DEVELOPMENT AND VALIDATION OF ANALYTICAL …shodhganga.inflibnet.ac.in/bitstream/10603/9593/12/... · · 2014-05-27DEVELOPMENT AND VALIDATION OF ANALYTICAL ... THEIR DOSAGE FORMS

DEVELOPMENT AND VALIDATION OF ANALYTICAL

METHODS FOR NEW CHEMICAL ENTITIES AND

THEIR DOSAGE FORMS BY USING HIGH

PERFORMANCE LIQUID CHROMATOGRAPHY

A THESIS

Submitted

in the partial fulfillment of the requirements for

the award of the degree of

DOCTOR OF PHILOSOPHY

in

FACULTY OF PHARMACEUTICAL SCIENCES

By

RAVI KUMAR KONDA[Reg. No. 0900PH1396]

RESEARCH AND DEVELOPMENT CELLJAWAHARLAL NEHRU TECHNOLOGICAL UNIVERSITY ANANTAPUR

ANANTAPUR – 515 002A.P., INDIA

SEPTEMBER- 2012

ii

DEDICATED TOMY BELOVED PARENTS

iii

JAWAHARLAL NEHRU TECHNOLOGICAL UNIVERSITY ANANTAPUR

ANANTAPUR – 515 002,

A.P., INDIA

CERTIFICATE

This is to certify that the thesis entitled “DEVELOPMENT AND VALIDATION

OF ANALYTICAL METHODS FOR NEW CHEMICAL ENTITIES AND

THEIR DOSAGE FORMS BY USING HIGH PERFORMANCE LIQUID

CHROMATOGRAPHY” that is being submitted by Sri RAVI KUMAR KONDA

in partial fulfillment for the award of Ph.D. in Pharmaceutical sciences to the

Jawaharlal Nehru Technological University, Anantapur, is a record of bonafide work

carried out by him under our guidance and supervision.

The results embodied in this thesis have not been submitted to any other

University or Institute for the award of any degree or diploma.

Research Co-Supervisor Research Supervisor

Dr.K.B. CHANDRA SEKHAR Dr.CH.BABU RAOProfessor in Chemistry & Professor & Director,Director of Evaluation Don Bosco P G College of PharmacyJ N T University Anantapur, 5ThMile, PulladiGunta (Po),Anantapur, Guntur (D.t.)A.P, INDIA A.P, INDIA

iv

DECLARATION

I here by declare that this thesis entitled “DEVELOPMENT AND

VALIDATION OF ANALYTICAL METHODS FOR NEW

CHEMICAL ENTITIES AND THEIR DOSAGE FORMS BY

USING HIGH PERFORMANCE LIQUID CHROMATOGRAPHY”which is being submitted by me in partial fulfillment of the requirement for the award

of the degree of Ph.D. to the Jawaharlal Nehru Technological University Anantapur,

Anantapur, A.P., India, is original and has not been submitted for any degree/diploma

of this or any other university.

(RAVI KUMAR KONDA)

Regd. No. 0900PH1396

v

ACKNOWLEDGEMENTS

I express my deep sense of gratitude and indebtedness to my

Supervisor, Dr.Ch.Babu Rao, Professor & Director, Don Bosco PG College of

Pharmacy, Pulladigunta, Guntur, Andhra Pradesh, India for his invaluable

guidance, co-operation and encouragement throughout the course of work extended

for the successful completion of this project which served as a great source of

inspiration. Through his spontaneous encouragement and inspiring guidance, my

sincere and heartful thanks to him for his sumptuous suggestions.

I am very much indebted to my Co-Supervisor Dr.K.B.Chandra Sekhar

Professor & Director of Evaluation, JNTUA Anantapur, Andhra Pradesh, India

for their timely suggestions and constant support during the entire period of my

research work.

It is my great privilege to express profound thanks and immense sense of

gratitude to my college Principal, Collegues and Management of Hindu College of

Pharmacy for their timely valuable kind co-operation and help.

I wish wholehearted thanks to my beloved Chairman Dr.Mannava Radha

Krishna Murthy garu, Secretary & Correspondent Sri Godavarthy Satya Narayana

garu for their constant encouragement and blessings to complete this work

successfully.

vi

I am thankful to my close friends Dr.M.RamaKotaiah,Dr.Ch.BalaSekhar

Reddy, Dr.P.Ramalingam, Ch.Nagabhushanam, Ch.M.M.PrasadRao,B.Chandra

Sekhar for kind co-operation, as the project would not have been as lively as it

turned out to be.

I wish wholehearted thanks to my beloved parents Sri Konda Appa Rao &

K.V.D.Naga Rani for their constant encouragement and blessings to complete this

work successfully. I am also grateful to my family member’s affection.

I wish wholehearted thanks to my beloved sister and family members

D.Jyothi Prasad, Thamogna sai and Dhruthi saranya for their encouragement to

complete this work successfully.

I am beholden by galvanizing inspiration of my better half,

Mrs.K.V.Sravani, for her unfailing support and I remain highly grateful to her

forever.

Once again I wish to thank one and all who helped me in accomplishing my

objective. This Project could have been produced with the help and co-operation of

many people with whom I came in contact with during the period of my Research.

My devoted thanks to the Almighty god for giving me the strength and the

favorable circumstances to make this accomplishment.

RAVI KUMAR KONDA

vii

CONTENTS

Chapter Contents PageNo.

List of Tables ixList of Figures xviList of Abbrevations xixAbstract xxii

1 Introduction1.1 Pharmaceutical Analysis 11.2 Extraction Procedures for Drugs and Metabolites from

Biological Samples 31.3 Method development and validation 6

1.4 Estimation of Drugs in Biological Sample by HPLC-MS1.5 Bioavailability and Bioequivalence Studies

2122

2 Aim and objectives of Present Research work 25

3 Analytical method development and validation of Rasagiline byHigh Performance Liquid Chromatography with massspectrometry3.1 Introduction 283.2 Experimental Investigations 313.3 Method Development 343.4 Method Validation 423.5 Application 703.6 Pharmacokinetic Studies 73

4 Analytical method development and validation of Almotriptan byHigh Performance Liquid Chromatography with massspectrometry

4.1 Introduction 76

4.2 Experimental Investigations 77

4.3 Method Development 79

viii

4.4 Method Validation 86

4.5 Application 117

4.6 Pharmacokinetic Studies 119

5 Analytical method development and validation of Valacyclovir by

High Performance Liquid Chromatography with mass

spectrometry

5.1 Introduction 122




5.5 Application 161


6 Analytical method development and validation of Memantine by

High Performance Liquid Chromatography with mass

spectrometry

6.1 Introduction 165




6.5 Application 209


7

8

Summary and Conclusion

Bibliography

214

218

Appendx: Research Publications.

ix

LIST OF TABLES

Table No. Description Page No.

3.1 Preparation of Reagents and Solvents 32

3.2 Preparation of Stock solutions 32

3.3 Screening of Different batches of blank matrix (HumanK2EDTA Plasma) for interference free Rasagiline blankplasma

47

3.4 Specificity of Different batches of blank matrix (HumanK2EDTA Plasma) for Rasagiline

48

3.5 Limit of Quantitation for analyte (Rasagiline) 49

3.6 Intra batch (Within-Batch) Accuracy and Precision fordetermination of Rasagiline levels in human plasma

50

3.7 Inter batch (Between-Batch) Accuracy and Precision fordetermination of Rasagiline levels in human plasma

51

3.8 Summary of calibration curve parameters for Rasagiline inhuman Plasma

52

3.9 Back-calculated standard concentrations from eachcalibration curve for Rasagiline in human plasma.

53

3.10 Recovery of analyte Rasagiline and Rasagiline-13C3 mesylatefrom human plasma

55

3.11 Assessment of Matrix Effect on determination of Rasagilineat LQC levels in human plasma

57

3.12 Assessment of Matrix Effect on determination of Rasagilineat HQC levels in human plasma

58

3.13 Assessment of Dilution integrity for Rasagiline at DQCConc (pg/mL)

59

3.14 Assessment of Whole Batch Re-injection Reproducibilityduring estimation of Rasagiline in human plasma

60

3.15 Ruggedness of the method for estimation of RasagilinePlasma levels in human plasma with different Analyst

61

x


3.16 Ruggedness of the method for estimation of RasagilinePlasma levels in human plasma with different Analyticalcolumn

62

3.17 Assessment of stability of Analyte (Rasagiline) in Biologicalmatrix at Room temperature

63

3.18 Assessment of Freeze-Thaw stability of Analyte(Rasagiline )at -30 ±5°C

64

3.19 Assessment of Auto sampler stability of Analyte (Rasagiline)at 2-8°C

65

3.20 Assessment of Long term plasma stability of Analyte(Rasagiline) at -30°C

66

3.21 Assessment of Short term stock solution stability of Analyte(Rasagiline) and Internal standard (Rasagiline-13C3 mesylate)at Room temperature

68

3.22 Assessment of Short term solution stability of Internalstandard Spiking solution (Rasagiline-13C3 mesylate) atrefrigerated conditions

69

3.23 Rasagiline mean concentration (pg/mL) data for the subjectsamples obtained from the LC-MS/MS

71

3.24 Rasagiline Pharmacokinetic data 74

3.25 Rasagiline Pharmacokinetic data (Test/Reference) 74



4.3 Screening of Different batches of blank matrix (HumanK2EDTA Plasma) for interference free Almotriptan blankplasma

95

4.4 Specificity of Different batches of blank matrix (HumanK2EDTA Plasma) for Almotriptan

96

4.5 Limit of Quantitation for analyte (Almotriptan) 97

4.6 Intra batch (Within-Batch) Accuracy and Precision fordetermination of Almotriptan levels in human plasma

98

xi


4.7 Inter batch (Between-Batch) Accuracy and Precision fordetermination of Almotriptan levels in human plasma

99

4.8 Summary of calibration curve parameters for Almotriptan inhuman Plasma

100

4.9 Back-calculated standard concentrations from eachcalibration curve for Almotriptan in human plasma

101

4.10 Recovery of analyte Almotriptan and Almotriptan- D6 fromhuman plasma

103

4.11 Assessment of Matrix Effect on determination ofAlmotriptan at LQC levels in human plasma

104

4.12 Assessment of Matrix Effect on determination ofAlmotriptan at HQC levels in human plasma

105

4.13 Assessment of Dilution integrity for Almotriptan at DQCConc (ng/mL)

106

4.14 Assessment of Whole Batch Re-injection Reproducibilityduring estimation of Almotriptan in human plasma

107

4.15 Ruggedness of the method for estimation of AlmotriptanPlasma levels in human plasma with different Analyst

108

4.16 Ruggedness of the method for estimation of AlmotriptanPlasma levels in human plasma with different Analyticalcolumn.

109

4.17 Assessment of stability of Analyte (Almotriptan) inBiological matrix at Room temperature.

110

4.18 Assessment of Freeze-Thaw stability of Analyte(Almotriptan) at -30 ±5°C

111

4.19 Assessment of Auto sampler stability of Analyte(Almotriptan) at 2-8°C

112

4.20 Assessment of Long term plasma stability of Analyte(Almotriptan) at -30°C

113

4.21 Assessment of Short term stock solution stability of Analyte(Almotriptan) and Internal standard (Almotriptan- D6) atRoom temperature

115

xii


4.22 Assessment of Short term solution stability of Internalstandard Spiking solution (Almotriptan- D6) at refrigeratedconditions

115

4.23 Almotriptan mean concentration (ng/mL) data for the subjectsamples obtained from the LC-MS/MS

118

4.24 Almotriptan Pharmacokinetic data 120

4.25 Almotriptan Pharmacokinetic data (Test/Reference) 120



5.3 Screening of Different batches of blank matrix (HumanK2EDTA Plasma) for interference free Valacyclovir blankplasma

139

5.4 Specificity of Different batches of blank matrix (HumanK2EDTA Plasma) for Valacyclovir

140

5.5 Limit of Quantitation for analyte (Valacyclovir) 141

5.6 Intra batch (Within-Batch) Accuracy and Precision fordetermination of Valacyclovir levels in human plasma

142

5.7 Inter batch (Between-Batch) Accuracy and Precision fordetermination of Valacyclovir levels in human plasma

143

5.8 Summary of calibration curve parameters for Valacyclovir inhuman Plasma

144

5.9 Back-calculated standard concentrations from eachcalibration curve for Valacyclovir in human plasma.

145

5.10 Recovery of analyte Valacyclovir and Valacyclovir- D8

from human plasma147

5.11 Assessment of Matrix Effect on determination ofValacyclovir at LQC levels in human plasma

148

5.12 Assessment of Matrix Effect on determination ofValacyclovir at HQC levels in human plasma

149

xiii


5.13 Assessment of Dilution integrity for Valacyclovir at DQCConc (ng/mL)

150

5.14 Assessment of Whole Batch Re-injection Reproducibilityduring estimation of Valacyclovir in human plasma

151

5.15 Ruggedness of the method for estimation of ValacyclovirPlasma levels in human plasma with different Analyst

152

5.16 Ruggedness of the method for estimation of ValacyclovirPlasma levels in human plasma with different Analyticalcolumn.

153

5.17 Assessment of stability of Analyte (Valacyclovir) inBiological matrix at Room temperature.

154

5.18 Assessment of Freeze-Thaw stability of Analyte(Valacyclovir) at -30 ±5°C

155

5.19 Assessment of Auto sampler stability of Analyte(Valacyclovir) at 2-8°C

156

5.20 Assessment of Long term plasma stability of Analyte(Valacyclovir) at -30°C

157

5.21 Assessment of Short term stock solution stability of Analyte(Valacyclovir) and Internal standard (Valacyclovir- D8) atRoom temperature

159

5.22 Assessment of Short term solution stability of Internalstandard Spiking solution (Valacyclovir- D8) at refrigeratedconditions

159

5.23 Valacyclovir mean concentration (ng/mL) data for thesubject samples obtained from the LC-MS/MS bioanalyticalmethod

162

5.24 Valacyclovir Pharmacokinetic data 164

5.25 Valacyclovir Pharmacokinetic data (Test/Reference) 164



xiv


6.3 Screening of Different batches of blank matrix (HumanK2EDTA Plasma) for interference free Memantine blankplasma

186

6.4 Specificity of Different batches of blank matrix (HumanK2EDTA Plasma) for Memantine

188

6.5 Limit of Quantitation for analyte (Memantine) 189

6.6 Intra batch (Within-Batch) Accuracy and Precision fordetermination of Memantine levels in human plasma

190

6.7 Inter batch (Between-Batch) Accuracy and Precision fordetermination of Memantine levels in human plasma

191

6.8 Summary of calibration curve parameters for Memantine inhuman Plasma

192

6.9 Back-calculated standard concentrations from eachcalibration curve for Memantine in human plasma.

193

6.10 Recovery of analyte Memantine and Memantine- D6 fromhuman plasma

195

6.11 Assessment of Matrix Effect on determination of Memantineat LQC levels in human plasma

196

6.12 Assessment of Matrix Effect on determination ofMemantine at HQC levels in human plasma

197

6.13 Assessment of Dilution integrity for Memantine at DQCConc (pg/mL)

198

6.14 Assessment of Whole Batch Re-injection Reproducibilityduring estimation of Memantine in human plasma

199

6.15 Ruggedness of the method for estimation of MemantinePlasma levels in human plasma with different Analyst

200

6.16 Ruggedness of the method for estimation of MemantinePlasma levels in human plasma with different Analyticalcolumn.

201

6.17 Assessment of stability of Analyte (Memantine) inBiological matrix at Room temperature.

202

xv


6.18 Assessment of Freeze-Thaw stability of Analyte(Memantine) at -30 ±5°C

203

6.19 Assessment of Auto sampler stability of Analyte(Memantine) at 2-8°C

204

6.20 Assessment of Long term plasma stability of Analyte(Memantine) at -30°C

205

6.21 Assessment of Short term stock solution stability of Analyte(Memantine) and Internal standard (Memantine- D6) atRoom temperature

207

6.22 Assessment of Short term solution stability of Internalstandard Spiking solution (Memantine- D6) at refrigeratedconditions

207

6.23 Memantine mean concentration (pg/mL) data for the subjectsamples obtained from the LC-MS/MS

210

6.24 Memantine Pharmacokinetic data 213

6.25 Memantine Pharmacokinetic data (Test/Reference) 213

xvi

LIST OF FIGURES

Figure No. Description Page No.

3.1 Chemical structures of Rasagiline, Rasagiline-13C3 mesylate 28

3.2 Parent ion mass spectra (Q1) of Rasagiline 35

3.3 Product ion mass spectra (Q3) of Rasagiline 36

3.4 Parent ion mass spectra (Q1) of Rasagiline-13C3 mesylate 37

3.5 Product ion mass spectra (Q3) of Rasagiline-13C3 mesylate 38

3.6 MRM Chromatogram of Blank Human Plasma Sample 42

3.7 Chromatogram of Blank + IS 43

3.8 Chromatogram of LOQ sample (Rasagiline & IS) 43

3.9 Chromatogram of ULOQ Sample (Rasagiline & IS) 44

3.10 Chromatogram of LLOQ Sample (Rasagiline & IS) 44

3.11 Chromatogram of LQC Sample (Rasagiline & IS ) 45

3.12 Chromatogram of MQC Sample (Rasagiline & IS) 45

3.13 Chromatogram of HQC Sample (Rasagiline& IS) 46

3.14 Calibration Curve of Rasagiline 46

3.15 Mean plasma concentration Vs time curve for Rasagiline 72

4.1 Chemical structures of Almotriptan, Almotriptan- D6 malate 76

4.2 Parent ion mass spectra (Q1) of Almotriptan 80

4.3 Product ion mass spectra (Q3) of Almotriptan 81

4.4 Parent ion mass spectra (Q1) of Almotriptan- D6 malate 82

4.5 Product ion mass spectra (Q3) of Almotriptan- D6 malate 83



4.8 Chromatogram of LOQ sample (Almotriptan & IS) 88

xvii


4.9 Chromatogram of ULOQ Sample (Almotriptan & IS ) 89

4.10 Chromatogram of LLOQ Sample (Almotriptan & IS) 90

4.11 Chromatogram of LQC Sample (Almotriptan & IS ) 91

4.12 Chromatogram of MQC Sample (Almotriptan & IS) 92

4.13 Chromatogram of HQC Sample (Almotriptan & IS) 93

4.14 Calibration Curve of Almotriptan 94

4.15 Mean plasma concentration Vs time curve for Almotriptan 118

5.1 Chemical structures of Valacyclovir, Valacyclovir- D8 122

5.2 Parent and Product ion mass spectra of Valacyclovir 127

5.3 Parent and Product ion mass spectra of Valacyclovir- D8 128



5.6 Chromatogram of LOQ sample (Valacyclovir & IS) 133

5.7 Chromatogram of ULOQ sample (Valacyclovir & IS) 134

5.8 Chromatogram of LLOQ Sample (Valacyclovir & IS) 135

5.9 Chromatogram of LQC Sample (Valacyclovir & IS) 136

5.10 Chromatogram of MQC Sample (Valacyclovir & IS) 137

5.11 Chromatogram of HQC Sample (Valacyclovir & IS) 138

5.12 Calibration Curve of Valacyclovir 138

5.13 Mean plasma concentration Vs time curve for Valacyclovir 162

6.1 Chemical structures of Memantine, Memantine- D6 165

6.2 Parent ion mass spectra (Q1) of Memantine 170

6.3 Product ion mass spectra (Q3) of Memantine 171

6.4 Parent ion mass spectra (Q1) of Memantine -D6 172

xviii


6.5 Product ion mass spectra (Q3) of Memantine- D6 173



6.8 Chromatogram of LOQ sample (Memantine & IS) 179

6.9 Chromatogram of ULOQ sample (Memantine & IS ) 180

6.10 Chromatogram of LLOQ Sample (Memantine & IS) 181

6.11 Chromatogram of LQC Sample (Memantine & IS) 182

6.12 Chromatogram of MQC Sample (Memantine & IS) 183

6.13 Chromatogram of HQC Sample (Memantine & IS) 184

6.14 Calibration Curve of Memantine 185

6.15 Mean plasma concentration Vs time curve for Memantine 211

xix

LIST OF ABBREVATIONS

RSG : Rasagiline

RSG-IS : Rasagiline -13C3 mesylate

ALM : Almotriptan

ALM- D6 : Almotriptan- D6

VL : Valacyclovir

VL- D8 : Valacyclovir- D8

ME : Memantine

ME-D6 : Memantine- D6

IS : Internal Standard

MP : Mobile Phase

SP : Stationary Phase

TP : Theoretical Plates

LC-MS/MS : Liquid Chromatography - Tandem Mass Spectrometry

HPLC : High Performance Liquid Chromatography

LLOQ : Lower Limit of Quantitation

LLOQC : Lower Limit of Quality Control

LQC

DQC

: Low Quality Control

: Dilution Quality Control

MQC : Medium Quality Control

HQC : High Quality Control

ULOQ : Upper Limit of Quantitation

LOD : Limit of Detection

xx

PPT : Precipitation

LLE : Liquid-liquid extraction

SPE : Solid phase extraction

CS : Calibration Standards

QC : Quality Control

mg : Milli Grams

µg : Micro Grams

µL : Micro Litre

ng : Nano Grams

pg : Pico Grams

mL : Milli Litre

Hrs : Hours

Min : Minutes

Sec : Seconds

rpm : Rotations per Minute

RT : Retention Time

Temp : Temperature

Conc : Concentration

DCM : Dichloro methane

SD : Standard Deviation

CV : Coefficient of Variation

Max. : Maximum

xxi

0C : Degrees Celsius

r : Correlation Coefficient

API : Active Pharmaceutical Ingredient

ACN : Acetonitrile

MEOH : Methanol

USFDA : United States Food and Drug Administration

ICH : International Conference on Harmonization

NDA : New Drug Application

EP : Entrance Potential

FP : Focussing Potential

NNC : invitro-invivo correlation

PK : Pharmacokinetic

BA : Bioavailability

BE : Bioequivalence

IEC : Institutional Ethics committee

MRM : Multiple Reaction Monitoring

ANOVA : Analysis of Variance

DP : Declustering Potential

CE : Collision Energy

CAD : Collisionally Activated Dissociation

CXP : Collision exist Potential

MBTE : Methyl Tertiary Butyl Ether

xxii

ABSTRACT

Analytical method development and validation is a good research in the field of

Pharmaceutical analysis, utilized to determine the drug content in bulk and

pharmaceutical dosage forms and in biological fluids like blood, serum, urine etc.

