

ACN 1 H3PO4 (5842) 10011 476 3316 133

ACN 1 H3PO4 (6238) 9865 408 3040 141

Flow rate (11mLmin) 9985 391 2866 140

Flow rate (09 mLmin) 10141 478 3264 135

H3PO4 Conc (09 ) 9955 428 3167 139

H3PO4 Conc (11 ) 9941 427 3114 133



ACN 1 H3PO4 (6040) 10069 633 4139 142

ACN 1 H3PO4 (5842) 9965 715 4267 141

ACN 1 H3PO4 (6238) 10025 595 3964 148

Flow rate (11mLmin) 10095 575 4040 145

Flow rate (09 mLmin) 9926 703 4220 142

H3PO4 Conc (09 ) 10068 635 3998 144

H3PO4 Conc (11 ) 10029 636 4002 139


162




Rosuvastatin 5 40 4052 10130 103

Ezetimibe 5 10 1021 10210 131


163

48 Conclusion









formulations




















164










human plasma














formulations


165

5 REFERENCES






Page 971



2008 14(2) 314-324


141108


Accessed on 141108


141108



141108


151108




New York 2003






166


2002 106 3240



278 313ndash321




















2619ndash2628



77 31ndash36



167



1146


2000 9 2653ndash2661


239ndash252




44 379ndash387




32B









1ndash34






168





27 107ndash114




638





25 1191ndash1199









1995 58 412ndash417






597ndash602


169


420









69B



170-89


Lipid Res 1997 38 2071-2078



135 249- 256





Anal 2006 41(2) 408-414







170


317ndash 326






951ndash956





29636ndash29639



Sci USA 1984 81 4203ndash 4207




79 95ndash99




Biol Chem 1999 274 32048ndash32054



275 36703ndash 36707




171




S45-S48



Pharmacol 2000 56 631-5



47(2) 73-104








2002 71 80


Disease 2004 3 22











172



283 157-163




Pharmacol 2001 134 409-417


79-84







729-741









Heart J 2005 149 464ndash473




173



2003 107 2409ndash 2415







1758 ndash1773



2125ndash2134






Proc 2004 79 620ndash 629



418ndash424







728


174



455









334









1997 80 608ndash613






47 1584 ndash1587


5th Edition 1994 23-25 51-53


175




01082009


on 01082009






03082009







992ndash1009

171 Khedr A J AOAC Int 2007 90(6) 1547-53


Sci 2007 30(18) 3143-53


766


(11-12) 737-741



44(2) 379-87


176


241-6



Life Sci 2007 846(1-2) 215-21











382(5) 1242-9


Anal 2003 33(5) 1017-23


Spectrom 1999 13(11) 1003-15


1999 10(1) 55-66







2008 81(3) 453-459


177


Chemistry 2007 62(6 ) 536-541







Chemistry 2006 61 (1 ) 63-66




2004 49 (4) 289-289


Sep Sci 2004 27(13) 1087-92


2002 29 (4) 715-721


18(3) 232-4





1992 10(9) 693-7


2008 22(5) 511-8


Anal 2008 46(4) 808-813


178


2008 20 399ndash410



2719ndash2725


Chem Soc 2005 50(4) 639-646




Biomed Life Sci 2002 768(2) 349-59


41


1999 20(1-2) 137-43


Biomed Anal 1988 6(3) 271-6






Biomed Life Sci 2007 856(1-2) 35-40


Biomed Anal 2007 44(2) 540-6






179


International 2005 88 (4) 1142-1147


Anal 2004 35(3) 609-14


Pharm Sci 2007 69 819-21





B 2003 791 161ndash170



Anal 2001 26(1) 7-14








197-202


60(5) 425-38



J Chromatogr A 2004 1046(1-2) 133-40



180


2000 742(2) 391-400




118(10) 447-55




2) 137-142


67(1-2) 101-107


SR Ind J Pharm Sci 2008 70(2) 258-260




853(1-2) 88-96



Biomed Anal 2006 41(3) 1037-40




B 2006 830(1)143-150




181



IFPMA Geneva 1994



1996








1995 76(2) 126A-128A











Title_pages_PhDpdf


Muhammad Ashfaq




Supervisor


To

Abbreviationspdf


List_of_Tablespdf

List of Tables

TAB DESCRIPTIONPAGE

List_of_Figurespdf

FIG DESCRIPTIONPAGE




Table of Contents

DESCRIPTION PAGE









3512 HPLC Set Up69


3612 HPLC Set Up73

3711 HPLC Set Up76


3812 HPLC Set Up80

3911 HPLC Set Up84

31011 HPLC Set Up88

31112 HPLC Set Up92


48S-1pdf









48S-2pdf



48S-3pdf

3 EXPERIMENTAL WORK




3512 HPLC Set Up



3612 HPLC Set Up

3711 HPLC Set Up


3812 HPLC Set Up

3911 HPLC Set Up

31011 HPLC Set Up


31112 HPLC Set Up

48S-4pdf




























ACN 1 H3PO4 (6040) 10025 430 3126 135


ACN 1 H3PO4 (6040) 10069 633 4139 142

DECLARATION














______

Dated Supervisor


Submitted Through









Dedicated

To



ACKNOWLEDGEMENTS








work


























forgetfulness

Muhammad Ashfaq

Abbreviations

Abbreviations




LIST OF TABLES

xiv

List of Tables





















LIST OF TABLES

xv
























LIST OF TABLES

xvi




LIST OF FIGURES

xvii

List of Figures


reference substance



LIST OF FIGURES

xviii






xiii











ABSTRACT

i

ABSTRACT






formulations







5 min








138









ABSTRACT

ii






fenofibrate

























ABSTRACT

iii












in each study

TABLE OF CONTENTS

iv

Table of Contents

DESCRIPTION PAGE

Abstract i-iii









141 Chylomicrons 03













161 Statins 06



162 Fibrates 09


TABLE OF CONTENTS

v











182 Simvastatin 18

183 Lovastatin 19


185 Gemfibrozil 22

186 Fenofibrate 23

187 Ezetimibe 24





1922 Column 27


1924 Eluent 27

1925 Flow Rate 27

1926 Peak 27

1927 Resolution 27



19210 Tailing 28


TABLE OF CONTENTS

vi



111 Statistics 30

1111 Average 30























31 Solvents 63

32 Chemicals 63



TABLE OF CONTENTS

vii




353 Linearity 66


355 Accuracy 67

356 Precision 67

357 Selectivity 67

358 Robustness 68




3512 HPLC Set Up 69




363 Linearity 70


365 Accuracy 70

366 Precision 71

367 Selectivity 71

368 Robustness 71




3612 HPLC Set Up 73




373 Linearity 74


TABLE OF CONTENTS

viii

375 Accuracy 74

376 Precision 75

377 Selectivity 75

378 Robustness 75



3711 HPLC Set Up 76




383 Linearity 77


385 Accuracy 77

386 Precision 78

387 Selectivity 78

388 Robustness 78




3812 HPLC Set Up 80




393 Linearity 81


395 Accuracy 81

396 Precision 82

397 Selectivity 82

398 Robustness 82



TABLE OF CONTENTS

ix

3911 HPLC Set Up 84




3103 Linearity 85


3105 Accuracy 86

3106 Precision 86

3107 Selectivity 86

3108 Robustness 86








3114 Linearity 89


3116 Accuracy 90

3117 Precision 90

3118 Selectivity 90

3119 Robustness 91








4121 Linearity 93

TABLE OF CONTENTS

x


4123 Accuracy 94

4124 Precision 94

4125 Selectivity 95


4127 Robustness 95






4221 Linearity 104


4223 Accuracy 106

4224 Precision 106



4227 Robustness 109






4321 Linearity 113


4323 Accuracy 113

4324 Precision 114



4327 Robustness 120


TABLE OF CONTENTS

xi




4421 Linearity 122


4423 Accuracy 123

4424 Precision 123



4427 Robustness 124








4323 Accuracy 134

4524 Precision 134



4527 Robustness 139





4621Linearity 143


4623 Accuracy 144

4624 Precision 144

TABLE OF CONTENTS

xii



4627 Robustness 149





4721 Linearity 153


4723 Accuracy 156

4724 Precision 156



4727 Robustness 160



48 Conclusion 163



1

1 INTRODUCTION




























2


























3






follows

141 Chylomicrons
















4


















population





5
























6
















161 Statins

162 Fibrates











7































8






























9








162 Fibrates





















10































11











pregnancy [104 108-109]











117]






12












placebo [126]















13


hyperlipidemia


























14






























15






























16














Synthetic

B Drug Category


C Chemical name





17


N

O-

OHOH

O

CH3CH3

O

NH

F

2

Ca+2

3H2O


E Molecular Formula


F Molecular Weight

120942

G Colour


H Solubility





18

182 Simvastatin


Semi-synthetic

B Drug Category


C Chemical name




O

CH3

CH3

O

O

CH3

CH3

CH3

OOH

H


E Molecular Formula

C25H38O5


19

F Molecular Weight

41857

G Colour


H Solubility



183 Lovastatin


Semi-synthetic

B Drug Category


C Chemical name




20


O

C H 3

CH 3

O

O

C H 3

HCH 3

OH

H

O


E Molecular Formula

C24H36O5

F Molecular Weight

40454

G Colour


H Solubility





Synthetic


21

B Drug Category


C Chemical name




N

N O-

CH3CH3

NS

CH3

O

O

OOHOH

FCH3

Ca+2+2

2


E Molecular Formula

C22H28FN3O6S

F Molecular Weight

100114

G Colour



22

H Solubility



185 Gemfibrozil


Synthetic

B Drug Category


C Chemical name



O

CH3

CH3OH

OCH3

CH3


E Molecular Formula

C15H22O3


23

F Molecular Weight

25033

G Colour


H Solubility


186 Fenofibrate


Synthetic

B Drug Category


C Chemical name



24


O

Cl

O

CH3

CH3

O

O CH3

CH3


E Molecular Formula

C20H21ClO4

F Molecular Weight

36083

G Colour


H Solubility


in alcohol

187 Ezetimibe


Synthetic


25

B Drug Category


C Chemical name




N

O

OH

F

OH F


E Molecular Formula

C24H21F2NO3

F Molecular Weight

4094

G Colour



26

H Solubility


and acetone


















the compounds


27


1921 Chromatogram



1922 Column



mobile phase




1924 Eluent



1925 Flow Rate



1926 Peak



called a peak

1927 Resolution







28

1929 Retention Time


retention time

19210 Tailing








Linearity

Accuracy

Precision

Specificity

Limits of detection


Robustness










29
























is used mostly


the formula


30





111 Statistics






1111 Average [165]



X- = X1 + X2 + X3 + X4 + n












31













Y= mX + C










32


















11221 Granulation





33




too hard or soft




proper granules











34











forced degradation




35

2 LITERATURE REVIEW




each drug























36






to ICH guidelines





















37




























38




























39





























40





























41


respectively








just 19 min


















42




























43






to be 03 mLmin




















acid


44






was 147 minutes






















45




























46
























were below 15






47







for lovastatin

















to 10049





48

























lovastatin


49






















oxidation products






50















than 6 minutes













51



























52






















LOQ





53




























54



between 99 and 101

























55










LOQ












fenofibrate







56





respectively
























57




















microgmL










58





























59












studied
















60





























61












precision

















62












acceptance range


63

3 EXPERIMENTAL WORK







31 Solvents





32 Chemicals















64












Model CP324S

Min 00001g

Max 320 g


Model 5986-62


Model DA-60D


Model PS 02000A


34 Glass Apparatus







65








j) Glass Funnel


66















353 Linearity
















67


accuracy

355 Accuracy









356 Precision





357 Selectivity













68

358 Robustness
























RSD






69







each

3512 HPLC Set Up



3 Detector UV

4 Wavelength 242 nm

5 Injector Rheodyne








70












363 Linearity





triplicate






accuracy

365 Accuracy







71

366 Precision





367 Selectivity












368 Robustness













72
























73

3612 HPLC Set Up



3 Detector UV

4 Wavelength 240 nm

5 Injector Rheodyne








74











373 Linearity





made in triplicate







375 Accuracy








75

376 Precision





days

377 Selectivity














378 Robustness










76












condition




3711 HPLC Set Up



3 Detector UV

4 Wavelength 237 nm

5 Injector Rheodyne








77














383 Linearity











385 Accuracy





78



386 Precision





days

387 Selectivity












388 Robustness






were calculated




79




























80

3812 HPLC Set Up


2 HPLC Pump LC-20AT


4 Wavelength 240 nm

5 Injector Rheodyne








81















393 Linearity












395 Accuracy




82




396 Precision





days

397 Selectivity













for lovastatin

398 Robustness






83


were calculated


















under each stress





84

3911 HPLC Set Up


2 HPLC Pump LC-20AT


4 Wavelength 240 nm

5 Injector Rheodyne








85














respectively

3103 Linearity












accuracy


86

3105 Accuracy






3106 Precision





3107 Selectivity














3108 Robustness





87






















88

31011 HPLC Set Up


2 HPLC Pump LC-20AT


4 Wavelength 240 nm

5 Injector Rheodyne








89

























3114 Linearity






90








3116 Accuracy






3117 Precision





days

3118 Selectivity











91



3119 Robustness

























92

31112 HPLC Set Up


2 HPLC Pump LC-20AT


4 Wavelength 240 nm

5 Injector Rheodyne


ratio of 4060 (vv)