In view of the industrial scenario and literature, it was noted that chromatographic

techniques like HPLC, LC–MS/MS methods have created revolutionary precision and

accuracy in quantification of drugs in Formulation and in Biological fluids even at low

concentration.

There is a rapid advancement and developments in the field of pharmaceutical

analysis where sensitive chromatographic and spectral techniques have been evoked for

the determination of drugs in pharmaceutical dosage forms and in biological fluids.

In pharmaceutical industry, the analyst or bioanalyst plays an important role in FDA

approval of newer potent drugs with respect to validation and determination of drugs.

The main goal of this research activity is selected based on the increasing needs

of the pharmaceutical/biopharmaceutical industry in developing suitable analytical

methods. Among the various other available techniques, the scope of this work was

focused on the modern chromatographic techniques such as LC-MS/MS which are

accurate, precise, sophisticated and are having wide spectrum of application in

pharmaceutical/biopharmaceutical industries.

Pharmacokinetic and bioequivalence studies require very precise and accurate

assay methods that are well validated to quantify drugs in biological samples. The assay

methods have to be sensitive enough to determine the biological sample concentration

of the drug and/or its metabolite(s) for a period of about five elimination half- life’s

xxiii

after dosage of the drug. The assay methods also have to be very selective to ensure

reliable data, free from interference of endogenous compounds and possible metabolites

in the biological samples.

Bioanalytical methods have to be simple, sensitive, selective, rugged and

reproducible for long run analysis in BA/BE studies. The Validation parameters like

Selectivity (matrix interference), Sensitivity (LOQ), Linearity, Precision and Accuracy

batches (minimum three), matrix effect, recovery, ruggedness, stock solution stability,

Plasma stabilities like bench top stability, freeze thaw stability, autosampler stability,

reinjection stability, long-term stability, dilution integrity were proved for selected

drugs.

Literature survey reveals that there is a need to develop new, simple, specific, reliable

Bioanalytical methods for determination of Rasagiline, Almotriptan, Valacyclovir and

Memantine in human plasma which are covered in Chapter 3 to Chapter 6.

In chapter 1 discussed about general introduction.

In chapter 2 discussed about aim and objectives of the present research

work.

In chapter 3 discussed and developed the good analytical method namely

“Analytical method development and validation of Rasagiline by High

Performance Liquid Chromatography with mass spectrometry.”

In chapter 4 discussed and developed the good analytical method namely

“Analytical method development and validation of Almotriptan by High

Performance Liquid Chromatography with mass spectrometry”

xxiv

In chapter 5 discussed and developed the analytical method namely

“Analytical method development and validation of Valacyclovir by High


In chapter 6 discussed and developed the analytical method namely

“Analytical method development and validation of Memantine by High


In chapter 7 Summarized the overall thesis and conclusion was drawn

In chapter 8 Summarized all the Bibliography.

CHAPTER 1

Introduction

Chapter 1 Introduction

1

1.1 Pharmaceutical Analysis

Analytical methods are used for product research, product

development, process control and chemical quality control purposes. Each of the

techniques used in chromatographic or spectroscopic, have their own special features

and deficiencies, which must be considered. Each step in the method must be

investigated to determine the extent to which environment, matrix, or procedural

variables can affect the estimation of analyte in the matrix from the time of collection

up to the time of analysis1-3.

Pharmaceutical analysis require very precise and accurate assay methods to

quantify drugs either in Pharmaceutical or biological samples. The assay methods

have to be sensitive, selective, rugged and reproducible3.

Analytical chemistry is the qualitative and quantitative analysis of drug

substances in biological fluids (mainly plasma and urine) or tissue. It plays a

significant role in the evaluation and interpretation of pharmacokinetic data2.The main

analytical phases comprise method development, method validation and sample

analysis (method application).

1.1.1 Need for pharmaceutical Analysis

New Drug Development.

Method Validation as for ICH Guidelines

Research in Pharmaceutical Sciences

Clinical Pharmacokinetic Studies


2

When promising results are obtained from explorative validation performed

during the method devlopment phase,then only full validation should be stared.The

process of validating a method cannot be saparated from the acutal devlopment of

method conditions.

1.1.2 Assay of Drugs and their Metabolites

A number of allusions have been made to analytical methods that distinguish

drugs from their metabolites. Drug metabolism reactions can be divided into phase I

and phase II categories. Phase I typically involves oxidation, reduction, and

hydrolysis reactions. In contrast, phase II transformations involve coupling or

condensation of drugs or their phase I metabolites with common body constituents

(e.g., sulfate, glucuronic acid). Except for reduction processes, most phase I and phase

II reactions yield metabolites that are more polar and hence more water soluble than

the parent drug. Assays must distinguish between drug and its metabolites5.

1.1.3 Analysis of Drugs from various samples

The most common samples obtained for pharmaceutical analysis are blood and

urine. Feces are also utilized, especially if the drug or metabolite is poorly absorbed or

extensively excreted in the bile. Other media that can be utilized include saliva,

breath, and tissue.

Detection of a drug or its metabolite in biological media is usually

complicated by the matrix.Because of this, various types of cleanup procedures

involving techniques such as solvent extraction and chromatography are employed to

effectively separate drug components from endogenous material.


3

1.2 Extraction Procedures for Drugs and Metabolites from

Biological Samples

After pre treating biological material, the next step is usually the extraction of

the drugs from the biological matrix. All separation procedures use one or more

treatments of matrix-containing solute with some fluid6-8. Different extraction

procedures include protein precipitation or denaturation, liquid-liquid extraction, solid

phase extraction and dehydration methods.

1.2.1 Protein Precipitation or Denaturation

Biological materials such as plasma, feces, and saliva contain significant

quantities of protein, which can bind a drug. The drug should be free from this protein

before further manipulation. Protein denaturation is important, because the presence

of proteins, lipids, salts, and other endogenous material in the sample can cause rapid

deterioration of HPLC columns and also interfere with the assay.

Protein denaturation procedures include the use of tungstic acid, ammonium

sulfate, heat, alcohol, trichloroacetic acid and percholric acid.

Methanol and acetonitrile frequently have been used as protein denaturants of

biological samples. Methanol sometimes is preferred because it produces a flocculent

precipitate and not the gummy mass obtained with acetonitrile. Methanol also gives a

clear supernate and may prevent the drug entrapment that can be observed after

acetonitrile precipitation.


4

1.2.2 Liquid-Liquid Extraction

Liquid-liquid extraction is the most widely used technique because

The analyst can remove a drug or metabolite from larger concentrations of

endogenous materials that might interfere with the final analytical

determination.

The technique is simple, rapid, and has a relatively small cost factor per

sample.

The extract containing the drug can be evaporated to dryness, and the residue

can be redissolved in a smaller volume of a more appropriate solvent. In this

manner, the sample becomes more compatible with a particular analytical

methodology in the measurement step, such as a mobile phase in HPLC

determinations.

The extracted material can be redissolved in small volumes(e.g., 100 to 500 µl

of solvent), thereby extending the sensitivity limits of an assay.

It is possible to extract more than one sample concurrently.

Near quantitative recoveries (90% or better) of most drugs can be obtained

through multiple or continuous extractions.

Partitioning or distribution of a drug between two possible immiscible liquid

phases can be expressed in terms of a partition or distribution coefficient. By knowing

the partition coefficient value for the extracted drug and the absolute volumes of the

two phases to be utilized, the quantity of drug extracted after a single extraction can

be obtained. In multiple extractions methodology, the original biological sample is

extracted several times with fresh volumes of organic solvent until as much drug as


5

possible is obtained. Because the combined extracts now contain the total extracted

drug, it is desirable to calculate the number of extractions necessary to achieve

maximum extraction.

1.2.3 Solid Phase Extraction

Liquid-solid extractions occur between a solid phase and a liquid phase.

Among the solids that have been used successfully in the extraction (usually via

adsorption) of drugs from liquid samples are XAD-2 resin, charcoal, alumina, silica

gel and aluminum silicate. Sometimes the drugs are contained in a solid phase, such

as in lyophilized specimens. Liquid-solid extraction is often particularly suitable for

polar compounds that would otherwise tend to remain in the aqueous phase. The

method could also be useful for amphoteric compounds that cannot be extracted easily

from water.

Factors governing the adsorption and elution of drugs from the resin column

include solvent polarity, flow rate of the solvent through the column, and the degree

of contact the solvent has with the resin beads.

In the adsorption process, the hydrophobic portion of the solute that has little

affinity for the water phase is preferentially adsorbed on the resin surface while the

hydrophilic portion of the solute remains in the aqueous phase. Alteration in the

lipophilic / hydrophilic balance within the solute or solvent mix and not within the

resin affects adsorption of the solute.

Biological samples can be prepared for cleanup by passing the sample through

the resin bed where drug (metabolite) components are adsorbed and finally eluted

with an appropriate solvent.


6

The liquid solid extraction method provides a convenient isolation procedure

for blood samples, thus avoiding solvent extraction, protein precipitation, drug losses,

and emulsion formulation.

1.2.4 Dehydration Methods

An aqueous biological sample is treated with a sufficient quantity of anhydrous

salt (sodium or magnesium sulfate) to create a dried mix. This mix is then extracted

with a suitable organic solvent to remove the desired drug or metabolite.

1.3 Method development and validation

1.3.1 Method development

Method development3,4-16 involves evaluation and optimization of the

various stages of sample preparation, chromatographic separation, detection and

quantification.

Prior to method development of selected drug it is important for extensive

literature survey regarding:

1. Choice of the instrument which is suitable for the analyte such as

Gas Chromatography(GC)

High Pressure Liquid Chromatography (HPLC)

Combined GC and LC Mass Spectrometry (GCMS)

HPLC-MS

LC-MS-MS

Choice of the mass parameters such as parent ion, product ion.


7

Choice of the ionization mode such as positive mode or negative.

Choice of the compound parameters such as DP, FP, CE and CXP.

Choice of the gas parameters such as curtain gas, nebulizer gas,

heater gas and CAD gas

2. Choice of the chromatographic conditions such as

Mobile Phase

Column

Autosampler conditions

Flowrate, injection volume

3. Choice of the internal standard.

4. Choice of extraction method.

5. Choice of regression methods.

The method development and establishment for a analytical method include

determination of selectivity, accuracy, precision, recovery, calibration curve, and

stability of analyte in spiked samples12, 16, 27.

Selectivity

It is the ability of an analytical method to differentiate and quantify the analyte

in the presence of other components in the sample. For selectivity, analyses of blank

samples of the appropriate biological matrix (plasma, urine, or other matrix) should be

obtained from at least six sources. Each blank sample should be tested for

interference, and selectivity should be ensured at the lower limit of quantification

(LLOQ). Potential interfering substances in a biological matrix include endogenous

matrix components, metabolites, decomposition products, and in the actual study,


8

concomitant medication and other exogenous xenobiotics. If the method is intended to

quantify more than one analyte, each analyte should be tested to ensure that there is

no interference.

Accuracy

The accuracy of an analytical method describes the closeness of mean test

results obtained by the method to the true value (concentration) of the analyte.

Accuracy is determined by replicate analysis of samples containing known amounts

of the analyte. Accuracy should be measured using a minimum of five determinations

per concentration. A minimum of three concentrations in the range of expected

concentrations is recommended. The mean value should be within 15% of the actual

value except at LLOQ, where it should not deviate by more than 20%. The deviation

of the mean from the true value serves as the measure of accuracy.

Precision

The precision of an analytical method describes the closeness of individual

measures of an analyte when the procedure is applied repeatedly to multiple aliquots

of a single homogeneous volume of biological matrix. Precision should be measured

using a minimum of five determinations per concentration. A minimum of three

concentrations in the range of expected concentrations is recommended. The precision

determined at each concentration level should not exceed 15% of the coefficient of

variation (CV) except for the LLOQ, where it should not exceed 20% of the CV.

Precision is further subdivided into:


9

i. Within-run or intra-batch precision: This assesses precision during a single

analytical run.

ii. Between-run or inter-batch precision: This measures precision with time, and

may involve different analysts, equipment, reagents, and laboratories.

Recovery

The recovery of an analyte in an assay is the detector response obtained from

an amount of the analyte added to and extracted from the biological matrix, compared

to the detector response obtained for the true concentration of the pure authentic

standard. Recovery pertains to the extraction efficiency of an analytical method within

the limits of variability. Recovery of the analyte need not be 100%, but the extent of

recovery of an analyte and of the internal standard should be consistent, precise, and

reproducible. Recovery experiments should be performed by comparing the analytical

results for extracted samples at three concentrations (low, medium, and high) with

unextracted standards that represent 100% recovery.

Calibration/Standard Curve

A calibration (standard) curve3,4,26-29 is the relationship between instrument

response and known concentrations of the analyte. It should be generated for each

analyte in the sample. A sufficient number of standards should be used to adequately

define the relationship between concentration and response. It should be prepared in the

same biological matrix as the samples in the intended study by spiking the matrix with

known concentrations of the analyte. The number of standards used in constructing a

calibration curve will be a function of the anticipated range of analytical values and the


10

nature of the analyte/response relationship. Concentrations of standards should be

chosen on the basis of the concentration range expected in a particular study.

A calibration curve should consist of

i) A blank sample (matrix sample processed without internal standard)

ii) A zero sample (matrix sample processed with internal standard)

iii) Six to eight non-zero samples covering the expected range, including LLOQ.

Lower Limit of Quantification (LLOQ)

The lowest standard on the calibration curve should be accepted as the limit of

quantification if the following conditions are met:

The analyte response at the LLOQ should be at least 5 times the response

compared to blank response.

Analyte peak (response) should be identifiable, discrete, and reproducible with

a precision of 20% and accuracy of 80-120%.

Calibration Curve/Standard Curve-Concentration-Response

The simplest model that adequately describes the concentration-response

relationship should be used. Selection of weighting and use of a complex regression

equation should be justified. The following conditions should be met in developing a

calibration curve:

20% deviation of the LLOQ from nominal concentration.

15% deviation of standards other than LLOQ from nominal concentration.

At least four out of six non-zero standards should meet the above criteria,

including the LLOQ and the calibration standard at the highest concentration.

The standards when excluded should not change the model used.


11

Stability in a Biological Fluid

Drug stability in a biological fluid3,4,10,14,24-27 is a function of the storage

conditions, the chemical properties of the drug, the matrix, and the container system.

The stability of an analyte in a particular matrix and container system is relevant only

to that matrix and container system and should not be extrapolated to other matrices

and container systems. Stability procedures should evaluate the stability of the

analytes during sample collection and handling, after long-term (frozen at the

intended storage temperature) and short-term (bench top, room temperature) storage,

and after going through freeze and thaw cycles and the analytical process. Conditions

used in stability experiments should reflect situations likely to be encountered during

actual sample handling and analysis. The procedure should also include an evaluation

of analyte stability in stock solution.

All stability determinations should use a set of samples prepared from a

freshly made stock solution of the analyte in the appropriate analyte-free,

interference-free biological matrix. Stock solutions of the analyte for stability

evaluation should be prepared in an appropriate solvent at known concentrations.

Freeze and Thaw Stability

Analyte stability should be determined after three freeze and thaw cycles. At

least three aliquots at each of the low and high concentrations should be stored at the

intended storage temperature for 24 hours and thawed unassisted at room temperature.

When completely thawed, the samples should be refrozen for 12 to 24 hours under the

same conditions. The freeze-thaw cycle should be repeated two more times, and then

analyzed on the third cycle. If an analyte is unstable at the intended storage


12

temperature, the stability sample should be frozen at -70°C during the three freeze and

thaw cycles.

Short-Term Temperature Stability

Three aliquots of each of the low and high concentrations should be thawed at

room temperature and kept at this temperature from 4 to 24 hours (based on the

expected duration that samples will be maintained at room temperature in the intended

study) and analyzed.

Long-Term Stability

The storage time in a long-term stability evaluation should exceed the duration

between the date of first sample collection and the date of last sample analysis.

Long-term stability should be determined by storing at least three aliquots of each of

the low and high concentrations under the same conditions as the study samples. The

volume of samples should be sufficient for analysis on three separate occasions. The

concentrations of all the stability samples should be compared to the mean of

back-calculated values for the standards at the appropriate concentrations from the

first day of long-term stability testing.

Stock Solution Stability

The stability of stock solutions of drug and the internal standard should be

evaluated at room temperature for at least 6 hours. If the stock solutions are

refrigerated or frozen for the relevant period, the stability should be documented.

After completion of the desired storage time, the stability should be tested by

comparing the instrument response with that of freshly prepared solutions.


13

Post-preparative Stability/Autosampler Stability

The stability of processed samples, including the resident time in the auto sampler,

should be determined. The stability of the drug and the internal standard should be

assessed over the anticipated run time for the batch size in validation samples by

determining concentrations on the basis of original calibration standards. Other

statistical approaches based on confidence limits for evaluation of analyte stability in

a biological matrix can be used.

1.3.2 Method Validation

Method Validation3,4,10,12,21-23,28,29 involves documenting, through the use of

specific laboratory investigations, that the performance characteristics of the method

are suitable and reliable for the intended analytical applications. The acceptability of

analytical data corresponds directly to the criteria used to validate the method.

Validation is categorized into full validation, partial validation, and cross- validation.

Full Validation

Full validation is important when developing and implementing a bioanalytical

method for the first time.

Full validation is important for a new drug entity.

A full validation of the revised assay is important if metabolites are added to an

existing assay for quantification.

Partial Validation

Partial validations are modifications of already validated analytical methods.

Partial validation can range from as little as one intra-assay accuracy and precision


14

determination to a nearly full validation. Typical bioanalytical method changes that

fall into this category include, but are not limited to:

Bioanalytical method transfers between laboratories or analysts.

Change in analytical methodology (e.g., change in detection systems).

Change in anticoagulant in harvesting biological fluid.

Change in matrix within species (e.g., human plasma to human urine).

Change in sample processing procedures.

Change in species within matrix (e.g., rat plasma to mouse plasma).

Change in relevant concentration range.

Changes in instruments and/or software platforms.

Limited sample volume (e.g., pediatric study).

Rare matrices.

Selectivity demonstration of an analyte in the presence of concomitant

medications.

Selectivity demonstration of an analyte in the presence of specific metabolites.

Cross -Validation

Cross-validation is a comparison of validation parameters when two or more

analytical methods are used to generate data within the same study or across different

studies. An example of cross-validation would be a situation where an original

validated analytical method serves as the reference and the revised analytical method

is the comparator. The comparisons should be done both ways. When sample analyses

within a single study are conducted at more than one site or more than one laboratory,

cross-validation with spiked matrix standards and subject samples should be


15

conducted at each site or laboratory to establish inter laboratory reliability. Cross-

validation should also be considered when data generated using different analytical

techniques (e.g., LC-MS-MS vs. ELISA) in different studies are included in a

regulatory submission.

Specific Recommendation for analytical Method Validation

The matrix-based standard curve should consist of a minimum of six standard

points, excluding blanks, using single or replicate samples. The standard curve

should cover the entire range of expected concentrations.

Standard curve fitting is determined by applying the simplest model that

adequately describes the concentration-response relationship using appropriate

weighting and statistical tests for goodness of fit.

LLOQ is the lowest concentration of the standard curve that can be measured with

acceptable accuracy and precision. The LLOQ should be established using at least

five samples independent of standards and determining the coefficient of variation

and/or appropriate confidence interval. The LLOQ should serve as the lowest

concentration on the standard curve and should not be confused with the limit of

detection and/or the low QC sample. The highest standard will define the upper

limit of quantification (ULOQ) of an analytical method.

For validation of the analytical method, accuracy and precision should be

determined using a minimum of five determinations per concentration level

(excluding blank samples). The mean value should be within 15% of the

theoretical value, except at LLOQ, where it should not deviate by more than 20%.

The precision around the mean value should not exceed 15% of the CV, except for


16

LLOQ, where it should not exceed 20% of the CV. Other methods of assessing

accuracy and precision that meet these limits may be equally acceptable.

The accuracy and precision with which known concentrations of analyte in

biological matrix can be determined should be demonstrated. This can be

accomplished by analysis of replicate sets of analyte samples of known

concentrations QC samples from an equivalent biological matrix. At a minimum,

three concentrations representing the entire range of the standard curve should be

studied: one within 3x the lower limit of quantification (LLOQ) (low QC sample),

one near the center (middle QC), and one near the upper boundary of the standard

curve (high QC).

Reported method validation data and the determination of accuracy and precision

should include all outliers. However, calculations of accuracy and precision

excluding values that are statistically determined as outliers can also be reported.

The stability of the analyte in biological matrix at intended storage temperatures

should be established. The influence of freeze-thaw cycles (a minimum of three

cycles at two concentrations in triplicate) should be studied.

The stability of the analyte in matrix at ambient temperature should be evaluated

over a time period equal to the typical sample preparation, sample handling, and

analytical run times.

Reinjection reproducibility should be evaluated to determine if an analytical run

could be reanalyzed in the case of instrument failure.

The specificity of the assay methodology should be established using a minimum

of six independent sources of the same matrix. For hyphenated mass


17

spectrometry-based methods, however, testing six independent matrices for

interference may not be important. In the case of LC-MS and LC-MS-MS-based

procedures, matrix effects should be investigated to ensure that precision,

selectivity, and sensitivity will not be compromised. Method selectivity should be

evaluated during method development and throughout method validation and can

continue throughout application of the method to actual study samples.

Acceptance/rejection criteria for spiked, matrix-based calibration standards and

validation QC samples should be based on the nominal (theoretical) concentration

of analytes. Specific criteria can be set up in advance and achieved for accuracy

and precision over the range of the standards, if so desired.

1.3.3 Application of Validated Method to Routine DrugAnalysis

Assay and analysis3,4,13,26-29 of all samples in a biological matrix should be

completed within the time period for which stability data are available. In general,

biological samples can be analyzed with a single determination without duplicate or

replicate analysis if the assay method has acceptable variability as defined by

validation data. This is true for procedures where precision and accuracy variabilities

routinely fall within acceptable tolerance limits. For a difficult procedure with a labile

analyte where high precision and accuracy specifications may be difficult to achieve,

duplicate or even triplicate analyses can be performed for a better estimate of analyte.

The following recommendations should be noted in applying a analytical

method to routine drug analysis:

A matrix-based standard curve should consist of a minimum of six standard

points, excluding blanks (either single or replicate), covering the entire range.


18

Response Function: Typically, the same curve fitting, weighting, and goodness

of fit determined during pre study validation should be used for the standard

curve within the study. Response function is determined by appropriate

statistical tests based on the actual standard points during each run in the

validation. Changes in the response function relationship between pre study

validation and routine run validation indicate potential problems.

The QC samples should be used to accept or reject the run. These QC samples

are matrix spiked with analyte.

System suitability: Based on the analyte and technique, a specific sample

should be identified to ensure optimum operation of the system used.

Any required sample dilutions should use like matrix (e.g., human to human)

obviating the need to incorporate actual within-study dilution matrix QC

samples.

Repeat Analysis: It is important to establish guideline for repeat analysis and

acceptance criteria. This guideline should explain the reasons for repeating

sample analysis. Reasons for repeat analyses could include repeat analysis of

clinical or preclinical samples for regulatory purposes, inconsistent replicate

analysis, samples outside of the assay range, sample processing errors,

equipment failure, poor chromatography, and inconsistent pharmacokinetic

data. Reassays should be done in triplicate if sample volume allows. The

rationale for the repeat analysis and the reporting of the repeat analysis should

be clearly documented.


19

Sample Data Reintegration: A guideline for sample data reintegration should

be established. This guideline should explain the reasons for reintegration and

how the reintegration is to be performed. The rationale for the reintegration

should be clearly described and documented. Original and reintegration data

should be reported.

Acceptance Criteria for the Analytical Run

The following acceptance criteria should be considered for accepting the

analytical run:

Standards and QC samples can be prepared from the same spiking stock

solution, provided the solution stability and accuracy have been verified.

A single source of matrix may also be used, provided selectivity has been

verified.

Standard curve samples, blanks, QCs, and study samples can be arranged as

considered appropriate within the run.

Placement of standards and QC samples within a run should be designed to

detect assay drift over the run.

Matrix-based standard calibration samples:

Seventy-five percent or a minimum of six standards, when back-

calculated (including ULOQ) should fall within 15%, except for LLOQ,

when it should be 20% of the nominal value.

Values falling outside these limits can be discarded, provided they do

not change the established model.

Quality Control Samples:


20

Quality control samples replicated (at least once) at a minimum of three

concentrations (one within 3x of the LLOQ [low QC], one in the midrange

[middle QC], and one approaching the high end of the range [high QC]

should be incorporated into each run. The results of the QC samples

provide the basis of accepting or rejecting the run.

At least 67% (four out of six) of the QC samples should be within 15% of

their respective nominal (theoretical) values.

33% of the QC samples (not all replicates at the same concentration) can

be outside the 15% of the nominal value.

A confidence interval approach yielding comparable accuracy and

precision is an appropriate alternative.

The minimum number of samples (in multiples of three) should be at least 5%

of the number of unknown samples or six total QCs, whichever is greater.

Samples involving multiple analytes should not be rejected based on the data

from one analyte failing the acceptance criteria.

The data from rejected runs need not be documented, but the fact that a run

was rejected and the reason for failure should be recorded.

1.4. Estimation of Drugs in Biological Sample by HPLC-MS

Most of the drugs in biological sample can be analysed byHP LC-MS method

because of several advantages like rapidity, specificity, accuracy, precision, ease of

automation, eliminates tedious extraction and isolation procedures19.Some of the

advantages are:

Speed (analysis can be accomplished in 10 minutes or less)


21

Greater sensitivity (various detectors can be employed)

Improved resolution (wide variety of stationary phases)

Reusable columns (expensive columns but can be used for many samples)

Ideal for the substances of low volatility.

Easy sample recovery, handling and maintenance.

Instrumentation provides itself to automation and quantitation (less time).

Precise and reproducible.

Calculations are done by integrator itself.

Suitable for preparative liquid chromatography on a much large scale.

There are different modes of separation in LC-MS. They are:

o Normal phase mode

o Reverse phase mode

o Reverse phase ion pair chromatography

o Ion-Exchange chromatography

o Affinity chromatography

o Size Exclusion chromatography (gel permeation and gel filtration

chromatography)

1.5. Bioavailability and Bioequivalence studies

1.5.1. Bioavailability Studies

Bioavailability is defined as "the rate and extent to which the active ingredient

or active moiety is absorbed from a drug product and becomes available at the site of

action. For drug products that are not intended to be absorbed into the bloodstream,

bioavailability may be assessed by measurements intended to reflect the rate and


22

extent to which the active ingredient or active moiety becomes available at the site of

action." This definition focuses on the processes by which the active ingredients or

moieties are released from an oral dosage form and move to the site of action20.

Bioavailability studies provide pharmacokinetic information related to the

effects of the drug absorption, distribution and elimination, dose proportionality,

linearity in pharmacokinetics of the active moieties and, inactive moieties.

Systemic exposure patterns reflect both release of the drug substance

from the drug product and a series of possible presystemic/systemic actions on the

drug substance after its release from the drug product20.

The systemic exposure profiles of clinical trial material can be used as a

benchmark for subsequent formulation changes and may thus be useful as a reference

for future bioequivalence studies.

1.5.2. Bioequivalence Studies

Bioequivalence is defined as "the absence of a significant difference in the rate

and extent to which the active ingredient or active moiety in pharmaceutical

equivalents or pharmaceutical alternatives becomes available at the site of drug action

when administered at the same molar dose under similar conditions in an

appropriately designed study20.

Bioequivalence is useful during the IND/NDA period to establish links

between:

Early and late clinical trial formulations.

Formulations used in clinical trial and stability studies, if different.

Clinical trial formulations and to-be-marketed drug product, and


23

Other comparisons, as appropriate.

In each comparison, the new formulation or new method of manufacture is the

test product and the prior formulation or method of manufacture is the reference

product. Where the test product generates plasma levels that are substantially above

those of the reference product, the regulatory concern is not therapeutic failure, but

the adequacy of the safety database from the test product. Where the test product has

levels that are substantially below those of the reference product, the regulatory

concern becomes therapeutic efficacy. When the variability of the test product rises,

the regulatory concern relates to both safety and efficacy, because it may suggest that

the test product does not perform as well as the reference product, and the test product

may be too variable to be clinically useful20.

Pharmacokinetic Studies

The statutory definitions of BA and BE, expressed in terms of rate and extent

of absorption of the active ingredient or moiety to the site of action, emphasize the use

of pharmacokinetic measures in an accessible biological matrix such as blood,

plasma, and/or serum to indicate release of the drug substance from the drug product

into the systemic circulation.

Both direct (e.g., rate constant, rate profile) and indirect (e.g., Cmax, Tmax, mean

absorption time, mean residence time, Cmax normalized to AUC). Parameters on

systemic exposure measures should reflect comparable rate and extent of absorption,

which in turn should achieve the underlying statutory and regulatory objective of

ensuring comparable therapeutic effects. Exposure measures are defined relative to


24

early exposure, peak exposure, and total exposure portions of the plasma, serum, or

blood concentration time profile20-29.

The pharmacokinetic, pharmacodynamic, clinical, and in vitro studies can be

used to measure product quality. BA and BE frequently rely on pharmacokinetic

measures such as AUC and Cmax that are reflective of systemic exposure20-29.

CHAPTER 2

Aim and Objectives of thePresent Research work

25

Chapter 2 Aim & Objectives of the Present Research work

2. Aim and objectives of the Present Research work

Analytical methods employed for the quantitative and qualitative

determination of drugs and their metabolites in Pharmaceutical formulations,

biological samples. The developed must generate reproducible and reliable data in

order to permit valid interpretation of the studies they support. It is essential to

employ well-characterized and fully validated analytical methods to yield reliable

results that can be satisfactorily interpreted. It is recognized that analytical methods

and techniques are constantly undergoing changes and improvements and in many

instances, they are at the cutting edge of the technology. It is also important to

emphasize that each analytical technique has its own characteristics, which will vary

from analyte to analyte.In these instances, specific validation criteria may need to be

developed for each analyte. Moreover, the appropriateness of the technique may also

be influenced by the ultimate objective of the study.

Analytical method validation employed for the quantitative determination of

drugs and their metabolites in biological fluids plays a significant role in the

evaluation and interpretation of bioavailability, bioequivalence, pharmacokinetic and

toxicokinetic study data. These studies generally support regulatory filings. The

quality of these studies is directly related to the quality of the underlying analytical

data. It is therefore important that guiding principles for the validation of these

analytical methods be established and disseminated to the pharmaceutical community.

Our aim is to conduct method development and method validation of the

selected drugs namely Rasagiline, Almotriptan, Valacyclovir and Memantine

Pharmaceutical formulations in human plasma by using high performance liquid

chromatography with mass spectrometry.

26


LIST OF DRUGS SELECTED FOR PRESENT RESEARCH WORK

S.NO Name of theDrug

SubstanceChemical Structure Classification

1.Rasagiline

mesylate

Parkinson's

disease

2.Almotriptan

malateRelief of migraine

3.Valacyclovir

HydrochlorideAnti viral drug

4.Memantine

Hydrochloride

Alzheimer's

disease

27


In method development, extensive literature survey, mass parameter

optimization, chromatographic parameters optimization, extraction methods

optimization, linearity range, regression model selection, sensitivity, recovery,

stability parameters optimization to be carried out.

In method validation, selectivity, specificity, sensitivity, intra-inter assay

precision and accuracy, recovery, stability parameters like short term stability, long

term stability, auto sampler stability, bench top stability, freeze-thaw stability, and

reinjection stability to be proved.

After method development and method validation of selected drugs there is a

need to be prove its application of Pharmaceutical formulations in biological

matrices.

CHAPTER 3

Analytical method development andvalidation of Rasagiline by High performance

Liquid chromatography with massspectrometry

28

Chapter 3 Rasagiline - Introduction

3.1 Introduction

Rasagiline is (1R)-N-prop-2-ynyl-2,3-dihydro-1H-inden-1-amine used as a

monotherapy in early Parkinson's disease or as an adjunct therapy in more advanced

cases. The empirical formula is C12H13N with its molecular weight 171.24.

The recommended initial dose is 0.5 mg administered once daily. If a sufficient

clinical response is not achieved, the dose may be increased to 1 mg administered

once daily. Rasagiline's pharmacokinetics are linear with doses over the range of

1 -10 mg. Its mean steady-state half life is 3 hours but there is no correlation of

pharmacokinetics with its pharmacological effect because of its irreversible inhibition

of MAO-B. Rasagiline is rapidly absorbed, reaching peak plasma concentration (Cmax)

in approximately 1 hour. The absolute bioavailability of rasagiline is about 36%. Food

does not affect the Tmax of rasagiline, although Cmax and exposure (AUC) are

decreased by approximately 60% and 20%, respectively, when the drug is taken with

a high fat meal. The mean volume of distribution at steady-state is 87 L, indicating

29


that the tissue binding of rasagiline is in excess of plasma protein binding. Plasma

protein binding ranges from 88-94% with mean extent of binding of 61-63% to

human albumin over the concentration range of 1-100 ng/mL. Rasagiline undergoes

almost complete biotransformation in the liver prior to excretion. The metabolism of

rasagiline proceeds through two main pathways: N-dealkylation and/or hydroxylation

to yield 1-aminoindan (AI), 3-hydroxy-N-propargyl-1 aminoindan (3-OH-PAI) and

3-hydroxy-1-aminoindan (3-OH-AI). In vitro experiments indicate that both routes of

rasagiline metabolism are dependent on the cytochrome P450 (CYP) system, with

CYP1A2 being the major isoenzyme involved in Rasagiline metabolism. Glucuronide

conjugation of rasagiline and its metabolites, with subsequent urinary excretion, is the

major elimination pathway. Half life of the Rasagiline is about 38-45 minutes.30-33

Literature survey reveals that only a few methods were reported to

quantification of rasagiline in human plasma and pharmaceutical analysis 34-38. These

include HPLC 34,35 crystallographic analysis 36, LC-MS/MS 37,38. Only two methods

were reported for quantification of rasagiline in human plasma with LC-MS/MS 37, 38.

They developed the method with long run time for analysis with large amount of

plasma sample. Min song et.al 37 reported the method both in human plasma and urine

at a concentration range 0.01-40ng/mL and 0.025 to 40ng/mL respectively. They used

Papavarin as internal standard to compare the drug. They developed LLE method at

5.5 min runtime for each sample. They have done pharmacokinetic study with 30

human volunteers. The main drawback from this method is longer runtime and also

suitable internal standard usage. The draw backs are overcome by Jinfei Ma et.al38

with shorter runtimes at 3.5 minutes for each sample at a concentration range

30


0.02 - 50 ng/mL. They used Pseudoephedrine as an internal standard. They have done

pharmacokinetic study with 12 human volunteers. The main drawbacks are Sensitivity

is not achieved when compared with Min song et.al37 and not used similar internal

standard like deuterated or analogs of Rasagiline.

The purpose of the present investigation is to explore rapid run analysis time,

(3 min) more sensitive method (5pg/mL). More over with small amount of plasma

sample (100µL Plasma) utilization during sample processing, simple extraction and

analyte comparision with isotope labeled internal standarad (Rasagiline-13C3

mesylate). This method can also be employed in Pharmacokinetic and

Bio-equivalence study of Rasagiline.

31

Chapter 3 Rasagiline - Experimental

3.2. Experimental Investigations

3.2.1 Materials and reagents

Rasagiline and Rasagiline- 13C3 mesylate were obtained by TLC Pharma chem,

Canada. (Figure 3.1). LC grade methanol, Methyl t-butyl ether and Dichloromethane

were purchased from J.T. Baker Inc. (Phillipsburg, NJ, USA). Analytical Reagent

grade formic acid and Na2CO3 were procured from Merck (Mumbai, India). Human

plasma (K2EDTA) was obtained from Doctors pathological Lab, Hyderabad. The

AZILECT® tablets, containing 1 mg Rasagiline per tablet, were obtained from Teva

Pharma (USA). Ultra pure water from Milli-Q system (Millipore, Bedford, MA,

USA) was used through the study. All other chemicals in this study were of analytical

grade.

3.2.2 Instrumentation and equipment

An API 4000 LC-MS/MS system, 1200 Series HPLC system (Agilent

Technologies, Waldbronn, Germany), and Applied Biosystems Analyst® Software

version 1.4.1 were used for the determination of Rasagiline in human plasma.

Micro balance (ME5 model Sartorius), variable range micro pipette

(Eppendorf), Autosampler vials ,variable size glass bottles, graduated cylinders,

volumetric flasks (Borosil), ultrasonic bath (Pharmatek Scientifics), Vortexer

(Spinix), deep freezer (-30±5°C) and (-80±15°C) (Sanyo), nitrogen evaporator (Turbo

Vap LV Caliper Life Sciences), refrigerator (LG), pipette tips 10µl-1000µl, Ria vial,

12×7.5mm (Tarson) polypropylene tubes, combitips (Eppendorff) and variable size

surgical gloves (Surgicare).

32


3.2.3 Preparation of Reagents and Solvents

The volumes and concentrations mentioned in the process are theoretical. The

volumes and concentrations could be corrected on the actual weights used.

Table 3.1 Preparation of Reagents and Solvents

Reagents and solvents preparation

50% Methanol Mix 500 mL of methanol with 500 mL of water.

0.1% Formic Acid Mix 100 L of Formic Acid with 100 mL of water

1 M Sodium carbonate Dissolve 26.5 g of anhydrous sodium carbonate into 1L of water.

Mobile phase

Mix 200 mL of Methanol with 800 mL 0.1% Formic Acid.

Filter through 0.45 m filter

Extraction SolventMix 750 L of Methyl tertiary butyl ether with 250 mL of Dichloromethane

Reconstitution solution Mix 200 mL of Methanol with 800 mL 0.1% Formic Acid.

(Autosampler wash)80% Methanol

Mix 800 mL of Methanol with 200 mL of water.