93
























4121 Linearity





94

















4123 Accuracy







4124 Precision






95



4125 Selectivity








given in Table 43





given in Table 44

4127 Robustness






96




97





200 2028 10140 029

240 2368 9867 042

280 2812 10043 124

Ezetimibe 160 1588 9925 057

200 1965 9825 086

240 2442 10175 168

280 2782 9936 011





320 5 3215 103 3248 151

480 5 4772 086 4861 125

Ezetimibe 160 5 1570 070 1633 135

320 5 3252 083 3158 089

480 5 4882 039 4802 110


98





Ezetimibe



32 3218 10056

32 3162 9881

32 3178 9931

32 3252 10162


RSD = 126

32 3251 10159

32 3186 9956

32 3158 9869

32 3224 10075


RSD = 128


99





160 1573 1582 1615 138

320 3148 3168 3150 035

480 4818 4798 4880 089

Ezetimibe

160 1632 1611 1630 074

320 3281 3242 3218 094

480 4772 4848 4820 114


100






Flow rate (04mLmin) 10184 383 3507 125

Flow rate (06 mLmin) 9858 255 3310 141

Buffer (pH 63) 10089 303 3401 120

Buffer (pH 67) 10154 302 3467 121

1RT Retention Time






Flow rate (04mLmin) 9802 558 5221 115

Flow rate (06 mLmin) 9915 372 5019 118

Buffer (pH 63) 10022 441 5186 110

Buffer (pH 67) 10005 443 5125 111

1RT Retention Time


101





















102





Ezetimibe



32 3262 10194

32 3215 10047

32 3168 9900


RSD = 146

32 3256 10175

32 3256 10056

32 3248 10203


RSD = 048


103









column life





















104


[Figure 43 and 44]






4221 Linearity












coefficient










105




106

4223 Accuracy







48]

4224 Precision






4225 Selectivity













107




Ezetimibe 50 3 200 2005 10025 138

100 3 400 3965 9912 068

150 3 600 6020 10033 086

Simvastatin 50 3 200 2030 10150 038

100 3 400 4025 10062 115

150 3 600 6060 10100 102




Ezetimibe 200 5 1986 110 1995 078

400 5 4012 105 3990 115

600 5 5996 028 6012 120

Simvastatin 200 5 2024 145 2010 056

400 5 4056 068 3975 132

600 5 5942 075 6025 088


108



Ezetimibe 10062 5 296 - 123 6781

Simvastatin 9943 5 980 1964 106 13752






Ezetimibe

200 2032 2009 1985 024

400 4076 3990 4040 043

600 5970 6025 6056 044

Simvastatin

200 2012 1995 2005 008

400 4035 4025 4020 008

600 6015 6025 5975 026


109

4227 Robustness
























formulations


110






Flow rate (14 mLmin) 9842 318 6810 125

Flow rate (16 mLmin) 9905 278 6566 127

Buffer (pH 48) 10022 299 6685 125

Buffer (pH 52) 10089 301 6628 124






Flow rate (14 mLmin) 10033 1052 13927 105

Flow rate (16 mLmin) 9915 919 13425 107

Buffer (pH 48) 10129 985 13564 108

Buffer (pH 52) 10086 984 13416 108


111




Ezetimibe 5 10 1012 10125 075

Simvastatin 5 10 1005 10050 115


112






























113





solutions

4321 Linearity










099997










4323 Accuracy





114





4324 Precision






416

4325 Selectivity








the proposed method







115



116




Gemfibrozil 50 5 1200 12022 10018 095

100 5 2400 23734 9889 043

150 5 3600 35421 9839 042

Simvastatin 50 5 20 202 10100 133

100 5 40 406 10150 119

150 5 60 593 9883 074




Gemfibrozil 1200 5 12125 078 11958 125

2400 5 24456 095 24258 102

3600 5 36521 124 36321 085

Simvastatin 20 5 202 144 201 106

40 5 396 111 395 058

60 5 607 036 602 131


117



118


Gemfibrozil



Simvastatin



240 23645 9852

240 24142 10059

240 24356 10148

240 23988 9995


RSD = 125

4 405 10125

4 396 9900

4 398 9950

4 393 9825


RSD = 128


119





Gemfibrozil

1200 11808 11788 11756 022

2400 24262 23943 23640 130

3600 35828 35641 35494 047

Simvastatin

20 201 199 197 101

40 398 395 391 089

60 602 595 591 093


120

4327 Robustness


















121






Flow rate (11mLmin) 9869 424 7118 128

Flow rate (09 mLmin) 10041 518 8002 122

Buffer (pH 52) 9889 465 7719 123

Buffer (pH 48) 10115 465 7662 123

1Retention Time

2Theoretical Plates






Flow rate (11mLmin) 9921 698 10220 129

Flow rate (09 mLmin) 9903 853 12515 122

Buffer (pH 52) 10069 765 11308 122

Buffer (pH 48) 10098 766 11015 122

1Retention Time

2Theoretical Plates


122




















solutions

4421 Linearity










123












4423 Accuracy







4424 Precision





Table 422

4425 Selectivity






124









4427 Robustness





















125




126




Ezetimibe 5 50 80 788 9850 095

5 100 160 1581 9881 031

5 150 240 2405 10021 033

Fenofibrate 5 50 1280 12924 10097 018

5 100 2560 25492 9958 051

5 150 3840 38850 10117 075




Ezetimibe 160 5 158 138 157 151

160 5 1608 095 1611 107

400 5 3995 055 3991 085

Fenofibrate 256 5 2550 096 2553 063

256 5 2548 033 2545 051

640 5 6373 022 6355 039


127


Ezetimibe



Fenofibrate



160 1611 10089

160 1593 9956

160 1588 9925

160 1590 9938


RSD = 076

2560 2538 9914

2560 2543 9934

2560 2581 10082

2560 2546 9945


RSD = 077


128





Ezetimibe

160 157 156 155 064

160 1618 1590 1576 134

400 3988 3942 3912 041

Fenofibrate

256 2484 2465 2456 058

2560 25512 25349 25215 059

6400 63841 63555 63373 037


129






Flow rate (14mLmin) 10098 261 6372 136

Flow rate (16 mLmin) 9962 234 4803 116

Buffer (pH 52) 10126 243 6005 122

Buffer (pH 48) 10085 244 6078 123






Flow rate (14mLmin) 10049 950 14337 121

Flow rate (16 mLmin) 9979 808 9991 107

Buffer (pH 52) 10021 876 12885 115

Buffer (pH 48) 9905 877 12687 116


130








131


Ezetimibe



Fenofibrate



16 1624 10150

16 1605 10031

16 1591 9944


RSD = 103

256 25894 10115

256 25536 9975

256 25748 10058


RSD = 070


132































133








shown)























134




4323 Accuracy








4524 Precision







4525 Selectivity








the proposed method


135



136




Ezetimibe 50 3 50 508 10160 102

100 3 100 988 9880 146

150 3 150 1541 10273 039

Lovastatin 50 3 100 1022 10220 063

100 3 200 1944 9720 119

150 3 300 2928 9760 093




Ezetimibe 080 5 082 122 081 163

250 5 2458 086 2443 138

1000 5 9869 074 9805 108

Lovastatin 16 5 156 111 155 151

500 5 5059 055 5046 149

2000 5 20241 032 19968 098


137



138


Ezetimibe



Lovastatin



100 1023 10230

100 992 9920

100 986 9860

100 1018 10180


RSD = 184

200 2054 10270

200 2036 10180

200 1978 9890

200 1986 9930


RSD = 185


139






4527 Robustness















140





Ezetimibe

080 081 078 079 193

250 2484 2466 2448 073

1000 10098 9922 9805 148

Lovastatin

160 163 161 159 124

500 4963 4921 4893 071

2000 20098 19852 19646 114


141






Flow rate (09 mLmin) 9946 453 5100 134

Flow rate (11 mLmin) 9905 370 4886 140

Buffer (pH 52) 10048 405 5454 139

Buffer (pH 48) 10215 404 5404 139






Flow rate (09 mLmin) 9932 1080 8004 126

Flow rate (11 mLmin) 10068 883 7575 136

Buffer (pH 52) 10046 967 7785 130

Buffer (pH 48) 9885 966 7715 130


142





























143






shown)