3.2.4 Preparation of Stock solutions

Table 3.2 Preparation of Stock solutions

Name of the solution Concentration Volume (mL) Diluent

Rasagiline stock solution 50.0 µg/mL 50 mL Methanol

Rasagiline-13C3 mesylate stock solution 50.0 µg/mL 50 mL Methanol

33


3.2.5 Preparation of standards and quality control (QC) samples

Standard stock solutions of Rasagiline (50.0µg/ mL) and Rasagiline-13C3

mesylate (50.0µg/ mL) were prepared in methanol. The IS spiking solutions

(30.0 ng /mL) were prepared in 50% methanol from Rasagiline-13C3 mesylate

standard stock solution. Standard stock solutions and IS spiking solutions were stored

in refrigerator conditions (2-8 °C) until analysis. Standard stock solution Rasagiline

was added to drug free human plasma to obtain Rasagiline concentration levels of 5.0,

10.0, 100.0, 600.0, 1200.0, 2400.0, 4800.0, 7200.0, 9600.0 and 12000 pg/mL for

analytical standards and 5.0, 15.0, 4500.0 and 900.0 pg/mL (LLOQ, LQC, MQC,

HQC) for quality control standards and stored in the freezer below -30°C until

analysis. The aqueous standards were prepared in reconstitution solution

(0.1% Formic Acid: Methanol (80:20 v/v) and stored in the refrigerator (2-8°C) for

validation experiments until analysis.

34

Chapter 3 Rasagiline – Method Development

3.3 Method Development

The goal of this research is to develop and validate a simple, selective,

sensitive, rapid, rugged and reproducible assay method for the quantitative

determination of Rasagiline from plasma samples. In the way to develop a simple and

easy applicable method for Rasagiline assay in human plasma for pharmacokinetic

study, HPLC with MS detection was selected as the method of choice.

Mass parameter Optimization,Chromatographic optimization and Extraction

optimization to be optimized carefully to achieve the best results.

The mass parameter optimization was performed by direct infusion of

solutions of both Rasagiline and Rasagiline -13C3 mesylate into the ESI source of the

mass spectrometer. Other parameters, such as the nebulizer and the heater gases and

Declustering potential(DP), Entrance potential(EP),Collision energy(CE) was

optimized to obtain a better spray shape, resulting in better ionization and droplet

drying to form the protonated ionic Rasagiline and Rasagiline-13C3 mesylate

molecules. A CAD product ion spectrum for Rasagiline and Rasagiline-13C3 mesylate

yielded high-abundance fragment ions of m/z(amu) 117.1 and m/z(amu) 117.2

respectively (Figure 3.2 - Figure 3.5) from its parent ion mass spectra.

35


Figure 3.2 Parent ion mass spectra (Q1) of Rasagiline

36


Figure 3.3 Product ion mass spectra (Q3) of Rasagiline

37


Figure 3.4 Parent ion mass spectra (Q1) of Rasagiline -13C3 mesylate

38


Figure 3.5 Product ion mass spectra (Q3) of Rasagiline- 13C3 mesylate

Initially, a mobile phase consisting of ammonium acetate and acetonitrile in varying

combinations was tried, but a low response was observed. The mobile phase

containing acetic acid: acetonitrile (20:80 v/v) and acetic acid: methanol (20:80 v/v)

gives the better response, but poor peak shape was observed. A mobile phase of 0.1%

formic acid in water in combination with methanol and acetonitrile with varying

combinations were tried. Using a mobile phase containing 0.1% formic acid in water

39


in combination with methanol (20:80 v/v), the best signal along with a marked

improvement in the peak shape was observed for Rasagiline and Rasagiline- 13C3

mesylate.Short length columns, such as Symmetry Shield RP18 (50mm x 2.1 mm, 3.5

μm), Inertsil ODS-2V (50mm x 4.6 mm,5μm), Hypurity C18 (50mm x 4.6 mm, 5 μm)

and Hypurity Advance (50 mm x 4.0 mm, 5 μm), YMC basic (50 mm x2 mm, 5μm),

Zorbax Eclipse Plus C18, (2.1mm x 50 mm, 3.5 m) were tried during the method

development. The best signal and good peak shape was obtained using the Zorbax

Eclipse Plus C18, 2.1 x 50 mm, 3.5 m, column. It gave satisfactory peak shapes for

both Rasagiline and Rasagiline- 13C3 mesylate.Flow rate of 0.3mL/min without splitter

was used and reduced the run time to 3.0 min. Both Drug and IS were eluted with

shorter time at 2.0 min. For an LC-MS/MS analysis, utilization of stable isotope-

labeled or suitable analog drugs as an internal standard proves helpful when a

significant matrix effect is possible. In our case, Rasagiline- 13C3 mesylate was found

to be best for the present purpose. The column oven temperature was kept at a

constant temperature of about 45°C. Injection volume of 10µL sample is adjusted for

better ionization and chromatography.

Prior to load the sample for LC injection, the co-extracted proteins should be removed

from the prepared solution. For this purpose, initially we tested with different

extraction procedures like PPT (Protein Precipitation),LLE (Liquid Liquid extraction),

and SPE (Solid Phase extraction). We found ion suppression effect in protein

precipitation method for drug and internal standard. Further, we tried with SPE and

LLE. Out of all, we observed LLE is suitable for extraction of drug and IS. We tried

40


with several organic solvents (ethyl acetate, chloroform, n-hexane, dichloro methane

and methyl tertiary butyl ether) individually as well with combinations in LLE to

extract analyte from the plasma sample. In our case methyl tertiary butyl ether:

dichloromethane (75:25) combination served as good extraction solvent. Autosampler

wash is optimized as 80% methanol. Several compounds were investigated to find a

suitable IS, and finally Rasagiline-13C3 mesylate found the most appropriate internal

standard for the present purpose. There was no significant effect of IS on analyte

recovery, sensitivity or ion suppression. High recovery and selectivity was observed

in the Liquid-Liquid extraction method. These optimized detection parameters,

chromatographic conditions and extraction procedure resulted in reduced analysis

time with accurate and precise detection of Rasagiline in human plasma.

Chromatographic conditions

Zorbax Eclipse Plus C18, 2.1 x 50 mm, 3.5 m, was selected as the analytical

column. Column temperature was set at 45°C. Mobile phase composition was 0.1%

formic acid: methanol (80:20 v/v). Source flow rate 300 µL/min without split.

Injection volume of 10 µL. Rasagiline and Rasagiline-13C3 mesylate were eluted

at 1.2 ± 0.2 min, with a total run time of 3.0 min for each sample.

Sample preparation

Liquid-Liquid extraction procedure was used for isolation of Rasagiline from

the plasma samples. For this purpose, 50µL of Rasagiline- 13C3 mesylate (IS)

concentration of 10ng/mL) 100 µL plasma (respective concentration of plasma

sample) was added into ria vials then vortexed approximately. Followed by 200 µl of

41


1M Na2CO3 solution, 3mL of Extraction solvent (MTBE: DCM (3:1,v/v) was added

to each tube and vortexed for 10 minutes. After that, the samples were centrifuged at

4000 rpm for approximately 10 minutes at 20°C temperature and transfer the

supernatant into respective ria vials. These samples were allowed to evaporate until

dryness under nitrogen stream at 25°C. Finally, the residue was reconstituted with 200

L of reconstitution solution (MeOH:0.1% formic acid (1:4). Further samples were

centrifuged at 4000 rpm for approximately 2 minutes and at 20°C and supernatant

were transferred into auto sampler vials with caps and 10 µL of sample was injected

onto the LC-MS/MS system.

Calibration curve parameters and regression model

The analytical curves of Rasagiline were constructed in the concentrations

ranging from 5.0 -12000.0 pg/mL in human plasma. Calibration curves were obtained

by weighted linear regression (weighing factor: 1/x2). The ratio of Rasagiline peak

area to Rasagiline- 13C3 mesylate peak area was plotted against the ratio of

Rasagiline concentration in ng/mL. The fitness of calibration curve was confirmed by

back-calculating the concentrations of calibration standards.

Method Development Conclusion

The developed method is suitable for estimation of plasma concentrations of

Rasagiline as a single analytical run, in clinical samples from Bioequivalence and

Pharmacokinetic studies. This was followed by method validation.

42

Chapter 3 Rasagiline – Method Validation

3.4 Method Validation

The objective of the work is to validate specific HPLC-MS method for the

determination of Rasagiline in human plasma for bioavailability and pharmacokinetic

study.

Chromatography

Representative chromatograms of Plasma blank, blank +IS, LOQ, ULOQ,

LLOQ, LQC, MQC, HQC samples, Calibration curve are represented in

Figure 3.6 to 3.14.

Figure 3.6 MRM Chromatogram of Blank Human Plasma Sample

43


Figure 3.7 Chromatogram of Blank + IS

Figure 3.8 Chromatogram of LOQ Sample (Rasagiline & IS)

44


Figure 3.9 Chromatogram of ULOQ Sample (Rasagiline & IS)

Figure 3.10 Chromatogram of LLOQ Sample (Rasagiline & IS)

45


Figure 3.11 Chromatogram of LQC Sample (Rasagiline & IS)

Figure 3.12 Chromatogram of MQC Sample (Rasagiline & IS)

46


Figure 3.13 Chromatogram of HQC Sample (Rasagiline & IS)

Figure 3.14 Calibration Curve of Rasagiline

47


Blank Matrix Screening

During validation, blank plasma samples from 10 different lots were processed

according to the extraction procedure and evaluate the interference at the retention

times of analyte and internal standard. The 6 free interference lots were selected from

the 10 lots. Results are presented in Table 3.3.

Table 3.3 Screening of Different batches of blank matrix(Human K2EDTA Plasma) for interference free Rasagiline blank plasma

Matrix identification

Blank plasma Area at

Analyte(Rasagiline) RT

Internalstandard RT

22-10342 A 0 0

22-10343 A 0 022-10344 A 0 022-10345 A 0 022-10346 A 14 022-10347 A 0 022-10348 A 0 022-10349 A 23 8922-10350 A 0 1222-10351 A 0 0

Blank+IS with PL Blank(Blank Plasma Lot-1) 27 32264

LOQ with PL Blank (Blank Plasma Lot-1) 1345 31792

Blank Matrix Specificity and Limit of Quantification

During specificity run, prepare the LLOQ standard in one of the screened

blank plasma including the spiking of working range of internal standard. Blank

plasma samples from 10 different lots, 6 LLOQ standards were processed according

to the extraction procedure. The responses for the blank plasma from 10 different lots

48


were compared to the LLOQ standard of the analyte and internal standard. No

significant response (≤ 20% for the analyte response and ≤ 5% of the internal standard

response.) was observed at the retention times of the analyte or the internal standard

in blank plasma as compared to the LLOQ standard. Results are presented in

Table 3.5

The specificity experiment shall be considered for calculation of LOQ

Experiment. Results are presented in Table 3.4

Table 3.4 Specificity of Different batches of blank matrix

(Human K2EDTA Plasma) for Rasagiline

Matrix IdentificationLLOQArea

Internalstandard(IS) Area

Interferencewith Analyte

(% ofLLOQ

Response)

Interferencewith IS

(% of ISResponse)

22-10342 A 0 12 0 0.003

22-10343 A 0 0 0 0

22-10344 A 32 0 0.81 0

22-10345 A 0 0 0 0

22-10346 A 0 0 0 0

22-10347 A 0 0 0 0

Acceptance criteria:

1. Analyte response should be ≤ 20% of LOQ Response in at least 75% of the blank.

2. Internal standard response should be ≤ 5% of mean internal standard response in

at least 75% of the blank.

49


Table 3.5 Limit of Quantitation for analyte (Rasagiline)

Matrix identificationBlank plasma

area atAnalyte RT

LLOQresponse

LLOQ S/NRATIO

AP/22-10342 A

0 3481 14.00 3858 12.60 3923 12.70 3935 12.60 3965 14.90 4864 18.4

N 6 6 6Mean 4004.33 14.20SD 457.67 2.26

CV% 11.43 15.92

LLOQ was spiked in 22-10342 A


1. Mean S/N ratio of LLOQ should be ≥ 5.

2. S/N ratio is Analyst software generated data.

Intra Batch Accuracy and precision

Intra batch accuracy and precision evaluation were assessed by analyzing 1

calibration curve and 6 replicate each of the LLOQ, LQC, MQC, HQC, from

precision and accuracy batch-1.

The Intra batch percentage of nominal concentrations for Rasagiline was

ranged between 98.0% and 101.4%.

The Intra batch percentage of coefficient of variation is 1.1% to 4.6% for

Rasagiline.

Results are presented in Table 3.6

50


Table 3.6 Intra batch (Within-Batch) Accuracy and Precision for determinationof Rasagiline levels in human plasma

AnalyticalRun ID

LLOQ 5.00pg/mL

Low QC 15.00pg/mL

Mid QC 4500.00pg/mL

High QC 9000.00pg/mL

Conc.Found

%Nominal

Conc.Found

%Nominal

Conc.Found

%Nominal

Conc.Found

%Nominal

P&ABatch 1

5.10 102 14.9 99.3 4450.60 98.9 9012.50 100.14.80 96 15.2 101.3 4612.40 102.5 8976.60 99.74.70 94 14.8 98.7 4521.30 100.5 8956.80 99.54.80 96 14.7 98.0 4531.20 100.7 9876.40 109.75.20 104 14.6 97.3 4456.50 99.0 8897.50 98.94.80 96 15.3 102.0 4412.70 98.1 9056.70 100.6

N 6 6 6 6Mean 4.90 14.90 4497.50 9129.40SD (±) 0.10 0.68 61.20 98.90CV (%) 2.70 4.60 1.40 1.10%Nominal 98.00 99.40 99.90 101.40Acceptance criteria:

1. % CV ≤ 15 % except LLOQ for which it is ≤ 20%.

2. Mean % Nominal (100 ±15% & for LLOQ 100±20%).

Inter Batch Accuracy and Precision

Inter batch accuracy and precision evaluation were assessed by analyzing 5

sets of calibration curves for Rasagiline and 5 sets of QC samples, 6 replicates each of

the LLOQ, LQC, MQC and HQC.

The inter batch percentage of nominal concentrations for Rasagiline was


The Inter batch percentage of coefficient of variation is 1.00% to 3.80% for

Rasagiline.


51


Table 3.7 Inter batch (Between-Bach) Accuracy and Precision for determination

of Rasagiline levels in human plasma

AnalyticalRun ID

LLOQ 5.00pg/mL

Low QC 15.00pg/mL

Mid QC 4500.00pg/mL

High QC 9000.00pg/mL

Conc.Found

%Nominal

Conc.Found

%Nominal

Conc.Found

%Nominal

Conc.Found

%Nominal

P&A Batch 1

5.10 102 14.90 99.3 4450.60 98.9 9012.50 100.14.80 96 15.20 101.3 4612.40 102.5 8976.60 99.74.70 94 14.80 98.7 4521.30 100.5 8956.80 99.54.80 96 14.70 98.0 4531.20 100.7 9876.40 109.75.20 104 14.60 97.3 4456.50 99.0 8897.50 98.94.80 96 15.30 102.0 4412.70 98.1 9056.70 100.6

P&A Batch 2

4.90 98 15.10 100.7 4220.60 93.8 9123.10 101.45.20 104 14.50 96.7 4572.40 101.6 8879.40 98.74.90 98 14.20 94.7 4545.20 101.0 8959.30 99.55.30 106 14.80 98.7 4678.50 104.0 9132.40 101.54.60 92 15.20 101.3 4478.20 99.5 8997.50 100.04.90 98 14.70 98.0 4476.20 99.5 9032.30 100.4

P&A Batch 3

4.90 98.0 14.40 96.0 4390.10 97.6 9117.30 101.35.10 102.0 15.50 103.3 4662.20 103.6 8876.10 98.64.70 94.0 14.60 97.3 4491.20 99.8 8976.10 99.74.80 96.0 14.30 95.3 4501.10 100.0 9143.20 101.64.90 98.0 14.10 94.0 4489.10 99.8 8997.20 100.05.10 102.0 14.90 99.3 4466.10 99.2 9156.40 101.7

P&A Batch 4

4.90 98.0 13.80 92.0 4467.20 99.3 9121.10 101.34.60 92.0 14.60 97.3 4665.10 103.7 8898.10 98.94.30 86.0 14.60 97.3 4489.20 99.8 8944.10 99.44.20 84.0 14.80 98.7 4509.10 100.2 9256.10 102.84.50 90.0 15.20 101.3 4467.10 99.3 8877.10 98.65.40 108.0 14.70 98.0 4498.10 100.0 9126.10 101.4

P&A Batch 5

4.70 94.0 14.20 94.7 4350.10 96.7 9112.10 101.24.60 92.0 15.00 100.0 4652.60 103.4 8871.40 98.64.80 96.0 14.60 97.3 4531.10 100.7 8929.50 99.25.10 102.0 14.10 94.0 4631.30 102.9 9776.80 108.64.80 96.0 14.30 95.3 4446.70 98.8 8997.40 100.04.90 98.0 14.10 94.0 4467.80 99.3 9159.80 101.8

N 30 29 30 30Mean 4.90 14.60 4504.40 9074.50SD (±) 0.19 0.56 59.40 93.30CV (%) 3.80 3.80 1.30 1.00

%Nominal 98.00 97.30 100.10 100.80

Acceptance criteria: Same as presented inTable 3.6

52


Calibration Curve

Calibration curves are found to be consistently accurate and precise for

Rasagiline over 5.0 to 12000.0 pg/mL for calibration range. The correlation

coefficient is greater than or equal to 0.9980 for Rasagiline. Back calculations were

made from the calibration curves to determine Rasagiline concentrations of each

calibration standard.

Results are presented in Tables 3.8 & 3.9.

Table 3.8 Summary of calibration curve parameters for Rasagiline in humanPlasma

Analytical Run

IDslope intercept r-squared

P&A Batch-1 0.1142 0.0002108 0.9995

P&A Batch-2 0.1089 0.0001224 0.9981

P&A Batch-3 0.1107 0.0003502 0.9988

P&A Batch-4 0.1120 0.001779 0.9994

P&A Batch-5 0.1154 -0.0006144 0.9997

N 5 5 5

Mean 0.1122 0.0003696 0.9991

SD(±) 0.002618 0.0008720 0.0007

% CV 2.3 235.9 0.1


1. Coefficient of regression (r) ≥ 0.9980.

53


Table 3.9 Back-calculated standard concentrations from each calibration curvefor Rasagiline in human plasma.

AnalyticalRun ID

Nominal Concentration (ng/mL)CS1 CS2 CS3 CS4 CS55.00

pg/mL10.00

pg/mL100.00pg/mL

600.00pg/mL

1200.00pg/mL

P&A Batch-1 4.90 9.90 98.30 597.40 1204.80P&A Batch-2 5.00 9.66 96.60 579.60 1189.20P&A Batch-3 4.90 9.03 90.30 541.80 1183.60P&A Batch-4 5.10 9.82 98.20 569.20 1158.40P&A Batch-5 4.90 9.45 94.50 567.00 1184.00

N 5 5 5 5 5Mean 4.96 9.57 95.58 571.00 1184.00SD(±) 0.10 0.30 3.30 20.30 16.70% CV 1.80 3.64 3.48 3.55 1.41

%Nominal 99.20 95.70 95.60 95.20 98.70

Analyticalrun ID

Nominal Concentration(ng/mL)CS6 CS7 CS8 CS9 CS10

2400.00pg/mL

4800.00pg/mL

7200.00pg/mL

9600.00pg/mL

12000.00pg/mL


N 5 5 5 5 5Mean 2408.00 4676.00 6904.00 9032.00 11893.40SD(±) 63.00 152.70 254.80 210.90 139.20% CV 2.620 3.27 3.69 2.33 1.17

% Nominal 100.30 97.40 95.90 94.10 99.10


1. Mean %Nominal (100±15%) except lowest calibration standard.

2. Mean %Nominal (100±20%) for lowest calibration standard (CS1).

3. % CV≤ 15% except lowest calibration standard (CS1) for which it is ≤ 20%.

54


Recovery

The percentage recovery of Rasagiline was determined by comparing the

mean peak area of Rasagiline in extracted LQC, MQC, HQC samples with freshly

prepared unextracted LQC, MQC, HQC samples respectively.

The mean % recovery for LQC, MQC, HQC samples of Rasagiline were

96.5%, 97.3% and 97.0% respectively.

The mean recovery of Rasagiline across QC levels is 96.9%.

The mean recovery of % CV recovery of Rasagiline across QC levels is 0.4%.

For the internal standard, mean peak area of 18 extracted samples was

compared to the mean peak area of 18 unextracted IS solution. The mean %

recovery is 96.7%.

The % CV recovery of IS Rasagiline- 13C3 mesylate for extracted is 3.40%.


55


Table 3.10 Recovery of Analyte Rasagiline and Rasagiline- 13C3mesylate from humanplasma

Standard UnextractedRasagiline

peak response

ExtractedRasagiline

peakresponse

UnextractedRasagiline

13C3mesylatepeak response

ExtractedRasagiline

13C3 mesylatepeak

response

Low QC:15.0 pg/ml

765 720 47921 46993758 720 47927 41485787 721 47934 45674742 743 46921 46876765 743 46856 46981732 741 47678 46323

N 6 6Mean 758.2 731.3SD(±) 19.3 12.1% CV 2.6 1.7

% Recovery 96.5

MediumQC:

4500.0pg/ml

282875 276543 46999 46321272657 265686 47078 42456262543 267656 47221 46567302653 270675 48437 46486282789 279978 47890 46098282321 279876 48967 46798

N 6 6Mean 280973.0 273402.3SD(±) 13323.5 6245.2% CV 4.7 2.3

% Recovery 97.3

High QC:9000.0pg/ml

508665 487654 47543 46769508654 497657 47231 46786488894 489876 47543 44643508679 489876 46789 46935499876 488312 47156 45679507658 478212 46984 46568

N 6 6Mean 503737.7 488597.8SD(±) 8043.4 6231.8% CV 1.6 1.3

% Recovery 97.0Meanrecovery

96.90 96.70

Mean % CV 0.40 3.40

56



1. The coefficient of variation for mean recovery across LQC, MQC and HQC

shall not exceed 25 %.

2. The coefficient of variation for mean recovery of IS shall not exceed 25%.

Matrix Effect

Samples were prepared at LQC & HQC level in triplicate in each of 6 different

lots of human plasma. A calibration curve and 6 replicates of LQC & HQC samples in

triplicate for each matrix were freshly prepared and analyzed in single run.

Percentage bias was calculated for each matrix.

No significant matrix effect found in different sources of human plasma tested

for Rasagiline, Rasagiline- 13C3 mesylate.

Results are presented in Tables 3.11 and 3.12.

57


Table 3.11 Assessment of Matrix Effect on determination of Rasagiline at LQClevels in human plasma

Identification ofmatrix

Drug responsein Matrix atLQC Level

Internalstandardresponse

Matrix factor

22-10342 A 5081 473287 0.01033822-10342 A 5214 542008 0.00926422-10342 A 5355 534628 0.00964622-10343A 5300 472358 0.01080522-10343 A 5231 453924 0.01109822-10343 A 5367 465418 0.01110522-10344 A 5364 480406 0.01075222-10344 A 5425 480328 0.01087622-10344 A 5292 470720 0.01082622-10345 A 4599 465392 0.00951622-10345 A 4525 454854 0.0095822-10345 A 4604 397888 0.01114322-10346 A 6417 382925 0.01613822-10346 A 5653 440819 0.01234922-10346 A 6565 467155 0.01353322-10347 A 5342 479270 0.01073422-10347 A 5431 431460 0.01212222-10347A 5248 465182 0.010864

N 18 18 18Grand Mean 0.011149

SD(±) 0.0016% CV 14.63


1. Mean % Nominal 100 ±15% of nominal value.

2. % CV ≤ 15%.

58


Table 3.12 Assessment of Matrix Effect on determination of Rasagiline atHQC levels in human plasma

Identification of matrix Drug responsein Matrix atHQC Level


Matrix factor

22-10342 A 3048670 410486 7.15217922-10342 A 3128522 442261 6.81219222-10342 A 3213086 445791 6.94092522-10343A 3179976 442288 6.92380722-10343 A 3138658 440469 6.86206722-10343 A 3220102 454488 6.82297122-10344 A 3218510 448930 6.90402822-10344 A 3254700 452154 6.93187722-10344 A 3174996 437936 6.98166222-10345 A 2759532 397455 6.68611422-10345 A 2715030 393713 6.64081222-10345 A 2762552 396455 6.71031422-10346 A 3850490 540626 6.85875622-10346 A 3391652 528000 6.18591122-10346 A 3939020 516830 7.33950522-10347 A 3205338 447335 6.90028822-10347 A 3258824 446898 7.0222922-10347A 3148910 411973 7.360677

N 18 18 18Grand Mean 6.89091

SD(±) 0.26% CV 3.80



2. % CV ≤ 15%.

59


Dilution Integrity

Dilution integrity experiment was carried out at six replicate of two times

diluted (1 in 2 dilution) and four times diluted of approx 1.5 × ULOQ (1 in 4 dilution)

samples were prepared and concentrations were calculated including the dilution

factor against the freshly prepared calibration curve.

The % accuracy of Rasagiline nominal concentrations ranged between 96.11%

to 101.11% and 97.78% to 101.11% for 1 in 2 dilutions and 1 in 4 dilutions

respectively.

The % CV is 1.64% to 1.32% for 1 in 2 dilutions and 1 in 4 dilutions

respectively.


Table 3.13 Assessment of Dilution integrity for Rasagiline at DQC Concentration(pg/mL)

DQCDilution factor: ½

Nominal conc.: 18000.00 pg/mL

DQCDilution factor: ¼

Nominal conc.: 18000.00 pg/mLConc. Found % Nominal Conc. Found %Nominal17800.00 98.89 18100.00 100.5617800.00 98.89 18200.00 101.1118200.00 101.11 18000.00 100.0017300.00 96.11 17800.00 98.8917900.00 99.44 17600.00 97.7817900.00 99.44 17700.00 98.33

N 6 6Mean 17816.67 17900.00SD(±) 292.69 236.64% CV 1.64 1.32

Acceptance criteria

1. % CV ≤ 15%.2. Mean % Nominal (100 ±15%).

60


Whole Batch Reinjection Reproducibility

To evaluate the whole batch reinjection reproducibility experiment, samples of

P and A batch-2 were kept on bench at room temperature for approx 26hr after the

initial analysis and were re-injected again after approx 26 hr. Concentrations were

calculated to determine precision and accuracy after reinjection.

% Accuracy of Rasagiline LQC, HQC samples in reinjection was 101.63%

and 94.98%.

The Precision (%CV) of Rasagiline QC samples in reinjection was between

1.97 % and 12.7%.

Rasagiline was found to be stable at room temperature post extraction (in

reconstitution solution) for approx 26 hrs and reproducible after reinjection.

Results are presented in tables 3.14

Table 3.14 Assessment of Whole Batch Re-injection Reproducibility duringestimation of Rasagiline in human plasma

AnalyticalRun ID

Low QC 1.5 pg/mL High QC 9000.0 pg/mLcomp

sampleRe-inj sample comp

sampleRe-inj sample

13.69 13.79 8573.46 8342.6815.21 13.78 8725.71 8076.7515.09 14.23 8716.07 8683.6114.54 18.70 8694.75 8281.7114.41 14.10 9070.18 8292.6415.48 15.30 8874.75 8333.36

N 6 6 6 6Mean 14.74 14.98 8775.82 8335.13SD(±) 0.65 1.91 173.23 196.43% CV 4.44 12.72 1.97 2.36

%Accuracy 101.63 94.98Acceptance criteria:

1. % CV≤ 15% Except LLOQ for which it is ≤ 20%.2. Mean % Nominal (100±15% & for LLOQ 100±20%).3. 67% of the re-injected QCs at each level shall be within ± 20% of their previous

concentration.

61


Ruggedness with Different Analyst

To evaluate ruggedness experiment with different analysts, one P&A batch

(P&A-3) was processed by different analyst. The run consisted of a calibration curve

standards and 6 replicates of each LLOQ, LQC, MQC, HQC samples.

The Accuracy of Rasagiline QC samples within the range of 98.10% to

100.34%.

The Precision of Rasagiline QC samples within the range of 1.24% to 4.08%.

These results indicated that the method is rugged and reproducible by different

analyst.

Results are presented in Table 3.15.

Table 3.15 Ruggedness of the method for estimation of Rasagiline Plasma levelsin human plasma with different Analyst

AnalyticalRun ID

LLOQ 5.00pg/mL Low QC 15.00 pg/mLMid QC 4500.00

pg/mLHigh QC 9000.00

pg/mLAnalyst

ID 1Analyst

ID 2Analyst

ID 1Analyst

ID 2Analyst

ID 1Analyst

ID 2Analyst

ID 1Analyst

ID 2

P&A Batch1 Analyst A3 Analyst B

5.10 4.90 14.90 14.40 4450.60 4390.10 9012.50 9117.304.80 5.10 15.20 15.50 4612.40 4662.20 8976.60 8876.104.70 4.70 14.80 14.60 4521.30 4491.20 8956.80 8976.104.80 4.80 14.70 14.30 4531.20 4501.10 9876.40 9143.205.20 4.90 14.60 14.10 4456.50 4489.10 8897.50 8997.204.80 5.10 15.30 14.90 4412.70 4466.10 9056.70 9156.40

N 6 6 6 6 6 6 6 6

Mean 4.90 4.92 14.92 14.63 4497.45 4499.97 9129.42 9044.38

SD(±) 0.20 0.16 0.28 0.50 72.08 89.14 369.83 112.10

CV (%) 4.08 3.26 1.87 3.45 1.60 1.98 4.05 1.24

%Accuracy 100.34 98.10 100.06 99.07



2. Mean % Nominal (100±15% & for LLOQ 100 ±20%)

62


Ruggedness with different column

To evaluate ruggedness experiment with different column, samples of P&A

batch-5 were re-injected on different columns with same and specifications,

Concentrations were calculated to determine precision and accuracy.

The Accuracy of Rasagiline QC samples within the range of 96.42% to 100.35%.

The Precision of Rasagiline QC samples within the range of 1.87% to 4.08%.


analyst.

Results are presented in tables 3.16.

Table 3.16 Ruggedness of the method for estimation of Rasagiline in humanplasma with different Analytical column

AnalyticalRun ID

LLOQ 5.00pg/mLLow QC 15.00

pg/mLMid QC 4500.00

pg/mLHigh QC 9000.00

pg/mLColumn

IDLC/102

ColumnID

LC/115

ColumnID

LC/102

ColumnID

LC/115

ColumnID

LC/102

ColumnID

LC/115

ColumnID

LC/102

ColumnID

LC/115

P&ABatch 5

5.10 4.70 14.90 14.20 4450.60 4350.10 9012.50 9112.104.80 4.60 15.20 15.00 4612.40 4652.60 8976.60 8871.404.70 4.80 14.80 14.60 4521.30 4531.10 8956.80 8929.504.80 5.10 14.70 14.10 4531.20 4631.30 9876.40 9776.805.20 4.80 14.60 14.30 4456.50 4446.70 8897.50 8997.404.80 4.90 15.30 14.10 4412.70 4467.80 9056.70 9159.80

N 6 6 6 6 6 6 6 6

Mean 4.90 4.82 14.92 14.38 4497.45 4513.27 9129.42 9141.17

SD (±) 0.20 0.17 0.28 0.35 72.08 115.57 369.83 329.69

CV (%) 4.08 3.58 1.87 2.46 1.60 2.56 4.05 3.61

%NOM 98.30 96.42 100.35 100.13Acceptance criteria:


2. Mean % Nominal (100 ±15% & for LLOQ 100 ±20%).

63


Bench Top Stability (At Room Temp for 24.0 Hrs)

Spiked LQC and HQC samples were retrieved from deep freezer and were

kept at room temperature for 24.0 hrs and were processed and analyzed along with

freshly prepared calibration standards, comparison LQC and HQC samples.

Concentrations were calculated to determine mean % change during stability period.

The mean Accuracy for LQC and HQC samples of Rasagiline from

comparison samples were 99.93% and 106.35% respectively.

The plasma samples of Rasagiline were found to be stable for approximately

24.0 hrs min at room temperature.

Results are present in table 3.17.

Table 3.17 Assessment of stability of Analyte (Rasagiline) in biological matrix atRoom temperature

Low QC 15.00 pg/mL High QC 9000.00 pg/mLComparison

samples (0.00 hr)Stability samples

(24.0 hrs)Comparison


(24.0 hrs)Conc.found

%nominal

Conc.found

%nominal

Conc.found

%nominal

Conc.found

%nominal

P&ABatch 5

15.96 106.42 14.13 94.17 8846.25 98.29 10178.68 113.1015.56 103.75 15.10 100.67 8906.46 98.96 9023.68 100.2614.99 99.92 15.03 100.17 9977.25 110.86 9378.75 104.2115.73 104.83 16.09 107.25 9252.00 102.80 9777.11 108.6315.30 102.00 15.48 103.17 8920.71 99.12 9416.57 104.6316.26 108.42 14.13 94.17 9105.64 101.17 9656.36 107.29

N 6 6 6 6Mean 15.63 14.99 9168.05 9571.86SD(±) 0.46 0.77 424.01 394.82CV (%) 2.93 5.12 4.62 4.12Accuracy 104.22 99.93 101.87 106.35Acceptance criteria:1. % change should be ± 15 or % Ratio (stability/comparison) should be within

85-115 %.2. % CV ≤ 15%.

3. Mean % Nominal (100 ±15%).

64


FREEZE AND THAW STABILITY (after 3rd cycle at -30°C)

Samples were prepared at LQC and HQC levels, aliquoted and frozen at

-30 ± 5°C six samples from each concentration were subjected to three freeze and

thaw cycles (stability samples). These samples were processed and analyzed along

with freshly prepared calibration standards, LQC and HQC samples (comparison

samples). Concentrations were calculated to determine mean % change after 3 cycles.



The plasma samples of Rasagiline e were found to be stable after 3 cycles at -

30±5°C

Results are present in Table 3.18.

Table 3.18 Assessment of Freeze-Thaw stability of Analyte (Rasagiline) at -30 ± 5°C

Low QC 15.00 pg/ml High QC 9000.00 pg/mlComparison

samplesStability sample at

4th cycleComparison


4th cycleConc.found

%nominal

Conc.found

%nominal

Conc.found

%nominal

Conc.found

%nominal

P&ABatch 5

15.96 106.42 16.59 110.58 8846.25 98.29 9784.29 108.7115.56 103.75 16.24 108.25 8906.46 98.96 10030.71 111.4514.99 99.92 16.84 112.25 9977.25 110.86 8677.39 96.4215.73 104.83 15.53 103.50 9252.00 102.80 9145.82 101.6215.30 102.00 15.91 106.08 8920.71 99.12 9916.29 110.1816.26 108.42 16.06 107.08 9105.64 101.17 9364.07 104.05

N 6 6 6 6Mean 15.63 16.19 9168.05 9486.43SD (±) 0.46 0.47 424.01 520.63% CV 2.93 2.92 4.62 5.49

%NOM 104.22 107.96 101.87 105.40Acceptance criteria: % change should be ± 15 or % Ratio (stability/comparison)should be within 85-115 %.

1. % CV ≤ 15%.2. Mean % Nominal (100 ±15%).

65


Autosampler stability at 2-8°C in autosampler

LQC and HQC samples were prepared and processed. These processed

samples were analyzed and kept in autosampler for 55 hrs at 2-8°C and analyzed

along with freshly prepared calibration standard samples. Concentrations were

calculated to determine mean % change during stability period.



Rasagiline samples were stable for 55 hrs at 2-8°C in autosampler.


Table 3.19 Assessment of Auto sampler stability of Analyte (Rasagiline) at 2-8°CLow QC 15.00 pg/mL High QC 9000.00 pg/mL

Comparisonsamples (0.0 hr)

Stability samples(55hrs)


Stability samples(55 hrs)

Conc.found

%nominal

Conc.found

%nominal

Conc.found

%nominal

Conc.found

%nominal

P&ABatch 5

13.69 91.25 14.83 98.83 8573.46 95.26 8343.64 92.7115.21 101.42 14.36 95.75 8725.71 96.95 8662.29 96.2515.09 100.58 14.86 99.08 8716.07 96.85 8444.25 93.8314.54 96.92 15.28 101.83 8694.75 96.61 8196.21 91.0714.41 96.08 14.76 98.42 9070.18 100.78 8873.79 98.6015.48 103.17 14.66 97.75 8874.75 98.61 8793.21 97.70

N 6 6 6 6

Mean 14.74 14.79 8775.82 8552.23SD(±) 0.65 0.30 173.23 266.65% CV 4.44 2.01 1.97 3.12%NOM 98.24 98.61 97.51 95.02


1. % change should be ± 15 or % Ratio (stability/comparison) should be within 85-

115 %.

2. % CV ≤ 15%.

3. Mean % Nominal (100 ±15%).

66


Long Term Stability (at -30°C Temp for 64 days)

Spiked LQC and HQC samples were retrieved from deep freezer after 78 days

and were processed and analyzed along with freshly prepared calibration standards,

comparison LQC and HQC samples. Concentrations were calculated to determine

mean % change during stability period.



The plasma samples of Rasagiline were found to be stable for approximately

78days at -30°C temp.

Results are present in Table 3.20

Table 3.20 Assessment of Long term plasma stability of Analyte(Rasagiline) at -30°C.



(64days)Comparison


(64 days)Conc.found

%nominal

Conc.found

%nominal

Conc.found

%nominal

Conc.found

%nominal

P&ABatch 5

14.84 98.92 15.63 104.17 8455.82 93.95 8576.14 95.2914.26 95.08 14.80 98.67 8786.36 97.63 8458.50 93.9814.10 94.00 15.16 101.08 9116.68 101.30 8428.82 93.6514.28 95.17 15.49 103.25 9141.32 101.57 8921.79 99.1314.06 93.75 15.98 106.50 9232.29 102.58 8823.32 98.0415.00 100.00 15.68 104.50 8573.04 95.26 8267.57 91.86

N 6 6 6 6Mean 14.42 15.45 8884.25 8579.36SD(±) 0.40 0.42 325.95 249.51

CV (%) 2.75 2.69 3.67 2.91%NOM 96.15 103.03 98.71 95.33


1. % change should be ± 15 or % Ratio (stability/comparison) should be within 85-

115 %.

2. % CV ≤ 15%.

3. Mean % Nominal (100 ±15%).

67


Short term stock solution stability of Rasagiline, Rasagiline -13C3 mesylate and

spiking solution of internal standard (Rasagiline-13C3 mesylate) at room

temperature

Stock solution stability was determined by comparing the peak areas of freshly

prepared stock solutions (comparison samples) with stability stock solutions. Main

Stock solutions of Rasagiline and Rasagiline 13 C3 mesylate were freshly prepared and

aliquots of stocks were kept at room temperature for 9.0 hrs (stability samples).

Aqueous equivalent highest calibration standard of Rasagiline and solution of

Rasagiline 13 C3 mesylate were prepared from the stability samples and analyzed.

Areas of stability samples and freshly prepared samples were compared to determine


The %CV for Rasagiline stock solution from comparison samples was 0.4%

and %Ratio (stability/comparison) was 100.78.

The %CV for Rasagiline 13 C3 mesylate stock solution from comparison

samples was 1.7% and %Ratio (stability/comparison) was 101.27.

The %CV for Rasagiline 13 C3 mesylate working solution (Internal standard

spiking solution) from comparison samples was 1.1% and %Ratio

(stability/comparison) was 100.63.

Rasagiline, Rasagiline13 C3 mesylate stock solutions and Rasagiline13 C3

mesylate working solutions were found to be stable at room temperature for

9 hrs.

Results are present in Tables 3.21 and 3.22.

68


Table 3.21 Assessment of Short term stock solution stability of Analyte(Rasagiline) and internal standard (Rasagiline- 13 C3 mesylate) at Room

temperature

Analyte Internal standardComparison

standard stocksolutionresponse(0.0 hrs)

Stability stocksolutionresponse

(9hrs)

Comparisonstock solution

response(0.0 hrs)

Stabilitystandard stock

Response(9hrs)

Curve 6

567282 580642 137431 138144585470 579028 139566 139479565590 576135 139400 139391576170 574545 136221 143864568253 580468 140427 138147578242 577151 137731 142314

N 6 6 6 6Mean 573501.17 577994.83 138462.67 140223.17SD (±) 7767.35 2460.23 1586.14 2345.507CV (%) 1.40 0.40 1.10 1.70% Ratio 100.78 101.27


1. % Ratio (stability/comparison) should be within 95 -105%.