of solutions

4621 Linearity



















144




4623 Accuracy








4624 Precision






435

4625 Selectivity








the proposed method


145



146




Atorvastatin 50 3 40 406 10150 163

100 3 80 789 9863 126

150 3 120 1212 10100 069

Gemfibrozil 50 3 2400 23658 9858 101

100 3 4800 48863 10180 065

150 3 7200 73356 10188 053




Atorvastatin 05 5 052 198 051 223

80 5 795 086 786 155

200 5 1984 063 1982 141

Gemfibrozil 300 5 2963 101 2951 129

4800 5 48212 073 47871 122

12000 5 118648 088 118002 125


147



148


Atorvastatin



Gemfibrozil



80 808 10100

80 796 9950

80 805 10063

80 793 9912


RSD = 090

4800 47222 9838

4800 47805 9959

4800 48215 10045

4800 47329 9860


RSD = 097


149






4627 Robustness














products





414


150





Atorvastatin

05 052 051 052 112

80 794 797 790 044

200 2022 1995 1990 086

Gemfibrozil

300 3046 2983 2955 156

4800 48258 47626 47298 069

12000 119239 118658 118022 051


151






Flow rate (14 mLmin) 10169 380 3310 136

Flow rate (16 mLmin) 10043 334 2866 141

Buffer (pH 48) 9932 354 3164 141

Buffer (pH 52) 9978 355 3214 140






Flow rate (14 mLmin) 9911 1255 4002 131

Flow rate (16 mLmin) 10009 1098 3687 135

Buffer (pH 48) 9969 1169 3998 134

Buffer (pH 52) 9955 1169 4008 135


152



Ca2+

3H2O

N

O

NH

CH3

CH3

F

OHOH

O-

O CH3OH H2O2

Room TempO

O

NH

O

OH

OCH3

OHCH3


153


























4721 Linearity





154




















155




156

4723 Accuracy








4724 Precision






4725 Selectivity













157




Rosuvastatin 50 3 400 4069 10172 145

100 3 800 7888 9860 115

150 3 1200 11821 9851 096

Ezetimibe 50 3 100 981 9810 139

100 3 200 2048 10240 121

150 3 300 2928 9760 111




Rosuvastatin 50 5 505 144 503 189

800 5 7925 119 7805 169

1600 5 16228 095 16059 128

Ezetimibe 125 5 123 151 122 205

200 5 2051 076 2028 128

400 5 3965 105 3921 156


158


Rosuvastatin



Ezetimibe



800 8089 10111

800 7866 9832

800 8129 10161

800 8052 10065


RSD = 145

200 2048 10240

200 1963 9815

200 1983 9915

200 1972 9860


RSD = 193


159





Rosuvastatin

50 497 498 495 031

800 7942 7881 7885 043

1600 16152 15922 15905 086

Ezetimibe

125 123 121 122 082

200 1982 1975 1955 071

400 4008 3928 3911 131


160

4727 Robustness
























161



ACN 1 H3PO4 (6040) 10025 430 3126 135

ACN 1 H3PO4 (5842) 10011 476 3316 133

ACN 1 H3PO4 (6238) 9865 408 3040 141

Flow rate (11mLmin) 9985 391 2866 140

Flow rate (09 mLmin) 10141 478 3264 135

H3PO4 Conc (09 ) 9955 428 3167 139

H3PO4 Conc (11 ) 9941 427 3114 133



ACN 1 H3PO4 (6040) 10069 633 4139 142

ACN 1 H3PO4 (5842) 9965 715 4267 141

ACN 1 H3PO4 (6238) 10025 595 3964 148

Flow rate (11mLmin) 10095 575 4040 145

Flow rate (09 mLmin) 9926 703 4220 142

H3PO4 Conc (09 ) 10068 635 3998 144

H3PO4 Conc (11 ) 10029 636 4002 139


162




Rosuvastatin 5 40 4052 10130 103

Ezetimibe 5 10 1021 10210 131


163

48 Conclusion









formulations




















164










human plasma














formulations


165

5 REFERENCES






Page 971



2008 14(2) 314-324


141108


Accessed on 141108


141108



141108


151108




New York 2003






166


2002 106 3240



278 313ndash321




















2619ndash2628



77 31ndash36



167



1146


2000 9 2653ndash2661


239ndash252




44 379ndash387




32B









1ndash34






168





27 107ndash114




638





25 1191ndash1199









1995 58 412ndash417






597ndash602


169


420









69B



170-89


Lipid Res 1997 38 2071-2078



135 249- 256





Anal 2006 41(2) 408-414







170


317ndash 326






951ndash956





29636ndash29639



Sci USA 1984 81 4203ndash 4207




79 95ndash99




Biol Chem 1999 274 32048ndash32054



275 36703ndash 36707




171




S45-S48



Pharmacol 2000 56 631-5



47(2) 73-104








2002 71 80


Disease 2004 3 22











172



283 157-163




Pharmacol 2001 134 409-417


79-84







729-741









Heart J 2005 149 464ndash473




173



2003 107 2409ndash 2415







1758 ndash1773



2125ndash2134






Proc 2004 79 620ndash 629



418ndash424







728


174



455









334









1997 80 608ndash613






47 1584 ndash1587


5th Edition 1994 23-25 51-53


175




01082009


on 01082009






03082009







992ndash1009

171 Khedr A J AOAC Int 2007 90(6) 1547-53


Sci 2007 30(18) 3143-53


766


(11-12) 737-741



44(2) 379-87


176


241-6



Life Sci 2007 846(1-2) 215-21











382(5) 1242-9


Anal 2003 33(5) 1017-23


Spectrom 1999 13(11) 1003-15


1999 10(1) 55-66







2008 81(3) 453-459


177


Chemistry 2007 62(6 ) 536-541







Chemistry 2006 61 (1 ) 63-66




2004 49 (4) 289-289


Sep Sci 2004 27(13) 1087-92


2002 29 (4) 715-721


18(3) 232-4





1992 10(9) 693-7


2008 22(5) 511-8


Anal 2008 46(4) 808-813


178


2008 20 399ndash410



2719ndash2725


Chem Soc 2005 50(4) 639-646




Biomed Life Sci 2002 768(2) 349-59


41


1999 20(1-2) 137-43


Biomed Anal 1988 6(3) 271-6






Biomed Life Sci 2007 856(1-2) 35-40


Biomed Anal 2007 44(2) 540-6






179


International 2005 88 (4) 1142-1147


Anal 2004 35(3) 609-14


Pharm Sci 2007 69 819-21





B 2003 791 161ndash170



Anal 2001 26(1) 7-14








197-202


60(5) 425-38



J Chromatogr A 2004 1046(1-2) 133-40



180


2000 742(2) 391-400




118(10) 447-55




2) 137-142


67(1-2) 101-107


SR Ind J Pharm Sci 2008 70(2) 258-260




853(1-2) 88-96



Biomed Anal 2006 41(3) 1037-40




B 2006 830(1)143-150




181



IFPMA Geneva 1994



1996








1995 76(2) 126A-128A











Title_pages_PhDpdf


Muhammad Ashfaq




Supervisor


To

Abbreviationspdf


List_of_Tablespdf

List of Tables

TAB DESCRIPTIONPAGE

List_of_Figurespdf

FIG DESCRIPTIONPAGE




Table of Contents

DESCRIPTION PAGE









3512 HPLC Set Up69


3612 HPLC Set Up73

3711 HPLC Set Up76


3812 HPLC Set Up80

3911 HPLC Set Up84

31011 HPLC Set Up88

31112 HPLC Set Up92


48S-1pdf









48S-2pdf



48S-3pdf

3 EXPERIMENTAL WORK




3512 HPLC Set Up



3612 HPLC Set Up

3711 HPLC Set Up


3812 HPLC Set Up

3911 HPLC Set Up

31011 HPLC Set Up


31112 HPLC Set Up

48S-4pdf




























ACN 1 H3PO4 (6040) 10025 430 3126 135


ACN 1 H3PO4 (6040) 10069 633 4139 142






______

Dated Supervisor


Submitted Through









Dedicated

To



ACKNOWLEDGEMENTS








work


























forgetfulness

Muhammad Ashfaq

Abbreviations

Abbreviations




LIST OF TABLES

xiv

List of Tables





















LIST OF TABLES

xv
























LIST OF TABLES

xvi




LIST OF FIGURES

xvii

List of Figures


reference substance



LIST OF FIGURES

xviii






xiii











ABSTRACT

i

ABSTRACT






formulations







5 min








138









ABSTRACT

ii






fenofibrate

























ABSTRACT

iii












in each study

TABLE OF CONTENTS

iv

Table of Contents

DESCRIPTION PAGE

Abstract i-iii









141 Chylomicrons 03













161 Statins 06



162 Fibrates 09


TABLE OF CONTENTS

v











182 Simvastatin 18

183 Lovastatin 19


185 Gemfibrozil 22

186 Fenofibrate 23

187 Ezetimibe 24





1922 Column 27


1924 Eluent 27

1925 Flow Rate 27

1926 Peak 27

1927 Resolution 27



19210 Tailing 28


TABLE OF CONTENTS

vi



111 Statistics 30

1111 Average 30























31 Solvents 63

32 Chemicals 63



TABLE OF CONTENTS

vii




353 Linearity 66


355 Accuracy 67

356 Precision 67

357 Selectivity 67

358 Robustness 68




3512 HPLC Set Up 69




363 Linearity 70


365 Accuracy 70

366 Precision 71

367 Selectivity 71

368 Robustness 71




3612 HPLC Set Up 73




373 Linearity 74


TABLE OF CONTENTS

viii

375 Accuracy 74

376 Precision 75

377 Selectivity 75

378 Robustness 75



3711 HPLC Set Up 76




383 Linearity 77


385 Accuracy 77

386 Precision 78

387 Selectivity 78

388 Robustness 78




3812 HPLC Set Up 80




393 Linearity 81


395 Accuracy 81

396 Precision 82

397 Selectivity 82

398 Robustness 82



TABLE OF CONTENTS

ix

3911 HPLC Set Up 84




3103 Linearity 85


3105 Accuracy 86

3106 Precision 86

3107 Selectivity 86

3108 Robustness 86








3114 Linearity 89


3116 Accuracy 90

3117 Precision 90

3118 Selectivity 90

3119 Robustness 91








4121 Linearity 93

TABLE OF CONTENTS

x


4123 Accuracy 94

4124 Precision 94

4125 Selectivity 95


4127 Robustness 95






4221 Linearity 104


4223 Accuracy 106

4224 Precision 106



4227 Robustness 109






4321 Linearity 113


4323 Accuracy 113

4324 Precision 114



4327 Robustness 120


TABLE OF CONTENTS

xi




4421 Linearity 122


4423 Accuracy 123

4424 Precision 123



4427 Robustness 124








4323 Accuracy 134

4524 Precision 134



4527 Robustness 139





4621Linearity 143


4623 Accuracy 144

4624 Precision 144

TABLE OF CONTENTS

xii



4627 Robustness 149





4721 Linearity 153


4723 Accuracy 156

4724 Precision 156



4727 Robustness 160



48 Conclusion 163



1

1 INTRODUCTION




























2


























3






follows

141 Chylomicrons
















4


















population





5
























6
















161 Statins

162 Fibrates











7































8






























9








162 Fibrates





















10































11











pregnancy [104 108-109]











117]






12












placebo [126]















13


hyperlipidemia


























14






























15






























16














Synthetic

B Drug Category


C Chemical name





17


N

O-

OHOH

O

CH3CH3

O

NH

F

2

Ca+2

3H2O


E Molecular Formula


F Molecular Weight

120942

G Colour


H Solubility





18

182 Simvastatin


Semi-synthetic

B Drug Category


C Chemical name




O

CH3

CH3

O

O

CH3

CH3

CH3

OOH

H


E Molecular Formula

C25H38O5


19

F Molecular Weight

41857

G Colour


H Solubility



183 Lovastatin


Semi-synthetic

B Drug Category


C Chemical name




20


O

C H 3

CH 3

O

O

C H 3

HCH 3

OH

H

O


E Molecular Formula

C24H36O5

F Molecular Weight

40454

G Colour


H Solubility





Synthetic


21

B Drug Category


C Chemical name




N

N O-

CH3CH3

NS

CH3

O

O

OOHOH

FCH3

Ca+2+2

2


E Molecular Formula

C22H28FN3O6S

F Molecular Weight

100114

G Colour



22

H Solubility



185 Gemfibrozil


Synthetic

B Drug Category


C Chemical name



O

CH3

CH3OH

OCH3

CH3


E Molecular Formula

C15H22O3


23

F Molecular Weight

25033

G Colour


H Solubility


186 Fenofibrate


Synthetic

B Drug Category


C Chemical name



24


O

Cl

O

CH3

CH3

O

O CH3

CH3


E Molecular Formula

C20H21ClO4

F Molecular Weight

36083

G Colour


H Solubility


in alcohol

187 Ezetimibe


Synthetic


25

B Drug Category


C Chemical name




N

O

OH

F

OH F


E Molecular Formula

C24H21F2NO3

F Molecular Weight

4094

G Colour



26

H Solubility


and acetone


















the compounds


27


1921 Chromatogram



1922 Column



mobile phase




1924 Eluent



1925 Flow Rate



1926 Peak



called a peak

1927 Resolution







28

1929 Retention Time


retention time

19210 Tailing








Linearity

Accuracy

Precision

Specificity

Limits of detection


Robustness










29
























is used mostly


the formula


30





111 Statistics






1111 Average [165]



X- = X1 + X2 + X3 + X4 + n












31













Y= mX + C










32


















11221 Granulation





33




too hard or soft




proper granules











34











forced degradation




35

2 LITERATURE REVIEW




each drug























36






to ICH guidelines





















37




























38




























39





























40





























41


respectively








just 19 min


















42




























43






to be 03 mLmin




















acid


44






was 147 minutes






















45




























46
























were below 15






47







for lovastatin

















to 10049





48

























lovastatin


49






















oxidation products






50















than 6 minutes













51



























52






















LOQ





53




























54



between 99 and 101

























55










LOQ












fenofibrate







56





respectively
























57




















microgmL










58





























59












studied
















60





























61












precision

















62












acceptance range


63

3 EXPERIMENTAL WORK







31 Solvents





32 Chemicals















64












Model CP324S

Min 00001g

Max 320 g


Model 5986-62


Model DA-60D


Model PS 02000A


34 Glass Apparatus







65








j) Glass Funnel


66















353 Linearity
















67


accuracy

355 Accuracy









356 Precision





357 Selectivity













68

358 Robustness
























RSD






69







each

3512 HPLC Set Up



3 Detector UV

4 Wavelength 242 nm

5 Injector Rheodyne








70












363 Linearity





triplicate






accuracy

365 Accuracy







71

366 Precision





367 Selectivity












368 Robustness













72
























73

3612 HPLC Set Up



3 Detector UV

4 Wavelength 240 nm

5 Injector Rheodyne








74











373 Linearity





made in triplicate







375 Accuracy








75

376 Precision





days

377 Selectivity














378 Robustness










76












condition




3711 HPLC Set Up



3 Detector UV

4 Wavelength 237 nm

5 Injector Rheodyne








77














383 Linearity











385 Accuracy





78



386 Precision





days

387 Selectivity












388 Robustness






were calculated




79




























80

3812 HPLC Set Up


2 HPLC Pump LC-20AT


4 Wavelength 240 nm

5 Injector Rheodyne








81















393 Linearity












395 Accuracy




82




396 Precision





days

397 Selectivity













for lovastatin

398 Robustness






83


were calculated


















under each stress





84

3911 HPLC Set Up


2 HPLC Pump LC-20AT


4 Wavelength 240 nm

5 Injector Rheodyne








85














respectively

3103 Linearity












accuracy


86

3105 Accuracy






3106 Precision





3107 Selectivity














3108 Robustness





87






















88

31011 HPLC Set Up


2 HPLC Pump LC-20AT


4 Wavelength 240 nm

5 Injector Rheodyne








89

























3114 Linearity






90








3116 Accuracy






3117 Precision





days

3118 Selectivity











91



3119 Robustness

























92

31112 HPLC Set Up


2 HPLC Pump LC-20AT


4 Wavelength 240 nm

5 Injector Rheodyne


ratio of 4060 (vv)