69


Table 3.22 Assessment of Short term solution stability of internal standard

spiking solution (Rasagiline- 13C3 mesylate) at refrigerated conditions

Comparison solution (Internalstandard Spiking solution)

Response (0.00 hrs)

Stability solution (Internalstandard Spiking solution)

Response (10 days atrefrigerated conditions)

Curve 6

140514 141485138527 141777142024 141138138838 142939144045 138636136663 139908

N 6 6

Mean 140101.83 140980.50

SD(±) 2657.67 1509.72

% CV 1.90 1.10

% Ratio 100.63Acceptance criteria:

1. % Ratio (stability/comparison) should be within 95-105%.

Method Validation Conclusion

As all the values obtained are within the acceptance criteria, the method stands

validated and is suitable for estimation of Rasagiline concentrations in plasma by

single analytical run. The rugged, efficient Liquid-Liquid extraction method, high

recovery, low limit of quantitation, and wide linearity range make this a suitable

method for use in clinical samples for bioequivalence and Pharmacokinetic studies.

70

Chapter 3 Rasagiline - Application

3.5 Application

The above described analytical method was applied to determine plasma

concentrations of Rasagiline following oral administration in healthy human

volunteers. These volunteers have informed consent before participation of study and

study protocol was approved by IEC (Institutional Ethics Committee) as per DCGI

(Drug Control General of India) guidelines. Each volunteer was administered 1 mg

dose (one 1 mg tablet) in 22 healthy human volunteers by oral administration with

240 mL of drinking water. The reference product AZILECT ® tablets 1 mg (Teva

Pharma, USA) and test product(APL Research Pvt.Ltd, India) Rasagiline tablet 1 mg

(Test tablet) was used. Blood samples were collected as a pre-dose (0 h) 5 min prior

to dosing followed by further samples at 0.083, 0.167, 0.25, 0.333, 0.417, 0.5, 0.667,

0.833, 1, 1.25, 1.5, 2, 2.5, 3, 3.75, 4.5, 5.5 and 6.5 hr. After dosing 5 mL blood was

collected each time in vacutainer containing K2EDTA. A total of 38 (19 time points

from test and reference respectively) time points were collected by using

centrifugation at 4000 rpm, 10°C, 10 min and stored below -30 °C until sample

analysis. Test and reference was administered to same human volunteers under fasting

conditions separately with a gap of 7 days washing period as per approved protocol.

The Mean Plasma concentration vs. time curve for 22 volunteers is shown in

Figure 3.15 and Table 3.23

71


Table 3.23 Rasagiline Mean concentration (pg/mL) data forsubject samples obtained from the LC-MS/MS

Time in hoursConcentration (pg/mL)

TEST REFERENCE

0 0.00 0.00

0.083 80.52 143.50

0.167 1006.95 1025.26

0.25 2657.68 3157.31

0.333 4094.30 4958.36

0.417 4529.87 5647.15

0.5 4505.71 5492.63

0.667 3511.61 3757.14

0.833 2762.70 2607.76

1 1850.04 1801.78

1.25 1091.91 1149.82

1.5 660.67 694.65

2 345.41 341.40

2.5 184.15 195.12

3 118.79 127.40

3.75 68.33 77.70

4.5 42.22 50.32

5.5 26.44 34.46

6.5 11.18 15.91

72


Figure 3.15 Mean plasma concentration Vs time curve for Rasagiline

73

Chapter 3 Rasagiline - Pharmacokinetic Studies

3.6 Pharmacokinetic Studies

Pharmacokinetics parameters from human plasma samples were calculated by

a non-compartmental statistic model using WinNon-Lin5.0 software (Pharsight,

USA). Blood samples were taken for a period of 3 to 5 times the terminal elimination

half-life (t1/2), and it was considered as the area under the concentration time curve

(AUC) ratio higher than 80% as per the FDA guidelines. Plasma Rasagiline

concentration-time profiles were visually inspected and Cmax and Tmax values were

determined. The AUC0–t was obtained by the trapezoidal method. AUC0–∞ was

calculated up to the last measureable concentration and extrapolations were obtained

by the last measureable concentration and the terminal elimination rate constant (Kel).

The Kel was estimated from the slope of the terminal exponential phase of the plasma

of the Rasagiline concentration-time curve using linear regression method. The t1/2

was then calculated as 0.693/Kel. The AUC0–t, AUC0–∞ and Cmax bioequivalence were

assessed by analysis of variance (ANOVA) and the standard 90% confidence intervals

(90% CIs) of the ratio’s test/reference. The bioequivalence was considered when the

ratio of averages of log transformed data was within 80-125% for AUC0–t, AUC0–∞

and Cmax.39-41

The above validated method was used in the determination of Rasagiline in plasma

samples for establishing the bioequivalence of a single 1 mg dose (one 1 mg tablet) in

22 healthy volunteers. Typical plasma concentration versus time profiles was shown

in Figure 3.15

74


All the plasma concentrations of Rasagiline were in the standard curve region and

remained above the 5 pg/ mL (LOQ) for the entire sampling period. Pharmacokinetic

data is shown in Table 3.24 and Table 3.25.

Table 3.24 Rasagiline Pharmacokinetic data

Rasagiline Pharmacokinetic data

Pharmacokinetic

Parameter

Test Reference

Mean±SD % CV Mean±SD % CV

Cmax (pg/mL)4529.87 ±

102.52.26 5647.15 ±

114.42.03

AUC 0-t (pg/mL)4005.11 ±

124.63.11 4407.78 ±

132.33.00

AUC 0-∞

(pg h/mL)

4022.33 ±113.2

2.81 4434.56 ±124.2

2.80

Tmax(h) 0.417 - 0.417 -

t 1/2 1.07 - 1.17 -

Table 3.25 Rasagiline Pharmacokinetic data (Test /Reference)

Pharmacokinetic

ParameterCmax AUC 0-t AUC 0-∞

Test/Reference 80.22 90.86 90.70

75


Pharmacokinetic Studies Conclusion

The present study provides firm evidence to support that the in house

Rasagiline 1 mg was bioequivalent with AZILECT ® (manufactured by Teva Pharma,

USA) tablets (Rasagiline) 1 mg tablet under fasting conditions.

In vivo data was predicted by using Liquid-Liquid Extraction procedure and

concentrations were found out through Liquid Chromatography Mass Spectroscopy

detection instrument. The Pharmacokinetic parameters assessed were AUC0-t, AUC0-

Cmax, Tmax, and t1/2. The bioequivalence criteria are based on the 90% confidence

intervals whose acceptance range is in between 80% -125%.

Therefore, it can be concluded that the two Rasagiline formulations

(reference and test) analyzed are bioequivalent in terms of rate and extent of

absorption.

CHAPTER 4

Analytical method development andvalidation of Almotriptan by High

performance Liquid chromatography withmass spectrometry

76

Chapter 4 Almotriptan - Introduction

4.1 Introduction

Almotriptan,N,N-dimethyl-2-{5-[(pyrrolidin-1-ylsulfonyl)methyl]-1H-indol-

3-yl}ehanamine is a novel 5-HT1B/1D receptor agonist used for the treatment of

symptomatic relief of migraines (Fig.1).42. Almotriptan is well absorbed orally, with

an absolute bioavailability of around 70%. The drug shows a dose linear

pharmacokinetics and a mean elimination half-life of 1.4-3.8 h. Approximately

40-50% of the dose is recovered unchanged in the urine; renal elimination probably

occurs via active tubular secretion. The balance of the dose is eliminated unchanged

in faecus (approximately 5%) or is metabolised 43,44

Fig.4.1.Chemical structures of Almotriptan malate, Almotriptan –D6 malate

As of now to our knowledge, several methods for the determination of

Almotriptan in biological matrixes42,45,46 pharmaceuticalcompounds47-50 by

LC–MS/MS 45,HPLC 47,48 HPTLC49and Fluorimetric and calorimetric 50 have been

reported.

However, Fleischhacker et.al 45concentrated more on pharmacokinetics part

rather than method development and validation part. They have not explained briefly

on extraction procedure, stability aspects, matrix factor effect, recovery for

determination of Almotriptan by LC–MS/MS. The purpose of this study was to

develop and validates a novel sensitive LC–MS/MS method to quantify Almotriptan

in human plasma.

77

Chapter 4 Almotriptan - Experimental

4.2 Experimental Investigations


Almotriptan malate was obtained from USP and Almotriptan malate- D6 was

obtained from clear synth Labs (P) Ltd, Mumbai, India. Human plasma (K2EDTA),

obtained from Navjeevan blood bank, Hyderabad. Formic acid, Ammonium formate,

sodium carbonate, acetonitrile, methanol obtained from SD- Fine chemicals, Mumbai.

MTBE (methyl-tertiary butyl ether) was obtained from Labscan,Mumbai. (Ultra pure

water obtained from Milli-Q System.


Refer Chapter - 3.2.2

4.2.3 Preparation of reagents and solvents


Reagents and Solvents preparation


10mM Ammoniumformate PH:4.5

Dissolve 1.26 g of ammonium formate into 2 L of waterand adjust PH with formic acid

0.5NSodiumcarbonate

Dissolve 26.5 g of anhydrous sodium carbonate into 1L ofwater.

Mobile phase10mM Ammonium formate pH:4.5: Acetonitrile in theratio of 50:50 and


(Autosampler wash)80% Acetonitrile

Mix 800 mL of Acetonitrile with 200 mL of water.

Reconstitutionsolution

Mix 500 mL of 10mM ammonium formate with 500 mL ofacetonitrile.

Extraction Solvent MTBE (methyl-tertiary butyl ether)

78

Chapter 4 Almotriptan - Experimental




Almotriptan stock solution 100.0 µg/mL 25 mL Methanol

Almotriptan-D6 stock solution 100.0 µg/mL 25 mL Methanol

4.2.5 Preparation of standards and quality control (QC) Samples

Standard stock solutions of Almotriptan (100.0µg/mL) and Almotriptan-D6

(100.0µg/mL) were prepared in methanol. The spiking solution for Almotriptan-D6

(80.0 ng/mL) was prepared in 50% methanol from respective standard stock solution.

Standard stock solutions and IS spiking solutions were stored in refrigerator

conditions (2-8°C) until analysis. Standard stock solutions were added to drug-free

human plasma to obtain Almotriptan concentration levels of 0.5, 1.0, 5.0, 15.0, 30.0,

45.0, 60.0, 90.0, 120.0 and 150.0 ng/mL for Analytical standards and 0.5, 1.5,75.0,

and 105.0 ng/mL for Quality control standards and stored in a -30°C set point freezer

until analysis.The Aqueous standards were prepared in reconstitution solution

(10mM ammonium formate: acetonitrile (50:50 v/v) for validation exercises until

analysis.

79

Chapter 4 Almotriptan - Method Development


The goal of this work was to develop and validate a simple, rapid and sensitive

assay method for the quantitative determination of Almotriptan from human plasma

samples by LC-MS/MS detection. We tested a wide spectrum of organic solvents

from different physicochemical categories with different volume fractions as well as

combinations. In terms of the analysis condition, various mobile phases, in different

proportions, buffered and non-buffered at various pH were attempted to provide the

best peak shape and less retention times. Also we tried different column packing, even

from normal phase. The MS optimization was performed by direct infusion of

solutions of both Almotriptan and Almotriptan-D6 into the ESI source of the mass

spectrometer. The critical parameters in the ESI source include the needle (ESI)

voltage, Other parameters, such as the nebulizer and the desolvation gases were

optimized to obtain a better spray shape, resulting in better ionization. A CAD

product ion spectrum for Almotriptan and Almotriptan-D6 yielded high-abundance

fragment ions at m/z 336.1201.3 and 342.2207.2 (Figure 4.2 - 4.5) in multiple

reaction monitoring (MRM) positive mode respectively. After the MRM channels

were tuned, the mobile phase was changed from an aqueous phase to a more organic

phase with acid dopant to obtain a fast and selective LC method.

The most accurate extraction method for analyte was selected as Liquid-Liquid

extraction. A good separation and elution were achieved using 10 mM ammonium

formate (pH 4.5.): acetonitrile (50:50 v/v) as the mobile phase, at a flow-rate of

0.5 mL/min and injection volume of 10 µL. The notable advantages of the developed

80


method are most sensitive and accurate methods over the developed methods based on

literature.

Figure 4.2 Parent ion mass spectra (Q1) of Almotriptan

81


Figure 4.3 Product ion mass spectra (Q3) of Almotriptan

82


Figure 4.4 Parent ion mass spectra (Q1) Almotriptan -D6 malate

83


Figure 4.5 Product ion mass spectra (Q3) of Almotriptan -D6

84



A good separation and elution were achieved using Zorbax, SB C18, 4.6 mm x 75

mm, 3.5 µm was selected as the analytical column. The mobile phase composition was

10mM Ammonium formate: acteonitrile (50:50 v/v) at a flow rate of 0.5 mL/min and

10L injection volume was used. Column temperature was set at 40°C.

Almotriptan-D6 was found to be appropriate internal standard. Retention time of

Almotriptan and Almotriptan-D6 were found to be 1.5 ± 0.2 min, with overall runtime

of 3.0 min.

Sample preparation

Liquid-liquid extraction was used to isolate Almotriptan and Almotriptan-D6

from human plasma. 100 µL of Almotriptan-D6 spiking solution (8.0 ng/mL) and

200 µL of plasma sample (respective concentration) were added respective ria vials

and vortexed 30 seconds followed by 100 µl of 0.5N sodium carbonate solution was

added and vortexed 10 minutes. Then, 2.5 mL of extraction solvent (Methyl Tertiary

butyl ether) was added and vortexed approximately for 20 min. This was followed by,

Centrifugation at 4000 rpm, 5 min at 20°C. Then samples were Flash freeze by using

dry-ice/Acetone. The supernatant from each ria vial was transferred into another set of

ria vials. These samples were evaporated at 40°C under nitrogen upto dryness.

Finally, the dried residue samples were reconstituted with 500 µL of reconstitution

solution (10mM Ammonium formate (pH 4.5): acetonitrile 50:50, v/v) and vortexed

briefly. These samples were transferred into auto sampler vials and injected in to

LC-MS/MS.

85



The analytical curves were constructed using values ranging from

0.5 -150.0 ng/mL of Almotriptan in human plasma. Calibration curves were obtained

by weighted 1/conc2 linear regression analysis.

y = ax + b

Where,

y = Peak area ratio (PAR) of Almotriptan to internal standard.

x = Concentration (ng/mL) of Almotriptan in plasma.

a = Slope

b = Intercept

r2= Coefficient of determination

The ratio of Almotriptan peak area to Almotriptan -D6 peak area was plotted

against the ratio of Almotriptan concentration in ng/ mL. Calibration curve standard

samples and quality control samples were prepared in replicates (n=6) for analysis.

Accuracy and precision for the back calculated concentrations of the calibration

points should be within ≤ 15 and ± 15% of their nominal values. However, for LLOQ,

the precision and accuracy should be within ≤ 20 and ± 20%.


The developed method is suitable for estimation of Almotriptan concentrations

in plasma as a single analytical run, in clinical samples from Pharmacokinetic studies.

This was followed by method validation.

86

Chapter 4 Almotriptan - Method Validation


The objective of the work is to validate specific HPLC- MS method for the

determination of Almotriptan in human plasma for clinical / bioavailability and

Pharmacokinetic study.

Chromatography


LLOQC, LQC, MQC, HQC, Calibration curve are shown in Figure 4.6 to Figure 4.14.


87



88


Figure 4.8 Chromatogram of LOQ Sample (Almotriptan & IS)

89


Figure 4.9 Chromatogram of ULOQ Sample (Almotriptan & IS)

90


Figure 4.10 Chromatogram of LLOQ Sample (Almotriptan & IS)

91


Figure 4.11 Chromatogram of LQC Sample (Almotriptan & IS)

92


Figure 4.12 Chromatogram of MQC Sample (Almotriptan & IS)

93


Figure 4.13 Chromatogram of HQC Sample (Almotriptan & IS)

94


Figure 4.14 Calibration Curve of Almotriptan

95






the 10 lots. Results are presented in Table 4.3

Table 4.3 Screening of Different batches of blank matrix(Human K2EDTA Plasma) for interference free Almotriptan blank plasma


Blank plasma AreaAnalyte

(Almotriptan)RT

Internal standardRT

AP/3271/07/10 0 0AP/3272/07/10 12 0AP/3273/07/10 0 0AP/3274/07/10 0 0AP/3275/07/10 0 0AP/3276/07/10 14 0AP/3277/07/10 0 0AP/3278/07/10 0 0AP/3279/07/10 16 0AP/3280/07/10 0 0

Blank+IS with AP/3271/07/10 0 511923LOQ with AP/3271/07/10 8174 520823


During specificity run, the LLOQ standard was prepared in one of the

screened blank plasma including the spiking of working range of internal standard.

Blank plasma samples from 10 different lots, 6 LLOQ standards were processed

according to the extraction procedure. The responses for the blank plasma from 10

different lots were compared to the LLOQ standard of the analyte and internal

96


standard. No significant response (≤ 20 % for the analyte response and ≤ 5% of the

internal standard response) was observed at the retention times of the analyte or the

internal standard in blank plasma as compared to the LLOQ standard. Results are

presented in Table 4.4.


experiment. Results are presented in Table 4.5


(Human K2EDTA Plasma) for Almotriptan

MatrixIdentification

LLOQArea

Internalstandard(IS) area

Interference withAnalyte(% of

LLOQ Response)

Interferencewith IS(% of IS

Response)PL Blank-(K2EDTA-

AP/3271/07/10)8174 511923 0 0

AP/3271/07/10 8234 520823 0 0

AP/3273/07/10 8078 561563 0 0

AP/3274/07/10 8127 566392 0 0

AP/3277/07/10 8133 556385 0 0

AP/3280/07/10 8243 510831 0 0


1. Analyte response should be ≤ 20% of LOQ Response in at least 75% of the

blank.

2. Internal standard response should be ≤5% of mean internal standard response

in at least 75% of the blank.

97


Table 4.5 Limit of Quantification for analyte (Almotriptan)

Matrix identificationBlank plasma area at

Analyte RTLLOQ

responseLLOQ S/N

RATIO

AP/3271/07/10

0 8174 70.6

0 8234 76.2

0 8078 62.8

0 8127 56.6

0 8133 58.0

0 8243 62.4

N 6 6 6

Mean 0 8164 64.43

LLOQ was spiked in AP/3271/07/10 Blank Plasma Lot



2. S/N ratio is analyst software generated data.


Intra batch accuracy and precision evaluation were assessed by analyzing 1

calibration curve and 6 replicate each of the LLOQ, LQC, MQC, HQC, from


The Intra batch percentage of nominal concentrations for Almotriptan was


The Intra batch percentage of coefficient of variation is 0.79% to 3.05% forAlmotriptan.


98


Table 4.6 Intra batch (Within-Batch) Accuracy and Precision for determinationof Almotriptan levels in human plasma

AnalyticalRun ID

LLOQ0.50 ng/mL

Low QC1.50 ng/mL

Mid QC75.00 ng/mL

High QC105.00 ng/mL

Conc.found

%nominal

Conc.found

%nominal

Conc.found

%nominal

Conc.found

%nominal

P&A Batch1

0.52 103.60 1.56 104.00 75.60 100.80 104.00 99.050.52 104.80 1.47 98.00 75.10 100.13 105.00 100.000.52 104.80 1.50 100.00 76.50 102.00 106.00 100.950.52 103.80 1.46 97.33 73.50 98.00 103.00 98.100.52 104.60 1.43 95.33 74.80 99.73 103.00 98.100.51 101.60 1.46 97.33 75.90 101.20 105.00 100.00

N 6 6 6 6Mean 0.52 1.48 75.23 104.33SD (±) 0.00 0.05 1.04 1.21CV (%) 0.79 3.05 1.38 1.16

%Accuracy 103.87 98.67 100.31 99.37



2. Mean % Nominal (100 ±15% and for LLOQ 100 ±20%).


Inter batch accuracy and precision evaluation were assessed by analyzing

5 sets of calibration curves for Almotriptan and 5 sets of QC samples, 6 replicates

each of the LLOQ, LQC, MQC and HQC.

The inter batch percentage of nominal concentrations for Almotriptan was



Almotriptan.