93
























4121 Linearity





94

















4123 Accuracy







4124 Precision






95



4125 Selectivity








given in Table 43





given in Table 44

4127 Robustness






96




97





200 2028 10140 029

240 2368 9867 042

280 2812 10043 124

Ezetimibe 160 1588 9925 057

200 1965 9825 086

240 2442 10175 168

280 2782 9936 011





320 5 3215 103 3248 151

480 5 4772 086 4861 125

Ezetimibe 160 5 1570 070 1633 135

320 5 3252 083 3158 089

480 5 4882 039 4802 110


98





Ezetimibe



32 3218 10056

32 3162 9881

32 3178 9931

32 3252 10162


RSD = 126

32 3251 10159

32 3186 9956

32 3158 9869

32 3224 10075


RSD = 128


99





160 1573 1582 1615 138

320 3148 3168 3150 035

480 4818 4798 4880 089

Ezetimibe

160 1632 1611 1630 074

320 3281 3242 3218 094

480 4772 4848 4820 114


100






Flow rate (04mLmin) 10184 383 3507 125

Flow rate (06 mLmin) 9858 255 3310 141

Buffer (pH 63) 10089 303 3401 120

Buffer (pH 67) 10154 302 3467 121

1RT Retention Time






Flow rate (04mLmin) 9802 558 5221 115

Flow rate (06 mLmin) 9915 372 5019 118

Buffer (pH 63) 10022 441 5186 110

Buffer (pH 67) 10005 443 5125 111

1RT Retention Time


101





















102





Ezetimibe



32 3262 10194

32 3215 10047

32 3168 9900


RSD = 146

32 3256 10175

32 3256 10056

32 3248 10203


RSD = 048


103









column life





















104


[Figure 43 and 44]






4221 Linearity












coefficient










105




106

4223 Accuracy







48]

4224 Precision






4225 Selectivity













107




Ezetimibe 50 3 200 2005 10025 138

100 3 400 3965 9912 068

150 3 600 6020 10033 086

Simvastatin 50 3 200 2030 10150 038

100 3 400 4025 10062 115

150 3 600 6060 10100 102




Ezetimibe 200 5 1986 110 1995 078

400 5 4012 105 3990 115

600 5 5996 028 6012 120

Simvastatin 200 5 2024 145 2010 056

400 5 4056 068 3975 132

600 5 5942 075 6025 088


108



Ezetimibe 10062 5 296 - 123 6781

Simvastatin 9943 5 980 1964 106 13752






Ezetimibe

200 2032 2009 1985 024

400 4076 3990 4040 043

600 5970 6025 6056 044

Simvastatin

200 2012 1995 2005 008

400 4035 4025 4020 008

600 6015 6025 5975 026


109

4227 Robustness
























formulations


110






Flow rate (14 mLmin) 9842 318 6810 125

Flow rate (16 mLmin) 9905 278 6566 127

Buffer (pH 48) 10022 299 6685 125

Buffer (pH 52) 10089 301 6628 124






Flow rate (14 mLmin) 10033 1052 13927 105

Flow rate (16 mLmin) 9915 919 13425 107

Buffer (pH 48) 10129 985 13564 108

Buffer (pH 52) 10086 984 13416 108


111




Ezetimibe 5 10 1012 10125 075

Simvastatin 5 10 1005 10050 115


112






























113





solutions

4321 Linearity










099997










4323 Accuracy





114





4324 Precision






416

4325 Selectivity








the proposed method







115



116




Gemfibrozil 50 5 1200 12022 10018 095

100 5 2400 23734 9889 043

150 5 3600 35421 9839 042

Simvastatin 50 5 20 202 10100 133

100 5 40 406 10150 119

150 5 60 593 9883 074




Gemfibrozil 1200 5 12125 078 11958 125

2400 5 24456 095 24258 102

3600 5 36521 124 36321 085

Simvastatin 20 5 202 144 201 106

40 5 396 111 395 058

60 5 607 036 602 131


117



118


Gemfibrozil



Simvastatin



240 23645 9852

240 24142 10059

240 24356 10148

240 23988 9995


RSD = 125

4 405 10125

4 396 9900

4 398 9950

4 393 9825


RSD = 128


119





Gemfibrozil

1200 11808 11788 11756 022

2400 24262 23943 23640 130

3600 35828 35641 35494 047

Simvastatin

20 201 199 197 101

40 398 395 391 089

60 602 595 591 093


120

4327 Robustness


















121






Flow rate (11mLmin) 9869 424 7118 128

Flow rate (09 mLmin) 10041 518 8002 122

Buffer (pH 52) 9889 465 7719 123

Buffer (pH 48) 10115 465 7662 123

1Retention Time

2Theoretical Plates






Flow rate (11mLmin) 9921 698 10220 129

Flow rate (09 mLmin) 9903 853 12515 122

Buffer (pH 52) 10069 765 11308 122

Buffer (pH 48) 10098 766 11015 122

1Retention Time

2Theoretical Plates


122




















solutions

4421 Linearity










123












4423 Accuracy







4424 Precision





Table 422

4425 Selectivity






124









4427 Robustness





















125




126




Ezetimibe 5 50 80 788 9850 095

5 100 160 1581 9881 031

5 150 240 2405 10021 033

Fenofibrate 5 50 1280 12924 10097 018

5 100 2560 25492 9958 051

5 150 3840 38850 10117 075




Ezetimibe 160 5 158 138 157 151

160 5 1608 095 1611 107

400 5 3995 055 3991 085

Fenofibrate 256 5 2550 096 2553 063

256 5 2548 033 2545 051

640 5 6373 022 6355 039


127


Ezetimibe



Fenofibrate



160 1611 10089

160 1593 9956

160 1588 9925

160 1590 9938


RSD = 076

2560 2538 9914

2560 2543 9934

2560 2581 10082

2560 2546 9945


RSD = 077


128





Ezetimibe

160 157 156 155 064

160 1618 1590 1576 134

400 3988 3942 3912 041

Fenofibrate

256 2484 2465 2456 058

2560 25512 25349 25215 059

6400 63841 63555 63373 037


129






Flow rate (14mLmin) 10098 261 6372 136

Flow rate (16 mLmin) 9962 234 4803 116

Buffer (pH 52) 10126 243 6005 122

Buffer (pH 48) 10085 244 6078 123






Flow rate (14mLmin) 10049 950 14337 121

Flow rate (16 mLmin) 9979 808 9991 107

Buffer (pH 52) 10021 876 12885 115

Buffer (pH 48) 9905 877 12687 116


130








131


Ezetimibe



Fenofibrate



16 1624 10150

16 1605 10031

16 1591 9944


RSD = 103

256 25894 10115

256 25536 9975

256 25748 10058


RSD = 070


132































133








shown)























134




4323 Accuracy








4524 Precision







4525 Selectivity








the proposed method


135



136




Ezetimibe 50 3 50 508 10160 102

100 3 100 988 9880 146

150 3 150 1541 10273 039

Lovastatin 50 3 100 1022 10220 063

100 3 200 1944 9720 119

150 3 300 2928 9760 093




Ezetimibe 080 5 082 122 081 163

250 5 2458 086 2443 138

1000 5 9869 074 9805 108

Lovastatin 16 5 156 111 155 151

500 5 5059 055 5046 149

2000 5 20241 032 19968 098


137



138


Ezetimibe



Lovastatin



100 1023 10230

100 992 9920

100 986 9860

100 1018 10180


RSD = 184

200 2054 10270

200 2036 10180

200 1978 9890

200 1986 9930


RSD = 185


139






4527 Robustness















140





Ezetimibe

080 081 078 079 193

250 2484 2466 2448 073

1000 10098 9922 9805 148

Lovastatin

160 163 161 159 124

500 4963 4921 4893 071

2000 20098 19852 19646 114


141






Flow rate (09 mLmin) 9946 453 5100 134

Flow rate (11 mLmin) 9905 370 4886 140

Buffer (pH 52) 10048 405 5454 139

Buffer (pH 48) 10215 404 5404 139






Flow rate (09 mLmin) 9932 1080 8004 126

Flow rate (11 mLmin) 10068 883 7575 136

Buffer (pH 52) 10046 967 7785 130

Buffer (pH 48) 9885 966 7715 130


142





























143






shown)