99


Table 4.7 Inter batch (Between-Batch) Accuracy and Precision for determinationof Almotriptan levels in human plasma

AnalyticalRun ID

LLOQ0.50 ng/mL

Low QC1.50 ng/mL

Mid QC75.00 ng/mL

High QC105.00 ng/mL

Conc.found

%nominal

Conc.found

%nominal

Conc.found

%nominal

Conc.found

%nominal

P&ABatch 1

0.52 103.60 1.56 104.00 75.60 100.80 104.00 99.050.52 104.80 1.47 98.00 75.10 100.13 105.00 100.000.52 104.80 1.50 100.00 76.50 102.00 106.00 100.950.52 103.80 1.46 97.33 73.50 98.00 103.00 98.100.52 104.60 1.43 95.33 74.80 99.73 103.00 98.100.51 101.60 1.46 97.33 75.90 101.20 105.00 100.00

P&ABatch 2

0.55 110.00 1.54 102.67 76.90 102.53 106.00 100.950.54 108.40 1.51 100.67 76.30 101.73 107.00 101.900.54 107.60 1.51 100.67 76.30 101.73 105.00 100.000.51 101.60 1.49 99.33 76.20 101.60 104.00 99.050.53 105.80 1.51 100.67 75.90 101.20 105.00 100.000.53 105.20 1.48 98.67 75.60 100.80 106.00 100.95

P&ABatch 3

0.53 105.00 1.48 98.67 77.10 102.80 108.00 102.860.50 100.60 1.50 100.00 77.40 103.20 107.00 101.900.54 107.80 1.46 97.33 75.70 100.93 109.00 103.810.52 104.20 1.50 100.00 76.00 101.33 105.00 100.000.52 104.00 1.47 98.00 75.70 100.93 106.00 100.950.52 103.00 1.46 97.33 74.80 99.73 106.00 100.95

P&ABatch 4

0.50 100.00 1.50 100.00 76.50 102.00 106.00 100.950.50 100.60 1.48 98.67 76.60 102.13 107.00 101.900.51 102.20 1.45 96.67 75.90 101.20 106.00 100.950.52 103.60 1.46 97.33 75.00 100.00 105.00 100.000.50 99.80 1.45 96.67 75.10 100.13 105.00 100.000.51 102.00 1.47 98.00 75.60 100.80 105.00 100.00

P&ABatch 5

0.51 102.00 1.51 100.67 76.30 101.73 107.00 101.900.53 106.60 1.47 98.00 75.10 100.13 107.00 101.900.50 100.60 1.47 98.00 74.90 99.87 105.00 100.000.51 102.40 1.48 98.67 74.90 99.87 107.00 101.900.51 102.00 1.40 93.33 73.40 97.87 104.00 99.050.50 100.60 1.45 96.67 74.80 99.73 103.00 98.10

N 30 30 30 30Mean 0.52 1.48 75.65 105.57SD(±) 0.01 0.03 0.93 1.45

CV (%) 2.65 2.15 1.23 1.38

%Nominal 103.63 98.62 100.86 100.54

Acceptance criteria: Same as Table 4.6

100


Calibration Curve


Almotriptan over 0.50 - 150.00ng/mL for calibration range. The correlation

coefficient is greater than 0.9980 for Almotriptan. Back calculations were made from

the calibration curves to determine Almotriptan concentrations of each calibration

standard.


Table 4.8 Summary of calibration curve parameters for Almotriptan in human

plasma

Analytical RunID Slope intercept

Coefficient ofregression (r2)

P&A Batch-1 0.03712 -0.0003300 0.9997P&A Batch-2 0.03677 -0.0003059 0.9997P&A Batch-3 0.03664 -0.0003384 0.9998P&A Batch-4 0.03742 -0.0005110 0.9995P&A Batch-5 0.03682 -0.0002150 0.9996

N 5 5 5Mean 0.03695 0.00008570 0.9997SD (±) 0.0003143 0.0003833 0.0001CV (%) 0.9 447.3 0.0

Regression Footnote (S): Resp.= Slope * Conc.+ Intercept



101


Table 4.9 Back-calculated standard concentrations from each calibration curve

for Almotriptan in human plasma

Analytical RunID


ng/mL1.00ng/mL

5.00ng/mL

15.00ng/mL

30.00ng/mL


N 5 5 5 5 5Mean 0.51 0.98 4.98 14.76 30.10SD(±) 0.00 0.01 0.05 0.05 0.25CV% 0.57 1.13 1.08 0.37 0.85

%Nominal 101.12 97.88 99.68 98.40 100.33

Analytical runID


ng/mL60.00

ng/mL90.00

ng/mL120.00ng/mL

150.00ng/mL


N 5 5 5 5 4Mean 44.42 61.56 90.76 121.60 148.60SD(±) 0.53 0.26 0.48 0.55 0.55CV% 1.18 0.42 0.53 0.45 0.37

%Nominal 98.71 102.60 100.84 101.33 99.07

Acceptance criteria

1. Mean % Nominal (100±15%) except lowest calibration standard.

2. Mean % Nominal (100±20%) for lowest calibration standard (CS1).

3. % CV ≤ 15% except lowest calibration standard (CS1) for which it is ≤ 20%.

102


Recovery

The percentage recovery of Almotriptan was determined by comparing the

mean peak area of Almotriptan in extracted LQC, MQC, HQC samples with freshly


The mean % recovery for LQC, MQC, HQC samples of Almotriptan were


The mean recovery of Almotriptan across QC levels is 94.02%.

The mean recovery of % CV recovery of Almotriptan across QC levels is

4.5%.



recovery is 84.35%.

The % CV recovery of IS Almotriptan D6 for extracted is 4.3%.


103


Table 4.10 Recovery of Analyte (Almotriptan) and Almotriptan-D6 from humanplasma

StandardExtracted peak

responseUnextracted peak

responseDrug IS Drug IS

Low QC:1.50 ng/ml

29360 537130 30540 63338831860 576252 30632 62605730295 561091 30226 62312527716 499819 30441 63039429954 551165 30257 63363029374 547313 30595 642957

N 6 6 6 6% Recovery 97.73

SD (±) 4.46%CV 4.6

Medium QC:75.00 ng/ml

1483019 525139 1624399 6272301421148 501126 1571217 6168921490415 537595 1576537 6242421427954 513112 1565064 6224991360767 490300 1575006 6285171457998 531803 1588431 639478

N 6 6 6 6% Recovery 90.97

SD(±) 3.04%CV 3.3

High QC:105.00 ng/ml

2046697 519018 2230351 6276352021415 514754 2144282 6053122044622 513242 2141492 6024622042496 530922 2167626 6164972039594 527580 2148138 6113951965355 504568 2193952 629811

N 6 6 6 6% Recovery 93.35

SD(±) 1.46%CV 1.6

Drug ISMean recovery 94.02 84.35Mean SD(±) 4.18 3.6Mean % CV 4.5 4.3Acceptance criteria:1. The coefficient of variation for mean recovery across LQC, MQC and HQC shall

not exceed 25%.2. The coefficient of variation for mean recovery of IS shall not exceed 25%.

104


Matrix Effect

Samples were prepared at LQC & HQC level in triplicate in each of 6 different lots of

human plasma. A calibration curve and 6 replicates of LQC & HQC samples in


No significant matrix effect found in different sources of human plasma tested for

Almotriptan, Almotriptan -D6.

Results are presented in Table 4.11 and 4.12

Table 4.11 Assessment of Matrix Effect on determination of Almotriptan at LQClevels in human plasma




Matrix factor

AP/3271/07/10 5557 712730 0.005214AP/3271/07/10 6081 682214 0.005398AP/3271/07/10 6159 721035 0.00555AP/3272/07/10 5766 710173 0.005188AP/3272/07/10 5833 711346 0.005332AP/3272/07/10 5502 700174 0.005166AP/3273/07/10 5698 681681 0.005341AP/3273/07/10 5691 682888 0.005249AP/3273/07/10 5422 693854 0.005148AP/3274/07/10 5635 674117 0.00525AP/3274/07/10 5922 687042 0.005425AP/3274/07/10 5847 698661 0.005584AP/3275/07/10 5287 670141 0.004979AP/3275/07/10 5619 679702 0.005142AP/3275/07/10 5592 699379 0.00529AP/3276/07/10 5164 676581 0.004985

AP/3276/07/10 5376 663070 0.005113AP/3276/07/10 5557 672930 0.005214

N 18 18 18Grand Mean 0.005254

SD(±) 0.002CV (%) 3.15

Acceptance criteria:1. Mean % Nominal 100 ±15% of nominal value.2. % CV ≤ 15%.

105


Table 4.12 Assessment of Matrix Effect on determination of Almotriptanat HQC levels in human plasma



Matrix factor

AP/3271/07/10 3781974 1009476 2.997178AP/3271/07/10 3853960 1027602 3.000352AP/3271/07/10 3726283 993973 2.999102AP/3272/07/10 3699126 976829 3.029498AP/3272/07/10 3829308 1019494 3.00487AP/3272/07/10 3818839 1027118 2.97441AP/3273/07/10 3767222 1001336 3.009756AP/3273/07/10 3855325 1029629 2.995507AP/3273/07/10 3679162 986086 2.984862AP/3274/07/10 3692219 992530 2.976007AP/3274/07/10 3731514 1003459 2.97492AP/3274/07/10 3713898 995875 2.983425AP/3275/07/10 3732461 993162 3.006527AP/3275/07/10 3727740 989657 3.01336AP/3275/07/10 3650347 971132 3.007085AP/3276/07/10 3638647 966077 3.013133AP/3276/07/10 3731021 1010666 2.953315AP/3276/07/10 3718867 997999 2.981058

N 18 18 18Grand Mean 2.99467

SD(±) 0.0186%CV 0.62


1. Mean % Nominal 100±15% of nominal value.

2. % CV ≤ 15%.

106


Dilution Integrity





The % accuracy of Almotriptan nominal concentrations ranged between

94.35% and 100.38% for 1 in 4 dilutions and 1 in 2 dilutions respectively.

The % CV is 1.54% to 2.01%.


Table 4.13 Assessment of Dilution integrity for Almotriptan at DQC Conc (ng/mL)


Nominal conc: 225.00 ng/mL


Nominal conc: 225.00 ng/mLConc. Found % Nominal Conc. Found %Nominal231.25 102.78 213.69 94.97228.77 101.68 219.89 97.73225.63 100.28 211.15 93.84223.39 99.28 210.05 93.36222.45 98.87 211.62 94.05223.61 99.38 207.30 92.13

N 6 6Mean

%Nominal 100.38 94.35SD (±) 3.4745935 4.275183CV (%) 1.54 2.01


1. % CV ≤ 15%.

2. Mean % Nominal (100 ± 15%).

107




P & A batch-2 were kept at auto sampler temperature for approx 26 hrs after the

initial analysis and were re-injected again after approx 26 hrs. Concentrations were


The Accuracy of Almotriptan QC samples in reinjection was between 98.22%

and 99.84%.

The Precision (% CV) of Almotriptan QC samples in reinjection was between1.16 % and 3.05%.

Almotriptan was found to be stable at autosampler temperature post extraction(in reconstitution solution) for approx 26 hrs and reproducible afterreinjection.

Results are presented in Table 4.14Table 4.14 Assessment of Whole Batch Re-injection Reproducibility during

estimation of Almotriptan in human plasma

AnalyticalRun ID

Low QC 6.0 ng/mL High QC 1750.0 ng/mL

Comp sample Reinjectionsample Comp sample Reinjection

sample1.56 1.51 104 1051.47 1.47 105 1061.5 1.48 106.00 1071.46 1.44 103 1031.43 1.48 103 1041.46 1.46 105 104

N 6 6 6 6Mean 1.48 1.47 104.33 104.83SD(±) 0.05 0.02 1.21 1.47%CV 3.05 1.59 1.16 1.40

%NOM 98.67 98.22 99.37 99.84


1. % CV≤ 15% Except LLOQ for which it is ≤ 20%.2. Mean % Nominal (100 ±15% and for LLOQ 100±20%).3. 67% 0f the re-injected QCs at each level shall be within ±20% of their previous

concentration.

108


Ruggedness-Different Analyst




The Accuracy of Almotriptan QC samples within the range of 98.56% to

104.33%.

The Precision of Almotriptan QC samples within the range of 0.79% to3.05%.


analyst.


Table 4.15 Ruggedness of the method for estimation of Almotriptan Plasmalevels in human plasma with different Analyst

AnalyticalRun ID

LLOQ0.50 ng/mL

Low QC1.50 ng/mL

Mid QC75.00 ng/mL

High QC105.00 ng/mL

AnalystID 1

AnalystID 2

AnalystID 1

AnalystID 2

AnalystID 1

AnalystID 2

AnalystID 1

AnalystID 2

P&A Batch3

0.52 0.53 1.56 1.48 75.60 77.10 104.00 108.000.52 0.50 1.47 1.50 75.10 77.40 105.00 107.000.52 0.54 1.50 1.46 76.50 75.70 106.00 109.000.52 0.52 1.46 1.50 73.50 76.00 103.00 105.000.52 0.52 1.43 1.47 74.80 75.70 103.00 106.000.51 0.52 1.46 1.46 75.90 74.80 105.00 106.00

N 6 6 6 6 6 6 6 6

Mean 0.52 0.52 1.48 1.48 75.23 76.12 104.33 106.83

SD (±) 0.00 0.01 0.05 0.02 1.04 0.97 1.21 1.47

CV (%) 0.79 2.55 3.05 1.24 1.38 1.27 1.16 1.38

%NOM 103.67 104.33 98.67 98.56 100.31 101.49 99.37 101.75

Acceptance criteria:1. % CV ≤ 15 % except LLOQ for which it is ≤ 20%.

2. Mean % Nominal (100±15% & for LLOQ 100 ± 20%).

109


Ruggedness-Different Column


batch-5 were reinjected on different columns with same and specifications,


The Accuracy of Almotriptan QC samples within the range of 97.56% to

103.67%.

The Precision of Almotriptan QC samples within the range of 0.79% to

3.05%.


analyst.


Table 4.16 Ruggedness of the method for estimation of Almotriptan Plasmalevels in human plasma with different Analytical column

AnalyticalRun ID

LLOQ0.50 ng/mL

Low QC1.50 ng/mL

Mid QC75.00 ng/mL

High QC105.00 ng/mL

ColumnID

LC/121

ColumnID

LC/145

ColumnID

LC/121

ColumnID

LC/145

ColumnID

LC/121

ColumnID

LC/145

ColumnID

LC/121

ColumnID

LC/145

P&ABatch 5

0.52 0.51 1.56 1.51 75.60 76.30 104.00 107.000.52 0.53 1.47 1.47 75.10 75.10 105.00 107.000.52 0.50 1.50 1.47 76.50 74.90 106.00 105.000.52 0.51 1.46 1.48 73.50 74.90 103.00 107.000.52 0.51 1.43 1.40 74.80 73.40 103.00 104.000.51 0.50 1.46 1.45 75.90 74.80 105.00 103.00

N 6 6 6 6 6 6 6 6Mean 0.52 0.51 1.48 1.46 75.23 74.90 104.33 105.50SD(±) 0.00 0.01 0.05 0.04 1.04 0.92 1.21 1.76

CV (%) 0.79 2.15 3.05 2.51 1.38 1.23 1.16 1.67%NOM 103.67 102.00 98.67 97.56 100.31 99.87 99.37 100.48

Acceptance criteria:1. % CV ≤ 15 % except LLOQ for which it is ≤ 20%.2. Mean % Nominal (100±15% & for LLOQ 100 ± 20%).

110


Bench Top Stability (at room temp for 26 hrs)


kept at room temperature for 26hrs and were processed and analyzed along with



The mean Accuracy for LQC & HQC samples of Almotriptan from


The plasma samples of Almotriptan were found to be stable for approximately

26 hrs min at room temperature.


Table 4.17 Assessment of stability of Analyte (Almotriptan) in Biological matrixat Room temperature

Low QC 1.50 ng/mL High QC 105.00 ng/mLComparison

samples(0.00 hr)

Stability samples(26hrs)

Comparisonsamples(0.00 hr)

Stability samples(26 hrs)

Conc.found

%nominal

Conc.found

%nominal

Conc.found

%nominal

Conc.found

%nominal

1.47 98.00 1.52 101.33 108.00 102.86 107.00 101.901.45 96.67 1.53 102.00 106.00 100.95 108.00 102.861.47 98.00 1.47 98.00 105.00 100.00 105.00 100.001.44 96.00 1.45 96.67 106.00 100.95 105.00 100.001.41 94.00 1.44 96.00 105.00 100.00 104.00 99.051.44 96.00 1.46 97.33 105.00 100.00 104.00 99.05

N 6 6 6 6Mean 1.45 1.48 105.83 105.50SD(±) 0.02 0.04 1.17 1.64

CV (%) 1.56 2.55 1.10 1.56%NOM 96.45 98.56 100.79 100.48

Acceptance criteria:1. % change should be ± 15 or % Ratio (stability/comparison) should be within

85-115 %.2. %CV ≤ 15%.3. Mean % Nominal (100 ±15%).

111


Freeze and Thaw Stability (after 3rd cycle at -30°C)


-30±5°C six samples from each concentration were subjected to three freeze and thaw

cycles (stability samples). These samples were processed and analyzed along with

freshly prepared calibration standards, LQC and HQC samples (comparison samples).

Concentrations were calculated to determine mean % change after 3 cycles.

The mean Accuracy for LQC & HQC samples of Almotriptan fromcomparison samples were 97.00% and 100.32% respectively.

The plasma samples of Almotriptan were found to be stable after 3 cycles at-30 ±5°C.

Results are present in Table 4.18Table 4.18 Assessment of Freeze-Thaw stability of Analyte (Almotriptan)

at -30±5°CLow QC 1.50 ng/mL High QC 105.00 ng/mL

Comparisonsamples

Stability sampleat 4th cycle

Comparisonsamples

Stability sample at4th cycle

Conc.found

%nominal

Conc.found

%nominal

Conc.found

%nominal

Conc.found

%nominal

1.47 98.00 1.5 100.00 108.00 102.86 107 101.901.45 96.67 1.49 99.33 106.00 100.95 106 100.951.47 98.00 1.44 96.00 105.00 100.00 105 100.001.44 96.00 1.43 95.33 106.00 100.95 105 100.001.41 94.00 1.46 97.33 105.00 100.00 104 99.051.44 96.00 1.41 94.00 105.00 100.00 105 100.00

N 6 6 6 6Mean 1.45 1.46 105.83 105.33SD(±) 0.02 0.04 1.17 1.03

CV (%) 1.56 2.41 1.10 0.98%Accuracy

96.45 97.00 100.79 100.32

Acceptance criteria

1. % change should be ± 15 or % Ratio (stability/comparison) should be within85-115 %.

2. % CV ≤ 15%.3. Mean % Nominal (100 ±15%)

112


Autosampler stability at 2-8°C in autosamplerLQC and HQC samples were prepared and processed. These processed

samples were analyzed and kept in auto sampler for 67 hrs at 2-8°C and analyzed



The mean Accuracy change for LQC & HQC samples of Almotriptan fromcomparison samples were 98.11% and 100.16% respectively.

Almotriptan samples were stable for 67 hrs at 2-8°C in autosampler. Results are present in table 4.19

Table 4.19 Assessment of Autosampler stability of Analyte (Almotriptan)at 2-8°C



Stability samples(57 hr)


Stability samples(57hr)

Conc.found

%nominal

Conc.found

%nominal

Conc.found

%nominal

Conc.found

%nominal

1.54 102.67 1.5 100.00 106 100.95 107.00 101.90

1.51 100.67 1.51 100.67 107 101.90 105 100.00

1.51 100.67 1.43 95.33 105 100.00 106 100.95

1.49 99.33 1.48 98.67 104 99.05 103 98.10

1.51 100.67 1.49 99.33 105 100.00 106 100.95

1.48 98.67 1.42 94.67 106 100.95 104 99.05

N 6 6 6 6

Mean 1.51 1.47 105.50 105.17

SD(±) 0.02 0.04 1.05 1.47

CV (%) 1.37 2.56 0.99 1.40

%NOM 100.45 98.11 100.48 100.16

Acceptance criteria:1.%change should be ± 15 or % ratio (stability/comparison) should be within

85-115 %.2.%CV ≤ 15%.3.Mean % Nominal (100 ±15%).

113


Long term stability (at -30°C temp for 65 days)





The mean Accuracy for LQC and HQC samples of Almotriptan from


The plasma samples of Almotriptan were found to be stable for approximately

65 days at -30°C temp.


Table 4.20 Assessment of Long term plasma stability of analyte (Almotriptan)at -30°C.