of solutions

4621 Linearity



















144




4623 Accuracy








4624 Precision






435

4625 Selectivity








the proposed method


145



146




Atorvastatin 50 3 40 406 10150 163

100 3 80 789 9863 126

150 3 120 1212 10100 069

Gemfibrozil 50 3 2400 23658 9858 101

100 3 4800 48863 10180 065

150 3 7200 73356 10188 053




Atorvastatin 05 5 052 198 051 223

80 5 795 086 786 155

200 5 1984 063 1982 141

Gemfibrozil 300 5 2963 101 2951 129

4800 5 48212 073 47871 122

12000 5 118648 088 118002 125


147



148


Atorvastatin



Gemfibrozil



80 808 10100

80 796 9950

80 805 10063

80 793 9912


RSD = 090

4800 47222 9838

4800 47805 9959

4800 48215 10045

4800 47329 9860


RSD = 097


149






4627 Robustness














products





414


150





Atorvastatin

05 052 051 052 112

80 794 797 790 044

200 2022 1995 1990 086

Gemfibrozil

300 3046 2983 2955 156

4800 48258 47626 47298 069

12000 119239 118658 118022 051


151






Flow rate (14 mLmin) 10169 380 3310 136

Flow rate (16 mLmin) 10043 334 2866 141

Buffer (pH 48) 9932 354 3164 141

Buffer (pH 52) 9978 355 3214 140






Flow rate (14 mLmin) 9911 1255 4002 131

Flow rate (16 mLmin) 10009 1098 3687 135

Buffer (pH 48) 9969 1169 3998 134

Buffer (pH 52) 9955 1169 4008 135


152



Ca2+

3H2O

N

O

NH

CH3

CH3

F

OHOH

O-

O CH3OH H2O2

Room TempO

O

NH

O

OH

OCH3

OHCH3


153


























4721 Linearity





154




















155




156

4723 Accuracy








4724 Precision






4725 Selectivity













157




Rosuvastatin 50 3 400 4069 10172 145

100 3 800 7888 9860 115

150 3 1200 11821 9851 096

Ezetimibe 50 3 100 981 9810 139

100 3 200 2048 10240 121

150 3 300 2928 9760 111




Rosuvastatin 50 5 505 144 503 189

800 5 7925 119 7805 169

1600 5 16228 095 16059 128

Ezetimibe 125 5 123 151 122 205

200 5 2051 076 2028 128

400 5 3965 105 3921 156


158


Rosuvastatin



Ezetimibe



800 8089 10111

800 7866 9832

800 8129 10161

800 8052 10065


RSD = 145

200 2048 10240

200 1963 9815

200 1983 9915

200 1972 9860


RSD = 193


159





Rosuvastatin

50 497 498 495 031

800 7942 7881 7885 043

1600 16152 15922 15905 086

Ezetimibe

125 123 121 122 082

200 1982 1975 1955 071

400 4008 3928 3911 131


160

4727 Robustness
























161



ACN 1 H3PO4 (6040) 10025 430 3126 135

ACN 1 H3PO4 (5842) 10011 476 3316 133

ACN 1 H3PO4 (6238) 9865 408 3040 141

Flow rate (11mLmin) 9985 391 2866 140

Flow rate (09 mLmin) 10141 478 3264 135

H3PO4 Conc (09 ) 9955 428 3167 139

H3PO4 Conc (11 ) 9941 427 3114 133



ACN 1 H3PO4 (6040) 10069 633 4139 142

ACN 1 H3PO4 (5842) 9965 715 4267 141

ACN 1 H3PO4 (6238) 10025 595 3964 148

Flow rate (11mLmin) 10095 575 4040 145

Flow rate (09 mLmin) 9926 703 4220 142

H3PO4 Conc (09 ) 10068 635 3998 144

H3PO4 Conc (11 ) 10029 636 4002 139


162




Rosuvastatin 5 40 4052 10130 103

Ezetimibe 5 10 1021 10210 131


163

48 Conclusion









formulations




















164










human plasma














formulations


165

5 REFERENCES






Page 971



2008 14(2) 314-324


141108


Accessed on 141108


141108



141108


151108




New York 2003






166


2002 106 3240



278 313ndash321




















2619ndash2628



77 31ndash36



167



1146


2000 9 2653ndash2661


239ndash252




44 379ndash387




32B









1ndash34






168





27 107ndash114




638





25 1191ndash1199









1995 58 412ndash417






597ndash602


169


420









69B



170-89


Lipid Res 1997 38 2071-2078



135 249- 256





Anal 2006 41(2) 408-414







170


317ndash 326






951ndash956





29636ndash29639



Sci USA 1984 81 4203ndash 4207




79 95ndash99




Biol Chem 1999 274 32048ndash32054



275 36703ndash 36707




171




S45-S48



Pharmacol 2000 56 631-5



47(2) 73-104








2002 71 80


Disease 2004 3 22











172



283 157-163




Pharmacol 2001 134 409-417


79-84







729-741









Heart J 2005 149 464ndash473




173



2003 107 2409ndash 2415







1758 ndash1773



2125ndash2134






Proc 2004 79 620ndash 629



418ndash424







728


174



455









334









1997 80 608ndash613






47 1584 ndash1587


5th Edition 1994 23-25 51-53


175




01082009


on 01082009






03082009







992ndash1009

171 Khedr A J AOAC Int 2007 90(6) 1547-53


Sci 2007 30(18) 3143-53


766


(11-12) 737-741



44(2) 379-87


176


241-6



Life Sci 2007 846(1-2) 215-21











382(5) 1242-9


Anal 2003 33(5) 1017-23


Spectrom 1999 13(11) 1003-15


1999 10(1) 55-66







2008 81(3) 453-459


177


Chemistry 2007 62(6 ) 536-541







Chemistry 2006 61 (1 ) 63-66




2004 49 (4) 289-289


Sep Sci 2004 27(13) 1087-92


2002 29 (4) 715-721


18(3) 232-4





1992 10(9) 693-7


2008 22(5) 511-8


Anal 2008 46(4) 808-813


178


2008 20 399ndash410



2719ndash2725


Chem Soc 2005 50(4) 639-646




Biomed Life Sci 2002 768(2) 349-59


41


1999 20(1-2) 137-43


Biomed Anal 1988 6(3) 271-6






Biomed Life Sci 2007 856(1-2) 35-40


Biomed Anal 2007 44(2) 540-6






179


International 2005 88 (4) 1142-1147


Anal 2004 35(3) 609-14


Pharm Sci 2007 69 819-21





B 2003 791 161ndash170



Anal 2001 26(1) 7-14








197-202


60(5) 425-38



J Chromatogr A 2004 1046(1-2) 133-40



180


2000 742(2) 391-400




118(10) 447-55




2) 137-142


67(1-2) 101-107


SR Ind J Pharm Sci 2008 70(2) 258-260




853(1-2) 88-96



Biomed Anal 2006 41(3) 1037-40




B 2006 830(1)143-150




181



IFPMA Geneva 1994



1996








1995 76(2) 126A-128A











Title_pages_PhDpdf


Muhammad Ashfaq




Supervisor


To

Abbreviationspdf


List_of_Tablespdf

List of Tables

TAB DESCRIPTIONPAGE

List_of_Figurespdf

FIG DESCRIPTIONPAGE




Table of Contents

DESCRIPTION PAGE









3512 HPLC Set Up69


3612 HPLC Set Up73

3711 HPLC Set Up76


3812 HPLC Set Up80

3911 HPLC Set Up84

31011 HPLC Set Up88

31112 HPLC Set Up92


48S-1pdf









48S-2pdf



48S-3pdf

3 EXPERIMENTAL WORK




3512 HPLC Set Up



3612 HPLC Set Up

3711 HPLC Set Up


3812 HPLC Set Up

3911 HPLC Set Up

31011 HPLC Set Up


31112 HPLC Set Up

48S-4pdf




























ACN 1 H3PO4 (6040) 10025 430 3126 135


ACN 1 H3PO4 (6040) 10069 633 4139 142








Dedicated

To



ACKNOWLEDGEMENTS








work


























forgetfulness

Muhammad Ashfaq

Abbreviations

Abbreviations




LIST OF TABLES

xiv

List of Tables





















LIST OF TABLES

xv
























LIST OF TABLES

xvi




LIST OF FIGURES

xvii

List of Figures


reference substance



LIST OF FIGURES

xviii






xiii











ABSTRACT

i

ABSTRACT






formulations







5 min








138









ABSTRACT

ii






fenofibrate

























ABSTRACT

iii












in each study

TABLE OF CONTENTS

iv

Table of Contents

DESCRIPTION PAGE

Abstract i-iii









141 Chylomicrons 03













161 Statins 06



162 Fibrates 09


TABLE OF CONTENTS

v











182 Simvastatin 18

183 Lovastatin 19


185 Gemfibrozil 22

186 Fenofibrate 23

187 Ezetimibe 24





1922 Column 27


1924 Eluent 27

1925 Flow Rate 27

1926 Peak 27

1927 Resolution 27



19210 Tailing 28


TABLE OF CONTENTS

vi



111 Statistics 30

1111 Average 30























31 Solvents 63

32 Chemicals 63



TABLE OF CONTENTS

vii




353 Linearity 66


355 Accuracy 67

356 Precision 67

357 Selectivity 67

358 Robustness 68




3512 HPLC Set Up 69




363 Linearity 70


365 Accuracy 70

366 Precision 71

367 Selectivity 71

368 Robustness 71




3612 HPLC Set Up 73




373 Linearity 74


TABLE OF CONTENTS

viii

375 Accuracy 74

376 Precision 75

377 Selectivity 75

378 Robustness 75



3711 HPLC Set Up 76




383 Linearity 77


385 Accuracy 77

386 Precision 78

387 Selectivity 78

388 Robustness 78




3812 HPLC Set Up 80




393 Linearity 81


395 Accuracy 81

396 Precision 82

397 Selectivity 82

398 Robustness 82



TABLE OF CONTENTS

ix

3911 HPLC Set Up 84




3103 Linearity 85


3105 Accuracy 86

3106 Precision 86

3107 Selectivity 86

3108 Robustness 86








3114 Linearity 89


3116 Accuracy 90

3117 Precision 90

3118 Selectivity 90

3119 Robustness 91








4121 Linearity 93

TABLE OF CONTENTS

x


4123 Accuracy 94

4124 Precision 94

4125 Selectivity 95


4127 Robustness 95






4221 Linearity 104


4223 Accuracy 106

4224 Precision 106



4227 Robustness 109






4321 Linearity 113


4323 Accuracy 113

4324 Precision 114



4327 Robustness 120


TABLE OF CONTENTS

xi




4421 Linearity 122


4423 Accuracy 123

4424 Precision 123



4427 Robustness 124








4323 Accuracy 134

4524 Precision 134



4527 Robustness 139





4621Linearity 143


4623 Accuracy 144

4624 Precision 144

TABLE OF CONTENTS

xii



4627 Robustness 149





4721 Linearity 153


4723 Accuracy 156

4724 Precision 156



4727 Robustness 160



48 Conclusion 163



1

1 INTRODUCTION




























2


























3






follows

141 Chylomicrons
















4


















population





5
























6
















161 Statins

162 Fibrates











7































8






























9








162 Fibrates





















10































11











pregnancy [104 108-109]











117]






12












placebo [126]















13


hyperlipidemia


























14






























15






























16














Synthetic

B Drug Category


C Chemical name





17


N

O-

OHOH

O

CH3CH3

O

NH

F

2

Ca+2

3H2O


E Molecular Formula


F Molecular Weight

120942

G Colour


H Solubility





18

182 Simvastatin


Semi-synthetic

B Drug Category


C Chemical name




O

CH3

CH3

O

O

CH3

CH3

CH3

OOH

H


E Molecular Formula

C25H38O5


19

F Molecular Weight

41857

G Colour


H Solubility



183 Lovastatin


Semi-synthetic

B Drug Category


C Chemical name




20


O

C H 3

CH 3

O

O

C H 3

HCH 3

OH

H

O


E Molecular Formula

C24H36O5

F Molecular Weight

40454

G Colour


H Solubility





Synthetic


21

B Drug Category


C Chemical name




N

N O-

CH3CH3

NS

CH3

O

O

OOHOH

FCH3

Ca+2+2

2


E Molecular Formula

C22H28FN3O6S

F Molecular Weight

100114

G Colour



22

H Solubility



185 Gemfibrozil


Synthetic

B Drug Category


C Chemical name



O

CH3

CH3OH

OCH3

CH3


E Molecular Formula

C15H22O3


23

F Molecular Weight

25033

G Colour


H Solubility


186 Fenofibrate


Synthetic

B Drug Category


C Chemical name



24


O

Cl

O

CH3

CH3

O

O CH3

CH3


E Molecular Formula

C20H21ClO4

F Molecular Weight

36083

G Colour


H Solubility


in alcohol

187 Ezetimibe


Synthetic


25

B Drug Category


C Chemical name




N

O

OH

F

OH F


E Molecular Formula

C24H21F2NO3

F Molecular Weight

4094

G Colour



26

H Solubility


and acetone


















the compounds


27


1921 Chromatogram



1922 Column



mobile phase




1924 Eluent



1925 Flow Rate



1926 Peak



called a peak

1927 Resolution







28

1929 Retention Time


retention time

19210 Tailing








Linearity

Accuracy

Precision

Specificity

Limits of detection


Robustness










29
























is used mostly


the formula


30





111 Statistics






1111 Average [165]



X- = X1 + X2 + X3 + X4 + n












31













Y= mX + C










32


















11221 Granulation





33




too hard or soft




proper granules











34











forced degradation




35

2 LITERATURE REVIEW




each drug























36






to ICH guidelines





















37




























38




























39





























40





























41


respectively








just 19 min


















42




























43






to be 03 mLmin




















acid


44






was 147 minutes






















45




























46
























were below 15






47







for lovastatin

















to 10049





48

























lovastatin


49






















oxidation products






50















than 6 minutes













51



























52






















LOQ





53




























54



between 99 and 101

























55










LOQ












fenofibrate







56





respectively
























57




















microgmL










58





























59












studied
















60





























61












precision

















62












acceptance range


63

3 EXPERIMENTAL WORK







31 Solvents





32 Chemicals















64












Model CP324S

Min 00001g

Max 320 g


Model 5986-62


Model DA-60D


Model PS 02000A


34 Glass Apparatus







65








j) Glass Funnel


66















353 Linearity
















67


accuracy

355 Accuracy









356 Precision





357 Selectivity













68

358 Robustness
























RSD






69







each

3512 HPLC Set Up



3 Detector UV

4 Wavelength 242 nm

5 Injector Rheodyne








70












363 Linearity





triplicate






accuracy

365 Accuracy







71

366 Precision





367 Selectivity












368 Robustness













72
























73

3612 HPLC Set Up



3 Detector UV

4 Wavelength 240 nm

5 Injector Rheodyne








74











373 Linearity





made in triplicate







375 Accuracy








75

376 Precision





days

377 Selectivity














378 Robustness










76












condition




3711 HPLC Set Up



3 Detector UV

4 Wavelength 237 nm

5 Injector Rheodyne








77














383 Linearity











385 Accuracy





78



386 Precision





days

387 Selectivity












388 Robustness






were calculated




79




























80

3812 HPLC Set Up


2 HPLC Pump LC-20AT


4 Wavelength 240 nm

5 Injector Rheodyne








81















393 Linearity












395 Accuracy




82




396 Precision





days

397 Selectivity













for lovastatin

398 Robustness






83


were calculated


















under each stress





84

3911 HPLC Set Up


2 HPLC Pump LC-20AT


4 Wavelength 240 nm

5 Injector Rheodyne








85














respectively

3103 Linearity












accuracy


86

3105 Accuracy






3106 Precision





3107 Selectivity














3108 Robustness





87






















88

31011 HPLC Set Up


2 HPLC Pump LC-20AT


4 Wavelength 240 nm

5 Injector Rheodyne








89

























3114 Linearity






90








3116 Accuracy






3117 Precision





days

3118 Selectivity











91



3119 Robustness

























92

31112 HPLC Set Up


2 HPLC Pump LC-20AT


4 Wavelength 240 nm

5 Injector Rheodyne


ratio of 4060 (vv)






93
























4121 Linearity





94

















4123 Accuracy







4124 Precision






95



4125 Selectivity








given in Table 43





given in Table 44

4127 Robustness






96




97





200 2028 10140 029

240 2368 9867 042

280 2812 10043 124

Ezetimibe 160 1588 9925 057

200 1965 9825 086

240 2442 10175 168

280 2782 9936 011





320 5 3215 103 3248 151

480 5 4772 086 4861 125

Ezetimibe 160 5 1570 070 1633 135

320 5 3252 083 3158 089

480 5 4882 039 4802 110


98





Ezetimibe



32 3218 10056

32 3162 9881

32 3178 9931

32 3252 10162


RSD = 126

32 3251 10159

32 3186 9956

32 3158 9869

32 3224 10075


RSD = 128


99





160 1573 1582 1615 138

320 3148 3168 3150 035

480 4818 4798 4880 089

Ezetimibe

160 1632 1611 1630 074

320 3281 3242 3218 094

480 4772 4848 4820 114


100






Flow rate (04mLmin) 10184 383 3507 125

Flow rate (06 mLmin) 9858 255 3310 141

Buffer (pH 63) 10089 303 3401 120

Buffer (pH 67) 10154 302 3467 121

1RT Retention Time






Flow rate (04mLmin) 9802 558 5221 115

Flow rate (06 mLmin) 9915 372 5019 118

Buffer (pH 63) 10022 441 5186 110

Buffer (pH 67) 10005 443 5125 111

1RT Retention Time


101





















102





Ezetimibe



32 3262 10194

32 3215 10047

32 3168 9900


RSD = 146

32 3256 10175

32 3256 10056

32 3248 10203


RSD = 048


103









column life





















104


[Figure 43 and 44]






4221 Linearity












coefficient










105




106

4223 Accuracy







48]

4224 Precision






4225 Selectivity













107




Ezetimibe 50 3 200 2005 10025 138

100 3 400 3965 9912 068

150 3 600 6020 10033 086

Simvastatin 50 3 200 2030 10150 038

100 3 400 4025 10062 115

150 3 600 6060 10100 102




Ezetimibe 200 5 1986 110 1995 078

400 5 4012 105 3990 115

600 5 5996 028 6012 120

Simvastatin 200 5 2024 145 2010 056

400 5 4056 068 3975 132

600 5 5942 075 6025 088


108



Ezetimibe 10062 5 296 - 123 6781

Simvastatin 9943 5 980 1964 106 13752






Ezetimibe

200 2032 2009 1985 024

400 4076 3990 4040 043

600 5970 6025 6056 044

Simvastatin

200 2012 1995 2005 008

400 4035 4025 4020 008

600 6015 6025 5975 026


109

4227 Robustness
























formulations


110






Flow rate (14 mLmin) 9842 318 6810 125

Flow rate (16 mLmin) 9905 278 6566 127

Buffer (pH 48) 10022 299 6685 125

Buffer (pH 52) 10089 301 6628 124






Flow rate (14 mLmin) 10033 1052 13927 105

Flow rate (16 mLmin) 9915 919 13425 107

Buffer (pH 48) 10129 985 13564 108

Buffer (pH 52) 10086 984 13416 108


111




Ezetimibe 5 10 1012 10125 075

Simvastatin 5 10 1005 10050 115


112






























113





solutions

4321 Linearity










099997










4323 Accuracy





114





4324 Precision






416

4325 Selectivity








the proposed method







115



116




Gemfibrozil 50 5 1200 12022 10018 095

100 5 2400 23734 9889 043

150 5 3600 35421 9839 042

Simvastatin 50 5 20 202 10100 133

100 5 40 406 10150 119

150 5 60 593 9883 074




Gemfibrozil 1200 5 12125 078 11958 125

2400 5 24456 095 24258 102

3600 5 36521 124 36321 085

Simvastatin 20 5 202 144 201 106

40 5 396 111 395 058

60 5 607 036 602 131


117



118


Gemfibrozil



Simvastatin



240 23645 9852

240 24142 10059

240 24356 10148

240 23988 9995


RSD = 125

4 405 10125

4 396 9900

4 398 9950

4 393 9825


RSD = 128


119





Gemfibrozil

1200 11808 11788 11756 022

2400 24262 23943 23640 130

3600 35828 35641 35494 047

Simvastatin

20 201 199 197 101

40 398 395 391 089

60 602 595 591 093


120

4327 Robustness


















121






Flow rate (11mLmin) 9869 424 7118 128

Flow rate (09 mLmin) 10041 518 8002 122

Buffer (pH 52) 9889 465 7719 123

Buffer (pH 48) 10115 465 7662 123

1Retention Time

2Theoretical Plates






Flow rate (11mLmin) 9921 698 10220 129

Flow rate (09 mLmin) 9903 853 12515 122

Buffer (pH 52) 10069 765 11308 122

Buffer (pH 48) 10098 766 11015 122

1Retention Time

2Theoretical Plates


122




















solutions

4421 Linearity










123












4423 Accuracy







4424 Precision





Table 422

4425 Selectivity






124









4427 Robustness





















125




126




Ezetimibe 5 50 80 788 9850 095

5 100 160 1581 9881 031

5 150 240 2405 10021 033

Fenofibrate 5 50 1280 12924 10097 018

5 100 2560 25492 9958 051

5 150 3840 38850 10117 075




Ezetimibe 160 5 158 138 157 151

160 5 1608 095 1611 107

400 5 3995 055 3991 085

Fenofibrate 256 5 2550 096 2553 063

256 5 2548 033 2545 051

640 5 6373 022 6355 039


127


Ezetimibe



Fenofibrate



160 1611 10089

160 1593 9956

160 1588 9925

160 1590 9938


RSD = 076

2560 2538 9914

2560 2543 9934

2560 2581 10082

2560 2546 9945


RSD = 077


128





Ezetimibe

160 157 156 155 064

160 1618 1590 1576 134

400 3988 3942 3912 041

Fenofibrate

256 2484 2465 2456 058

2560 25512 25349 25215 059

6400 63841 63555 63373 037


129






Flow rate (14mLmin) 10098 261 6372 136

Flow rate (16 mLmin) 9962 234 4803 116

Buffer (pH 52) 10126 243 6005 122

Buffer (pH 48) 10085 244 6078 123






Flow rate (14mLmin) 10049 950 14337 121

Flow rate (16 mLmin) 9979 808 9991 107

Buffer (pH 52) 10021 876 12885 115

Buffer (pH 48) 9905 877 12687 116


130








131


Ezetimibe



Fenofibrate



16 1624 10150

16 1605 10031

16 1591 9944


RSD = 103

256 25894 10115

256 25536 9975

256 25748 10058


RSD = 070


132































133








shown)























134




4323 Accuracy








4524 Precision







4525 Selectivity








the proposed method


135



136




Ezetimibe 50 3 50 508 10160 102

100 3 100 988 9880 146

150 3 150 1541 10273 039

Lovastatin 50 3 100 1022 10220 063

100 3 200 1944 9720 119

150 3 300 2928 9760 093




Ezetimibe 080 5 082 122 081 163

250 5 2458 086 2443 138

1000 5 9869 074 9805 108

Lovastatin 16 5 156 111 155 151

500 5 5059 055 5046 149

2000 5 20241 032 19968 098


137



138


Ezetimibe



Lovastatin



100 1023 10230

100 992 9920

100 986 9860

100 1018 10180


RSD = 184

200 2054 10270

200 2036 10180

200 1978 9890

200 1986 9930


RSD = 185


139






4527 Robustness















140





Ezetimibe

080 081 078 079 193

250 2484 2466 2448 073

1000 10098 9922 9805 148

Lovastatin

160 163 161 159 124

500 4963 4921 4893 071

2000 20098 19852 19646 114


141






Flow rate (09 mLmin) 9946 453 5100 134

Flow rate (11 mLmin) 9905 370 4886 140

Buffer (pH 52) 10048 405 5454 139

Buffer (pH 48) 10215 404 5404 139






Flow rate (09 mLmin) 9932 1080 8004 126

Flow rate (11 mLmin) 10068 883 7575 136

Buffer (pH 52) 10046 967 7785 130

Buffer (pH 48) 9885 966 7715 130


142





























143






shown)





of solutions

4621 Linearity



















144




4623 Accuracy








4624 Precision






435

4625 Selectivity








the proposed method


145



146




Atorvastatin 50 3 40 406 10150 163

100 3 80 789 9863 126

150 3 120 1212 10100 069

Gemfibrozil 50 3 2400 23658 9858 101

100 3 4800 48863 10180 065

150 3 7200 73356 10188 053




Atorvastatin 05 5 052 198 051 223

80 5 795 086 786 155

200 5 1984 063 1982 141

Gemfibrozil 300 5 2963 101 2951 129

4800 5 48212 073 47871 122

12000 5 118648 088 118002 125


147



148


Atorvastatin



Gemfibrozil



80 808 10100

80 796 9950

80 805 10063

80 793 9912


RSD = 090

4800 47222 9838

4800 47805 9959

4800 48215 10045

4800 47329 9860


RSD = 097


149






4627 Robustness














products





414


150





Atorvastatin

05 052 051 052 112

80 794 797 790 044

200 2022 1995 1990 086

Gemfibrozil

300 3046 2983 2955 156

4800 48258 47626 47298 069

12000 119239 118658 118022 051


151






Flow rate (14 mLmin) 10169 380 3310 136

Flow rate (16 mLmin) 10043 334 2866 141

Buffer (pH 48) 9932 354 3164 141

Buffer (pH 52) 9978 355 3214 140






Flow rate (14 mLmin) 9911 1255 4002 131

Flow rate (16 mLmin) 10009 1098 3687 135

Buffer (pH 48) 9969 1169 3998 134

Buffer (pH 52) 9955 1169 4008 135


152



Ca2+

3H2O

N

O

NH

CH3

CH3

F

OHOH

O-

O CH3OH H2O2

Room TempO

O

NH

O

OH

OCH3

OHCH3


153


























4721 Linearity





154




















155




156

4723 Accuracy








4724 Precision






4725 Selectivity













157




Rosuvastatin 50 3 400 4069 10172 145

100 3 800 7888 9860 115

150 3 1200 11821 9851 096

Ezetimibe 50 3 100 981 9810 139

100 3 200 2048 10240 121

150 3 300 2928 9760 111




Rosuvastatin 50 5 505 144 503 189

800 5 7925 119 7805 169

1600 5 16228 095 16059 128

Ezetimibe 125 5 123 151 122 205

200 5 2051 076 2028 128

400 5 3965 105 3921 156


158


Rosuvastatin



Ezetimibe



800 8089 10111

800 7866 9832

800 8129 10161

800 8052 10065


RSD = 145

200 2048 10240

200 1963 9815

200 1983 9915

200 1972 9860


RSD = 193


159





Rosuvastatin

50 497 498 495 031

800 7942 7881 7885 043

1600 16152 15922 15905 086

Ezetimibe

125 123 121 122 082

200 1982 1975 1955 071

400 4008 3928 3911 131


160

4727 Robustness
























161



ACN 1 H3PO4 (6040) 10025 430 3126 135

ACN 1 H3PO4 (5842) 10011 476 3316 133

ACN 1 H3PO4 (6238) 9865 408 3040 141

Flow rate (11mLmin) 9985 391 2866 140

Flow rate (09 mLmin) 10141 478 3264 135

H3PO4 Conc (09 ) 9955 428 3167 139

H3PO4 Conc (11 ) 9941 427 3114 133



ACN 1 H3PO4 (6040) 10069 633 4139 142

ACN 1 H3PO4 (5842) 9965 715 4267 141

ACN 1 H3PO4 (6238) 10025 595 3964 148

Flow rate (11mLmin) 10095 575 4040 145

Flow rate (09 mLmin) 9926 703 4220 142

H3PO4 Conc (09 ) 10068 635 3998 144

H3PO4 Conc (11 ) 10029 636 4002 139


162




Rosuvastatin 5 40 4052 10130 103

Ezetimibe 5 10 1021 10210 131


163

48 Conclusion









formulations




















164










human plasma














formulations


165

5 REFERENCES






Page 971



2008 14(2) 314-324


141108


Accessed on 141108


141108



141108


151108




New York 2003






166


2002 106 3240



278 313ndash321




















2619ndash2628



77 31ndash36



167



1146


2000 9 2653ndash2661


239ndash252




44 379ndash387




32B









1ndash34






168





27 107ndash114




638





25 1191ndash1199









1995 58 412ndash417






597ndash602


169


420









69B



170-89


Lipid Res 1997 38 2071-2078



135 249- 256





Anal 2006 41(2) 408-414







170


317ndash 326






951ndash956





29636ndash29639



Sci USA 1984 81 4203ndash 4207




79 95ndash99




Biol Chem 1999 274 32048ndash32054



275 36703ndash 36707




171




S45-S48



Pharmacol 2000 56 631-5



47(2) 73-104








2002 71 80


Disease 2004 3 22











172



283 157-163




Pharmacol 2001 134 409-417


79-84







729-741









Heart J 2005 149 464ndash473




173



2003 107 2409ndash 2415







1758 ndash1773



2125ndash2134






Proc 2004 79 620ndash 629



418ndash424







728


174



455









334









1997 80 608ndash613






47 1584 ndash1587


5th Edition 1994 23-25 51-53


175




01082009


on 01082009






03082009







992ndash1009

171 Khedr A J AOAC Int 2007 90(6) 1547-53


Sci 2007 30(18) 3143-53


766


(11-12) 737-741



44(2) 379-87


176


241-6



Life Sci 2007 846(1-2) 215-21











382(5) 1242-9


Anal 2003 33(5) 1017-23


Spectrom 1999 13(11) 1003-15


1999 10(1) 55-66







2008 81(3) 453-459


177


Chemistry 2007 62(6 ) 536-541







Chemistry 2006 61 (1 ) 63-66




2004 49 (4) 289-289


Sep Sci 2004 27(13) 1087-92


2002 29 (4) 715-721


18(3) 232-4





1992 10(9) 693-7


2008 22(5) 511-8


Anal 2008 46(4) 808-813


178


2008 20 399ndash410



2719ndash2725


Chem Soc 2005 50(4) 639-646




Biomed Life Sci 2002 768(2) 349-59


41


1999 20(1-2) 137-43


Biomed Anal 1988 6(3) 271-6






Biomed Life Sci 2007 856(1-2) 35-40


Biomed Anal 2007 44(2) 540-6






179


International 2005 88 (4) 1142-1147


Anal 2004 35(3) 609-14


Pharm Sci 2007 69 819-21





B 2003 791 161ndash170



Anal 2001 26(1) 7-14








197-202


60(5) 425-38



J Chromatogr A 2004 1046(1-2) 133-40



180


2000 742(2) 391-400




118(10) 447-55




2) 137-142


67(1-2) 101-107


SR Ind J Pharm Sci 2008 70(2) 258-260




853(1-2) 88-96



Biomed Anal 2006 41(3) 1037-40




B 2006 830(1)143-150




181



IFPMA Geneva 1994



1996








1995 76(2) 126A-128A











Title_pages_PhDpdf


Muhammad Ashfaq




Supervisor


To

Abbreviationspdf


List_of_Tablespdf

List of Tables

TAB DESCRIPTIONPAGE

List_of_Figurespdf

FIG DESCRIPTIONPAGE




Table of Contents

DESCRIPTION PAGE









3512 HPLC Set Up69


3612 HPLC Set Up73

3711 HPLC Set Up76


3812 HPLC Set Up80

3911 HPLC Set Up84

31011 HPLC Set Up88

31112 HPLC Set Up92


48S-1pdf









48S-2pdf



48S-3pdf

3 EXPERIMENTAL WORK




3512 HPLC Set Up



3612 HPLC Set Up

3711 HPLC Set Up


3812 HPLC Set Up

3911 HPLC Set Up

31011 HPLC Set Up


31112 HPLC Set Up

48S-4pdf




























ACN 1 H3PO4 (6040) 10025 430 3126 135


ACN 1 H3PO4 (6040) 10069 633 4139 142

ACKNOWLEDGEMENTS








work


























forgetfulness

Muhammad Ashfaq

Abbreviations

Abbreviations




LIST OF TABLES

xiv

List of Tables





















LIST OF TABLES

xv
























LIST OF TABLES

xvi




LIST OF FIGURES

xvii

List of Figures


reference substance



LIST OF FIGURES

xviii






xiii











ABSTRACT

i

ABSTRACT






formulations







5 min








138









ABSTRACT

ii






fenofibrate

























ABSTRACT

iii












in each study

TABLE OF CONTENTS

iv

Table of Contents

DESCRIPTION PAGE

Abstract i-iii









141 Chylomicrons 03













161 Statins 06



162 Fibrates 09


TABLE OF CONTENTS

v











182 Simvastatin 18

183 Lovastatin 19


185 Gemfibrozil 22

186 Fenofibrate 23

187 Ezetimibe 24





1922 Column 27


1924 Eluent 27

1925 Flow Rate 27

1926 Peak 27

1927 Resolution 27



19210 Tailing 28


TABLE OF CONTENTS

vi



111 Statistics 30

1111 Average 30























31 Solvents 63

32 Chemicals 63



TABLE OF CONTENTS

vii




353 Linearity 66


355 Accuracy 67

356 Precision 67

357 Selectivity 67

358 Robustness 68




3512 HPLC Set Up 69




363 Linearity 70


365 Accuracy 70

366 Precision 71

367 Selectivity 71

368 Robustness 71




3612 HPLC Set Up 73




373 Linearity 74


TABLE OF CONTENTS

viii

375 Accuracy 74

376 Precision 75

377 Selectivity 75

378 Robustness 75



3711 HPLC Set Up 76




383 Linearity 77


385 Accuracy 77

386 Precision 78

387 Selectivity 78

388 Robustness 78




3812 HPLC Set Up 80




393 Linearity 81


395 Accuracy 81

396 Precision 82

397 Selectivity 82

398 Robustness 82



TABLE OF CONTENTS

ix

3911 HPLC Set Up 84




3103 Linearity 85


3105 Accuracy 86

3106 Precision 86

3107 Selectivity 86

3108 Robustness 86








3114 Linearity 89


3116 Accuracy 90

3117 Precision 90

3118 Selectivity 90

3119 Robustness 91








4121 Linearity 93

TABLE OF CONTENTS

x


4123 Accuracy 94

4124 Precision 94

4125 Selectivity 95


4127 Robustness 95






4221 Linearity 104


4223 Accuracy 106

4224 Precision 106



4227 Robustness 109






4321 Linearity 113


4323 Accuracy 113

4324 Precision 114



4327 Robustness 120


TABLE OF CONTENTS

xi




4421 Linearity 122


4423 Accuracy 123

4424 Precision 123



4427 Robustness 124








4323 Accuracy 134

4524 Precision 134



4527 Robustness 139





4621Linearity 143


4623 Accuracy 144

4624 Precision 144

TABLE OF CONTENTS

xii



4627 Robustness 149





4721 Linearity 153


4723 Accuracy 156

4724 Precision 156



4727 Robustness 160



48 Conclusion 163



1

1 INTRODUCTION




























2


























3






follows

141 Chylomicrons
















4


















population





5
























6
















161 Statins

162 Fibrates











7































8






























9








162 Fibrates





















10































11











pregnancy [104 108-109]











117]






12












placebo [126]















13


hyperlipidemia


























14






























15






























16














Synthetic

B Drug Category


C Chemical name





17


N

O-

OHOH

O

CH3CH3

O

NH

F

2

Ca+2

3H2O


E Molecular Formula


F Molecular Weight

120942

G Colour


H Solubility





18

182 Simvastatin


Semi-synthetic

B Drug Category


C Chemical name




O

CH3

CH3

O

O

CH3

CH3

CH3

OOH

H


E Molecular Formula

C25H38O5


19

F Molecular Weight

41857

G Colour


H Solubility



183 Lovastatin


Semi-synthetic

B Drug Category


C Chemical name




20


O

C H 3

CH 3

O

O

C H 3

HCH 3

OH

H

O


E Molecular Formula

C24H36O5

F Molecular Weight

40454

G Colour


H Solubility





Synthetic


21

B Drug Category


C Chemical name




N

N O-

CH3CH3

NS

CH3

O

O

OOHOH

FCH3

Ca+2+2

2


E Molecular Formula

C22H28FN3O6S

F Molecular Weight

100114

G Colour



22

H Solubility



185 Gemfibrozil


Synthetic

B Drug Category


C Chemical name



O

CH3

CH3OH

OCH3

CH3


E Molecular Formula

C15H22O3


23

F Molecular Weight

25033

G Colour


H Solubility


186 Fenofibrate


Synthetic

B Drug Category


C Chemical name



24


O

Cl

O

CH3

CH3

O

O CH3

CH3


E Molecular Formula

C20H21ClO4

F Molecular Weight

36083

G Colour


H Solubility


in alcohol

187 Ezetimibe


Synthetic


25

B Drug Category


C Chemical name




N

O

OH

F

OH F


E Molecular Formula

C24H21F2NO3

F Molecular Weight

4094

G Colour



26

H Solubility


and acetone


















the compounds


27


1921 Chromatogram



1922 Column



mobile phase




1924 Eluent



1925 Flow Rate



1926 Peak



called a peak

1927 Resolution







28

1929 Retention Time


retention time

19210 Tailing








Linearity

Accuracy

Precision

Specificity

Limits of detection


Robustness










29
























is used mostly


the formula


30





111 Statistics






1111 Average [165]



X- = X1 + X2 + X3 + X4 + n












31













Y= mX + C










32


















11221 Granulation





33




too hard or soft




proper granules











34











forced degradation




35

2 LITERATURE REVIEW




each drug























36






to ICH guidelines





















37




























38




























39





























40





























41


respectively








just 19 min


















42




























43






to be 03 mLmin




















acid


44






was 147 minutes






















45




























46
























were below 15






47







for lovastatin

















to 10049





48

























lovastatin


49






















oxidation products






50















than 6 minutes













51



























52






















LOQ





53




























54



between 99 and 101

























55










LOQ












fenofibrate







56





respectively
























57




















microgmL










58





























59












studied
















60





























61












precision

















62












acceptance range


63

3 EXPERIMENTAL WORK







31 Solvents





32 Chemicals















64












Model CP324S

Min 00001g

Max 320 g


Model 5986-62


Model DA-60D


Model PS 02000A


34 Glass Apparatus







65








j) Glass Funnel


66















353 Linearity
















67


accuracy

355 Accuracy









356 Precision





357 Selectivity













68

358 Robustness
























RSD






69







each

3512 HPLC Set Up



3 Detector UV

4 Wavelength 242 nm

5 Injector Rheodyne








70












363 Linearity





triplicate






accuracy

365 Accuracy







71

366 Precision





367 Selectivity












368 Robustness













72
























73

3612 HPLC Set Up



3 Detector UV

4 Wavelength 240 nm

5 Injector Rheodyne








74











373 Linearity





made in triplicate







375 Accuracy








75

376 Precision





days

377 Selectivity














378 Robustness










76












condition




3711 HPLC Set Up



3 Detector UV

4 Wavelength 237 nm

5 Injector Rheodyne








77














383 Linearity











385 Accuracy





78



386 Precision





days

387 Selectivity












388 Robustness






were calculated




79




























80

3812 HPLC Set Up


2 HPLC Pump LC-20AT


4 Wavelength 240 nm

5 Injector Rheodyne








81















393 Linearity












395 Accuracy




82




396 Precision





days

397 Selectivity













for lovastatin

398 Robustness






83


were calculated


















under each stress





84

3911 HPLC Set Up


2 HPLC Pump LC-20AT


4 Wavelength 240 nm

5 Injector Rheodyne








85














respectively

3103 Linearity












accuracy


86

3105 Accuracy






3106 Precision





3107 Selectivity














3108 Robustness





87






















88

31011 HPLC Set Up


2 HPLC Pump LC-20AT


4 Wavelength 240 nm

5 Injector Rheodyne








89

























3114 Linearity






90








3116 Accuracy






3117 Precision





days

3118 Selectivity











91



3119 Robustness

























92

31112 HPLC Set Up


2 HPLC Pump LC-20AT


4 Wavelength 240 nm

5 Injector Rheodyne


ratio of 4060 (vv)






93
























4121 Linearity





94

















4123 Accuracy







4124 Precision






95



4125 Selectivity








given in Table 43





given in Table 44

4127 Robustness






96




97





200 2028 10140 029

240 2368 9867 042

280 2812 10043 124

Ezetimibe 160 1588 9925 057

200 1965 9825 086

240 2442 10175 168

280 2782 9936 011





320 5 3215 103 3248 151

480 5 4772 086 4861 125

Ezetimibe 160 5 1570 070 1633 135

320 5 3252 083 3158 089

480 5 4882 039 4802 110


98





Ezetimibe



32 3218 10056

32 3162 9881

32 3178 9931

32 3252 10162


RSD = 126

32 3251 10159

32 3186 9956

32 3158 9869

32 3224 10075


RSD = 128


99





160 1573 1582 1615 138

320 3148 3168 3150 035

480 4818 4798 4880 089

Ezetimibe

160 1632 1611 1630 074

320 3281 3242 3218 094

480 4772 4848 4820 114


100






Flow rate (04mLmin) 10184 383 3507 125

Flow rate (06 mLmin) 9858 255 3310 141

Buffer (pH 63) 10089 303 3401 120

Buffer (pH 67) 10154 302 3467 121

1RT Retention Time






Flow rate (04mLmin) 9802 558 5221 115

Flow rate (06 mLmin) 9915 372 5019 118

Buffer (pH 63) 10022 441 5186 110

Buffer (pH 67) 10005 443 5125 111

1RT Retention Time


101





















102





Ezetimibe



32 3262 10194

32 3215 10047

32 3168 9900


RSD = 146

32 3256 10175

32 3256 10056

32 3248 10203


RSD = 048


103









column life





















104


[Figure 43 and 44]






4221 Linearity












coefficient










105




106

4223 Accuracy







48]

4224 Precision






4225 Selectivity













107




Ezetimibe 50 3 200 2005 10025 138

100 3 400 3965 9912 068

150 3 600 6020 10033 086

Simvastatin 50 3 200 2030 10150 038

100 3 400 4025 10062 115

150 3 600 6060 10100 102




Ezetimibe 200 5 1986 110 1995 078

400 5 4012 105 3990 115

600 5 5996 028 6012 120

Simvastatin 200 5 2024 145 2010 056

400 5 4056 068 3975 132

600 5 5942 075 6025 088


108



Ezetimibe 10062 5 296 - 123 6781

Simvastatin 9943 5 980 1964 106 13752






Ezetimibe

200 2032 2009 1985 024

400 4076 3990 4040 043

600 5970 6025 6056 044

Simvastatin

200 2012 1995 2005 008

400 4035 4025 4020 008

600 6015 6025 5975 026


109

4227 Robustness
























formulations


110






Flow rate (14 mLmin) 9842 318 6810 125

Flow rate (16 mLmin) 9905 278 6566 127

Buffer (pH 48) 10022 299 6685 125

Buffer (pH 52) 10089 301 6628 124






Flow rate (14 mLmin) 10033 1052 13927 105

Flow rate (16 mLmin) 9915 919 13425 107

Buffer (pH 48) 10129 985 13564 108

Buffer (pH 52) 10086 984 13416 108


111




Ezetimibe 5 10 1012 10125 075

Simvastatin 5 10 1005 10050 115


112






























113





solutions

4321 Linearity










099997










4323 Accuracy





114





4324 Precision






416

4325 Selectivity








the proposed method







115



116




Gemfibrozil 50 5 1200 12022 10018 095

100 5 2400 23734 9889 043

150 5 3600 35421 9839 042

Simvastatin 50 5 20 202 10100 133

100 5 40 406 10150 119

150 5 60 593 9883 074




Gemfibrozil 1200 5 12125 078 11958 125

2400 5 24456 095 24258 102

3600 5 36521 124 36321 085

Simvastatin 20 5 202 144 201 106

40 5 396 111 395 058

60 5 607 036 602 131


117



118


Gemfibrozil



Simvastatin



240 23645 9852

240 24142 10059

240 24356 10148

240 23988 9995


RSD = 125

4 405 10125

4 396 9900

4 398 9950

4 393 9825


RSD = 128


119





Gemfibrozil

1200 11808 11788 11756 022

2400 24262 23943 23640 130

3600 35828 35641 35494 047

Simvastatin

20 201 199 197 101

40 398 395 391 089

60 602 595 591 093


120

4327 Robustness


















121






Flow rate (11mLmin) 9869 424 7118 128

Flow rate (09 mLmin) 10041 518 8002 122

Buffer (pH 52) 9889 465 7719 123

Buffer (pH 48) 10115 465 7662 123

1Retention Time

2Theoretical Plates






Flow rate (11mLmin) 9921 698 10220 129

Flow rate (09 mLmin) 9903 853 12515 122

Buffer (pH 52) 10069 765 11308 122

Buffer (pH 48) 10098 766 11015 122

1Retention Time

2Theoretical Plates


122




















solutions

4421 Linearity










123












4423 Accuracy







4424 Precision





Table 422

4425 Selectivity






124









4427 Robustness





















125




126




Ezetimibe 5 50 80 788 9850 095

5 100 160 1581 9881 031

5 150 240 2405 10021 033

Fenofibrate 5 50 1280 12924 10097 018

5 100 2560 25492 9958 051

5 150 3840 38850 10117 075




Ezetimibe 160 5 158 138 157 151

160 5 1608 095 1611 107

400 5 3995 055 3991 085

Fenofibrate 256 5 2550 096 2553 063

256 5 2548 033 2545 051

640 5 6373 022 6355 039


127


Ezetimibe



Fenofibrate



160 1611 10089

160 1593 9956

160 1588 9925

160 1590 9938


RSD = 076

2560 2538 9914

2560 2543 9934

2560 2581 10082

2560 2546 9945


RSD = 077


128





Ezetimibe

160 157 156 155 064

160 1618 1590 1576 134

400 3988 3942 3912 041

Fenofibrate

256 2484 2465 2456 058

2560 25512 25349 25215 059

6400 63841 63555 63373 037


129






Flow rate (14mLmin) 10098 261 6372 136

Flow rate (16 mLmin) 9962 234 4803 116

Buffer (pH 52) 10126 243 6005 122

Buffer (pH 48) 10085 244 6078 123






Flow rate (14mLmin) 10049 950 14337 121

Flow rate (16 mLmin) 9979 808 9991 107

Buffer (pH 52) 10021 876 12885 115

Buffer (pH 48) 9905 877 12687 116


130








131


Ezetimibe



Fenofibrate



16 1624 10150

16 1605 10031

16 1591 9944


RSD = 103

256 25894 10115

256 25536 9975

256 25748 10058


RSD = 070


132































133








shown)























134




4323 Accuracy








4524 Precision







4525 Selectivity








the proposed method


135



136




Ezetimibe 50 3 50 508 10160 102

100 3 100 988 9880 146

150 3 150 1541 10273 039

Lovastatin 50 3 100 1022 10220 063

100 3 200 1944 9720 119

150 3 300 2928 9760 093




Ezetimibe 080 5 082 122 081 163

250 5 2458 086 2443 138

1000 5 9869 074 9805 108

Lovastatin 16 5 156 111 155 151

500 5 5059 055 5046 149

2000 5 20241 032 19968 098


137



138


Ezetimibe



Lovastatin



100 1023 10230

100 992 9920

100 986 9860

100 1018 10180


RSD = 184

200 2054 10270

200 2036 10180

200 1978 9890

200 1986 9930


RSD = 185


139






4527 Robustness















140





Ezetimibe

080 081 078 079 193

250 2484 2466 2448 073

1000 10098 9922 9805 148

Lovastatin

160 163 161 159 124

500 4963 4921 4893 071

2000 20098 19852 19646 114


141






Flow rate (09 mLmin) 9946 453 5100 134

Flow rate (11 mLmin) 9905 370 4886 140

Buffer (pH 52) 10048 405 5454 139

Buffer (pH 48) 10215 404 5404 139






Flow rate (09 mLmin) 9932 1080 8004 126

Flow rate (11 mLmin) 10068 883 7575 136

Buffer (pH 52) 10046 967 7785 130

Buffer (pH 48) 9885 966 7715 130


142





























143






shown)





of solutions

4621 Linearity



















144




4623 Accuracy








4624 Precision






435

4625 Selectivity








the proposed method


145



146




Atorvastatin 50 3 40 406 10150 163

100 3 80 789 9863 126

150 3 120 1212 10100 069

Gemfibrozil 50 3 2400 23658 9858 101

100 3 4800 48863 10180 065

150 3 7200 73356 10188 053




Atorvastatin 05 5 052 198 051 223

80 5 795 086 786 155

200 5 1984 063 1982 141

Gemfibrozil 300 5 2963 101 2951 129

4800 5 48212 073 47871 122

12000 5 118648 088 118002 125


147



148


Atorvastatin



Gemfibrozil



80 808 10100

80 796 9950

80 805 10063

80 793 9912


RSD = 090

4800 47222 9838

4800 47805 9959

4800 48215 10045

4800 47329 9860


RSD = 097


149






4627 Robustness














products





414


150





Atorvastatin

05 052 051 052 112

80 794 797 790 044

200 2022 1995 1990 086

Gemfibrozil

300 3046 2983 2955 156

4800 48258 47626 47298 069

12000 119239 118658 118022 051


151






Flow rate (14 mLmin) 10169 380 3310 136

Flow rate (16 mLmin) 10043 334 2866 141

Buffer (pH 48) 9932 354 3164 141

Buffer (pH 52) 9978 355 3214 140






Flow rate (14 mLmin) 9911 1255 4002 131

Flow rate (16 mLmin) 10009 1098 3687 135

Buffer (pH 48) 9969 1169 3998 134

Buffer (pH 52) 9955 1169 4008 135


152



Ca2+

3H2O

N

O

NH

CH3

CH3

F

OHOH

O-

O CH3OH H2O2

Room TempO

O

NH

O

OH

OCH3

OHCH3


153


























4721 Linearity





154




















155




156

4723 Accuracy








4724 Precision






4725 Selectivity













157




Rosuvastatin 50 3 400 4069 10172 145

100 3 800 7888 9860 115

150 3 1200 11821 9851 096

Ezetimibe 50 3 100 981 9810 139

100 3 200 2048 10240 121

150 3 300 2928 9760 111




Rosuvastatin 50 5 505 144 503 189

800 5 7925 119 7805 169

1600 5 16228 095 16059 128

Ezetimibe 125 5 123 151 122 205

200 5 2051 076 2028 128

400 5 3965 105 3921 156


158


Rosuvastatin



Ezetimibe



800 8089 10111

800 7866 9832

800 8129 10161

800 8052 10065


RSD = 145

200 2048 10240

200 1963 9815

200 1983 9915

200 1972 9860


RSD = 193


159





Rosuvastatin

50 497 498 495 031

800 7942 7881 7885 043

1600 16152 15922 15905 086

Ezetimibe

125 123 121 122 082

200 1982 1975 1955 071

400 4008 3928 3911 131


160

4727 Robustness
























161



ACN 1 H3PO4 (6040) 10025 430 3126 135

ACN 1 H3PO4 (5842) 10011 476 3316 133

ACN 1 H3PO4 (6238) 9865 408 3040 141

Flow rate (11mLmin) 9985 391 2866 140

Flow rate (09 mLmin) 10141 478 3264 135

H3PO4 Conc (09 ) 9955 428 3167 139

H3PO4 Conc (11 ) 9941 427 3114 133



ACN 1 H3PO4 (6040) 10069 633 4139 142

ACN 1 H3PO4 (5842) 9965 715 4267 141

ACN 1 H3PO4 (6238) 10025 595 3964 148

Flow rate (11mLmin) 10095 575 4040 145

Flow rate (09 mLmin) 9926 703 4220 142

H3PO4 Conc (09 ) 10068 635 3998 144

H3PO4 Conc (11 ) 10029 636 4002 139


162




Rosuvastatin 5 40 4052 10130 103

Ezetimibe 5 10 1021 10210 131


163

48 Conclusion









formulations




















164










human plasma














formulations


165

5 REFERENCES






Page 971



2008 14(2) 314-324


141108


Accessed on 141108


141108



141108


151108




New York 2003






166


2002 106 3240



278 313ndash321




















2619ndash2628



77 31ndash36



167



1146


2000 9 2653ndash2661


239ndash252




44 379ndash387




32B









1ndash34






168





27 107ndash114




638





25 1191ndash1199









1995 58 412ndash417






597ndash602


169


420









69B



170-89


Lipid Res 1997 38 2071-2078



135 249- 256





Anal 2006 41(2) 408-414







170


317ndash 326






951ndash956





29636ndash29639



Sci USA 1984 81 4203ndash 4207




79 95ndash99




Biol Chem 1999 274 32048ndash32054



275 36703ndash 36707




171




S45-S48



Pharmacol 2000 56 631-5



47(2) 73-104








2002 71 80


Disease 2004 3 22











172



283 157-163




Pharmacol 2001 134 409-417


79-84







729-741









Heart J 2005 149 464ndash473




173



2003 107 2409ndash 2415







1758 ndash1773



2125ndash2134






Proc 2004 79 620ndash 629



418ndash424







728


174



455









334









1997 80 608ndash613






47 1584 ndash1587


5th Edition 1994 23-25 51-53


175




01082009


on 01082009






03082009







992ndash1009

171 Khedr A J AOAC Int 2007 90(6) 1547-53


Sci 2007 30(18) 3143-53


766


(11-12) 737-741



44(2) 379-87


176


241-6



Life Sci 2007 846(1-2) 215-21











382(5) 1242-9


Anal 2003 33(5) 1017-23


Spectrom 1999 13(11) 1003-15


1999 10(1) 55-66







2008 81(3) 453-459


177


Chemistry 2007 62(6 ) 536-541







Chemistry 2006 61 (1 ) 63-66




2004 49 (4) 289-289


Sep Sci 2004 27(13) 1087-92


2002 29 (4) 715-721


18(3) 232-4





1992 10(9) 693-7


2008 22(5) 511-8


Anal 2008 46(4) 808-813


178


2008 20 399ndash410



2719ndash2725


Chem Soc 2005 50(4) 639-646




Biomed Life Sci 2002 768(2) 349-59


41


1999 20(1-2) 137-43


Biomed Anal 1988 6(3) 271-6






Biomed Life Sci 2007 856(1-2) 35-40


Biomed Anal 2007 44(2) 540-6






179


International 2005 88 (4) 1142-1147


Anal 2004 35(3) 609-14


Pharm Sci 2007 69 819-21





B 2003 791 161ndash170



Anal 2001 26(1) 7-14








197-202


60(5) 425-38



J Chromatogr A 2004 1046(1-2) 133-40



180


2000 742(2) 391-400




118(10) 447-55




2) 137-142


67(1-2) 101-107


SR Ind J Pharm Sci 2008 70(2) 258-260




853(1-2) 88-96



Biomed Anal 2006 41(3) 1037-40




B 2006 830(1)143-150




181



IFPMA Geneva 1994



1996








1995 76(2) 126A-128A











Title_pages_PhDpdf


Muhammad Ashfaq




Supervisor


To

Abbreviationspdf


List_of_Tablespdf

List of Tables

TAB DESCRIPTIONPAGE

List_of_Figurespdf

FIG DESCRIPTIONPAGE




Table of Contents

DESCRIPTION PAGE









3512 HPLC Set Up69


3612 HPLC Set Up73

3711 HPLC Set Up76


3812 HPLC Set Up80

3911 HPLC Set Up84

31011 HPLC Set Up88

31112 HPLC Set Up92


48S-1pdf









48S-2pdf



48S-3pdf

3 EXPERIMENTAL WORK




3512 HPLC Set Up



3612 HPLC Set Up

3711 HPLC Set Up


3812 HPLC Set Up

3911 HPLC Set Up

31011 HPLC Set Up


31112 HPLC Set Up

48S-4pdf




























ACN 1 H3PO4 (6040) 10025 430 3126 135


ACN 1 H3PO4 (6040) 10069 633 4139 142

development and validation of liquid chromatographic

Documents