Low QC 1.50 ng/mL High QC 105.00 ng/mLComparisonsamples (0.0 hr)

Stability samples(65 days)


Stability samples(65 days)

Conc.found

%nominal

Conc.found

%nominal

Conc.found

%nominal

Conc.found

%nominal

1.48 98.67 1.49 99.33 108 102.86 107.00 101.901.44 96.00 1.45 96.67 105 100.00 106.00 100.951.47 98.00 1.49 99.33 105 100.00 103.00 98.101.43 95.33 1.41 94.00 106 100.95 105.00 100.001.42 94.67 1.46 97.33 105 100.00 104.00 99.051.43 95.33 1.41 94.00 106 100.95 107.00 101.90

N 6 6 6 6Mean 1.45 1.45 105.83 105.33SD(±) 0.02 0.04 1.17 1.63CV (%) 1.68 2.48 1.10 1.55%Accuracy 96.33 96.78 100.79 100.32

Acceptance criteria:1. % change should be ± 15 or % Ratio (stability/comparison) should be 85-115 %2. % CV ≤ 15%.3. Mean % Nominal (100 ±15%).

114


Short Term Stock Solution Stability of Almotriptan, and Almotriptan D6 at

Room Temperature



Stock solutions of Almotriptan and Almotriptan-D6 were freshly prepared and

aliquots of stocks were kept at room temperature for 9.5 hr (stability samples).

Aqueous equivalent highest calibration standard of Almotriptan and solution of

Almotriptan D6 were prepared from the stability samples and analyzed. Areas of

stability samples and freshly prepared samples were compared to determine mean %

change during stability period.

The % CV for Almotriptan stock solution from comparison samples was

1.19% and % Ratio (stability/comparison) was 101.63

The % CV for Almotriptan- D6 stock solution from comparison samples was


The % CV for Almotriptan- D6 working solution (Internal standard spiking

solution) from comparison samples was 1.53% and % Ratio (stability/

comparison) was 100.18

Almotriptan, Almotriptan- D6 stock solutions and Almotriptan D6 spiking

solutions were found to be stable at room temperature for 9.5 hr.

Results are present in Table 4.21 and 4.22

115


Table 4.21 Assessment of Short term stock solution stability of Analyte(Almotriptan) and Internal standard (Almotriptan- D6) at Room temperature


Standard stocksolution response

(0.0 hr)

Stability stocksolutionresponse(9.5 hr)

Comparison stocksolution response

(0.0 hr)

StabilityStandard stock

Response(9.5 hr)

1475785 1512936 596784 5869791471179 1500662 574940 5835571515412 1514800 575398 5705071471364 1514309 564602 5622771437948 1496336 545008 5489571491091 1468228 530397 556220

N 6 6 6 6Mean 1477129.83 1501211.83 564521.50 568082.83SD (±) 25558.25 17920.04 23719.09 15117.03CV (%) 1.73 1.19 4.20 2.66

% Ratio101.63 100.63


1. % change should be ± 5 %

Table 4.22 Assessment of short term solution stability of internal standardspiking solution (Almotriptan- D6) at refrigerated conditions


Response (0.0 hr)

Stability solution (Internalstandard spiking solution)

Response (9.5 hr)523207 543590538923 534802545832 535090538602 537538517929 519973528911 528185

N 6 6Mean 532234.00 533196.33SD (±) 10652.34 8161.04CV (%) 2.00 1.53

% Ratio100.18


1. % change should be ± 5%

116


Method validation Conclusion

As all the values obtained were within the Acceptance criteria. The method

stands validated and is suitable for estimation of Almotriptan concentrations in

plasma samples with a single analytical run. The rugged, efficient Liquid-liquid

extraction method provides exceptional sample clean up and constant recoveries using

200µl of plasma. The high extraction efficiency, low limit of quantification, and wide

linear dynamic range make this a suitable method for use in clinical samples from

Bioequivalence and pharmacokinetic studies following oral administration of

Almotriptan fixed dose (12.5 mg) tablets in healthy human subjects.

117

Chapter 4 Almotriptan - Application

4.5 Application

The analytical method described above was used to determine Almotriptan

concentrations in plasma following oral administration of healthy human volunteers.

Each volunteer obtained written informed consent before participating in this study.

Ten healthy volunteers were chosen as subjects and administered 12.5 mg dose

(one 12.5 mg tablet) by oral administration with 240 mL of drinking water. The

reference product, AXERT® (manufactured by Ortho-McNeil-Janssen

Pharmaceuticals, Inc., USA) 12.5 mg and test product, Almotriptan tablets (test tablet)

12.5 mg were used. Study protocol was approved by IEC (Institutional Ethical

committee) as per DCGI (Drug Control General of India). Blood samples were

collected as pre-dose (0) h, 5 min prior to dosing followed by further samples at 0.5,

1.0, 1.5, 2, 2.5, 3, 4, 6, 8, 12, 16 and and 24 hr. After dosing, 3 mL blood was

collected each time in vaccutainers containing K2EDTA. A total of 26 (13 time points

for test and 13 time points for reference) time points were collected from each

volunteer. The samples were centrifuged at 4000 rpm, 10°C, 10 min, and stored at -

30°C until sample analysis. Test and reference were administered to same human

volunteers under fasting conditions separately with proper washing periods (7days

gap between test and reference doses) as per approved protocol by IEC39-41.

The Mean Plasma concentration data for 18 volunteers is represented in

Table 4.25 with respective concentration-time curve is shown in Figure 4.15

118

Chapter 4 Almotriptan - Application

Table 4.23 Almotriptan Mean concentration (ng/mL) data for the subjectsamples obtained from the LC-MS/MS

Time in hoursMean Plasma Concentration data

Test Reference

0 0 0

0.5 25.134 23.876

1 32.543 31.987

1.5 45.654 42.987

2 48.345 45.876

2.5 50.765 49.756

3 45.342 43.987

4 35.653 35.234

6 20.324 17.765

8 12.435 11.564

12 5.875 3.987

16 0.5653 0.498

24 0.01 0.02

Figure 4.15 Mean plasma concentration Vs Time curve for Almotriptan

119

Chapter 4 Almotriptan - Pharmacokinetic Studies


Pharmacokinetic parameters from the human plasma concentration samples

were calculated by a non compartmental statistics model using WinNon-Lin5.0

software (Pharsight, USA). Blood samples were taken for a period of 3 to 5 times of

the terminal elimination half-life (t1/2) and it was considered as area under the

concentration time curve (AUC) ratio higher than 80% as per FDA guidelines38-40.

Plasma Almotriptan concentration-time profiles were visually inspected Cmax and Tmax

values were determined. The AUC0–t was obtained by trapezoidal method. AUC0-∞

was calculated up to the last measureable concentration and extrapolations were

obtained using the last measureable concentration and the terminal elimination rate

constant (Kel). The terminal elimination rate constant (Kel), was estimated from the

slope of the terminal exponential phase of the plasma of Almotriptan concentration–

time curve by means of the linear regression method. The terminal elimination half-

life, t1/2, was then calculated as 0.693/Kel. Regarding AUC0–t , AUC0-∞ and Cmax

bioequivalence was assessed by means of analysis of variance (ANOVA) and

calculating the standard 90% confidence intervals (90% CIs) of the ratios

test/reference (logarithmically transformed data). The bioequivalence was considered

when the ratio of averages of log-transformed data was within 80 to 125% for AUC0–t,

AUC0-∞ and Cmax. Pharmacokinetic data is shown in Table 4.24 and Table 4.25

120


Table 4.24 Almotriptan Pharmacokinetic data

Almotriptan Pharmacokinetic data

Pharmacokinetic

Parameter

Test Reference

Mean Mean

Cmax

(ng/ mL)50.76 49.75

AUC 0-t

(ng h/mL)293.55 272.24

AUC 0-∞

(ng h/mL)

293.55 272.24

Tmax(h) 2.5 2.5

t 1/2 2.01 2.03

Table 4.25 Almotriptan Pharmacokinetic data (Test/Reference)

Pharmacokinetic

Parameter

Cmax AUC 0-t AUC 0-∞

Test/Reference 102.02 107.83 107.82

121




Almotriptan 12.5 mg was bioequivalent with AXERT® (manufactured by Ortho-

McNeil-Janssen Pharmaceuticals, Inc., USA) 12.5 mg tablet under fasting conditions.

In vivo data was predicted by using Liquid Liquid Extraction procedure and

concentrations were found through Liquid Chromatography Tandem Mass

Spectroscopy detection. The Pharmacokinetic parameters assessed were AUC0-t,

AUC0-, Cmax, Tmax, t1/2. The bioequivalence criteria are based on the 90% confidence


The results obtained for Almotriptan was within the acceptance range.

Therefore, it can be concluded that the two Almotriptan formulations

(reference and test) analyzed were bioequivalent in terms of rate and extent of

absorption.

CHAPTER 5

Analytical method development andvalidation of Valacyclovir by High


122

Chapter 5 Valacyclovir - Introduction

5.1 Introduction

Valacyclovir is an antiviral drug. The chemical formula of Valacyclovir

hydrochloride is L-valine, 2-[(2-amino-1,6-dihydro-6oxo-9H-purin-9-yl) methoxy]

ethyl ester, mono hydrochloride with the molecular formula C13H20N6O4.HCl and a

molecular weight of 360.80 (Figure 5.1).

Figure 5.1 Chemical structures of Valacyclovir (A), Valacyclovir-D8 (B)

Valacyclovir hydrochloride is rapidly absorbed from the gastrointestinal tract and

nearly completely converted to acyclovir and L-valine by first-pass intestinal and

hepatic metabolism by enzymatic hydrolysis. Acyclovir is converted to a small extent

to inactive metabolites by aldehyde oxidase and by alcohol and aldehyde

dehydrogenase. Neither Valacyclovir nor acyclovir is metabolized by cytochrome P450

enzymes. Plasma concentrations of unconverted Valacyclovir are low and transient,

generally becoming non-quantifiable by 3 hours after administration. Peak plasma

concentrations of Valacyclovir are generally less than 0.5 µg/mL at all doses. The

absolute bioavailability of acyclovir after oral administration is 54.5% ± 9.1%. The

binding of Valacyclovir to human plasma proteins ranges from 13.5% to 17.9%.

123

Chapter 5 Valacyclovir - Introduction

The binding of acyclovir to human plasma proteins ranges from 9% to 33% 51

Several techniques such as HPLC and LC-MS/MS methods have been

reported in the literature for the quantitative estimation of Valacyclovir and Acyclovir

in biological fluids 52-58 and pharmaceutical dosage forms59-61,A number of methods

were developed in animals such as rabbit55 rat56 and horse57 plasma for quantification

of Valacyclovir and Acyclovir by LC-MS/MS. Only a few methods were reported in

human plasma for quantification of Valacyclovir and Acyclovir 52-54 by LC-MS/MS.

Among all Yadav M, Upadhyav V et al 52 achieved best results. They developed the

method with linearity between the concentration range of 5-1075 ng/mL for

Valacyclovir, 47.6-10225 ng/ mL for Acyclovir using the mobile phase ratio of 0.1%

formic acid: methanol (30:70) on Gemini C18 column . They compared the drug with

fluconazole as an internal standard. They have not achieved sensitive less than

5 ng/mL.52

In bioanalytical method development, usage of deuterated internal standard is

very helpful to find the exact matrix effect at analyte and internal standard retention

times. Till now, as of our knowledge, there is no method reported for comparision of

Valacyclovir with its deuterated internal standard.

124

Chapter 5 Valacyclovir - Experimental

5.2 Experimental Investigations5.2.1 Materials and reagents

Valacyclovir and Valacyclovir-D8 TFA salt, Acyclovir were obtained fromSynfine Research Canada. Formic acid, Acetic acid, Ammonium hydroxide, NH4OH,Ammonium formate were obtained from Merck Mumbai. Methanol, Acetonitrile(HPLC grade) were Obtained from J.T.Baker, Mumbai. Ammonium formate,Ammonia solution (NH4OH, 25%, Reagent grade), Formic acid, Glacial acetic acid(CH3COOH, reagent grade) were Obtained from sd.Fine chemicals Mumbai.Dichloromethane, were purchased from Merck Speciality Chemicals Ltd, Mumbai,India. Human plasma was procured from Navazeevan Blood bank, Hyderabad.Millipore water was used from Milli-Q system.

5.2.2 Instrumentation and equipmentRefer Chapter - 3.2.2

5.2.3 Preparation of Reagents and Solvents



0.1% Formic acid Dilute 1 mL of formic acid to 1000 mL with water.

30% Methanol in 0.1% formic acid Mix 300 mL of methanol with 700 mL of 0.1% formic acid.

1N Acetic acidDilute 60 mL of glacial acetic acid to 1000 mL with water (preparedaily).

30% Formic acid Dilute 30 mL of formic acid to 100 mL with water.

2.5% NH4OH in methanolMix 10 mL of ammonia solution with 90 mL of methanol (preparedaily).

10mM Ammonium formate, PH 5.0Dissolve 1.26 g of ammonium formate into 2 L of water. Adjust PH to5.0 ± 0.05 with formic acid.

10mM Ammonium formate, PH 3.1Dissolve 1.26 g of ammonium formate into 2 L of water. Adjust PH to3.1 ± 0.05 with formic acid.


Reconstitution solutionMix 800 mL of 10mM ammonium formate, PH 3.1 with 200 mL ofmethanol.

20% Methanol(Auto sampler wash)

Mix 200 mL of methanol with 800 mL of water.

Acidified plasma Add approximately 5 mL of 30% formic acid to 100 mL of plasma.

Mobile phaseMix 10mM Ammonium formate PH 5.0 : Methanol in the ratio of 80:20andFilter through 0.45 m filter

125

Chapter 5 Valacyclovir - Experimental




Valacyclovir stock solution 100.o µg/mL 100 mL Methanol

Valacyclovir- D8 stock solution 100.0 µg/mL 100 mL Methanol

Acyclovir stock solution 1000.0 µg/mL 10 mL Methanol


Standard stock solution of Valacyclovir (100 g/mL) was prepared in

50% methanol. From this stock, analytical standards were prepared at concentration

levels of 0.5, 1.0, 5.0, 35.0, 70.0, 140.0, 280.0, 420.0, 560.0 and 700.0 ng/mL by

appropriate dilution with human plasma.

From the standard stock solution, Quality control samples were prepared separately

at LLOQ (0.5 ng/mL) low (1.5ng/mL) medium, (210.0 ng/mL) and high

(490.0 ng/mL) concentrations. All the samples were stored in -80°C freezer until

analysis.

126

Chapter 5 Valacyclovir – Method Development


The goal of this research is to develop and validate a simple, selective,

sensitive, rapid, rugged and reproducible assay method for the quantitative

determination of Valacyclovir from plasma samples. In the way to develop a simple

and easy applicable method for Valacyclovir assay in human plasma for

pharmacokinetic study, HPLC with MS/MS detection was selected as the method of

choice.

Mass parameter Optimization,Chromatographic Optimization and Extraction

optimization to be optimized carefully to achieve the best results.

The MS optimization was performed by direct infusion of solutions of both

Valacyclovir and Valacyclovir-D8 into the ESI source of the mass spectrometer. Other

parameters, such as the nebulizer and the heater gases and Declustering potential(DP),

Entrance potential(EP),Collision energy(CE) was optimized to obtain a better spray

shape, resulting in better ionization and droplet drying to form the protonated ionic

Valacyclovir and Valacyclovir- D8 molecules.

A CAD product ion spectrum for Valacyclovir and Valacyclovir- D8 yielded

high-abundance fragment ions of m/z (amu) 152.0 and m/z (amu) 152.0 respectively

Shown in Figure 5.2 and Figure 5.3.

127


Figure 5.2 Parent and Product ion mass spectra of Valacyclovir

128


Figure 5.3 Parent and Product ion mass spectra of Valacyclovir-D8

Chromatographic conditions, especially, selection of column, composition and

nature of the mobile phase were optimized through several trials to achieve best

resolution and increase the signal of Valacyclovir and Valacyclovir- D8. Separation

was tried using various combinations of mobile phase with variety of columns like

YMC Pack pro C18, RP-Amide, Ascentis Express RP-amide, X-Bridge, Discovery

129


Cyano, Kromasil 100- 5CN. After the MRM channels were tuned, the mobile phase

was changed from more aqueous phase to organic phase to obtain a fast and selective

LC method. A good separation and elution were achieved using 10 mM ammonium

formate (pH 5.0): methanol (80:20 v/v) as the mobile phase, at a flow-rate of

0.25 mL/ min and injection volume of 5 µL. Chromatographic analysis of the analyte

and IS was initiated under isocratic conditions with an aim to develop a simple

separation process with a short run time.

Extraction was performed by different extraction techniques like SPE, LLE,

Precipitation methods. Finally a simple SPE technique was selected in the extraction

of Valacyclovir and Valacyclovir - D8 from the plasma samples.


Chromatographic separation was carried out on a reversed phase Zorbax, SB

C18, 4.6 x 75mm, 3.5 m column using a mixture of 10mM ammonium formate buffer

(PH 5) and methanol (80:20 v/v) as mobile phase with a flow-rate of 0.25 mL/min.

The column temperature was set at 45°C. Retention time of Valacyclovir and

Valacyclovir-D8 was found to be approximately 4.4 ± 0.2 min for both drug and IS.

Sample preparation

A Solid phase extraction procedure was used for extraction of drug and IS

from the plasma samples. For this purpose, 50µL of Valacyclovir- D8 (200ng/mL),

200 µL plasma ( respective concentration of plasma sample) was added into ria vials

then vortexed for 30 seconds followed by 200 µL of acetic acid solution was added

and vortexed briefly. SPE cartridges (Water Oasis, MCX LP, 3 cc, 60 mg) were

130


conditioned with 2 mL of dichloromethane, 1.5 mL of 2.5% NH4OH in methanol.

After that, samples from ria vials were loaded the onto SPE cartridge. Wash the

cartridges with 1.5 mL of water followed by 1.5 mL of methanol. Allowed to dry the

cartridge then eluted cartridges with 1.5 mL of 2.5% NH4OH in methanol into

prelabled ria vials. These samples were evaporated to dryness under the nitrogen

stream at 40°C. Finally, the residue was reconstituted with 400 µL of reconstitution

solution (10mM ammonium formate (PH 3.1): methanol 80:20) and vortexed briefly.

Then the samples were transferred into auto sampler vials injected into the

LC-MS/MS system.


The analytical curves of Valacyclovir were constructed in the concentrations

ranging from 0.5- 700.0 ng/mL in human plasma. Calibration curves were obtained by

weighted linear regression (weighing factor: 1/x2). The ratio of Valacyclovir peak area

to Valacyclovir-D8 peak area was plotted against the ratio of Valacyclovir

concentration in ng/mL. The fitness of calibration curve was confirmed by

back-calculating the concentrations of calibration standards.


The developed method is suitable for estimation of plasma concentrations for

Valacyclovir as a single analytical run, in clinical samples from Pharmacokinetic

studies. This was followed by method validation.

131

Chapter 5 Valacyclovir – Method Validation

5.4Method Validation

The objective of the work is to validate specific HPLC- MS method for the

determination of Valacyclovir in human plasma for clinical / Pharmacokinetic study.

Chromatography


LLOQC, LQC, MQC, HQC, Calibration curve are shown in Figure 5.6 to 5.14.


132



133


Figure 5.6 Chromatogram of LOQ Sample (Valacyclovir & IS)

134


Figure 5.7 Chromatogram of ULOQ Sample (Valacyclovir & IS)

135


Figure 5.8 Chromatogram of LLOQ Sample (Valacyclovir & IS)

136


Figure 5.9 Chromatogram of LQC Sample (Valacyclovir & IS)

137


Figure 5.10 Chromatogram of MQC Sample (Valacyclovir & IS)

138


Figure 5.11 Chromatogram of HQC Sample (Valacyclovir & IS)

Figure 5.12 Calibration Curve of Valacyclovir

139







Table 5.3 Screening of Different batches of blank matrix(Human K2EDTA Plasma) for interference free Valacyclovir blank plasma


Blank plasma AreaAnalyte

(Valacyclovir)RT

Internal standardRT

AP/3451/09/10 0 0AP/3452/09/10 0 0AP/3453/09/10 27 0AP/3454/09/10 0 0AP/3455/09/10 35 0AP/3456/09/10 0 0AP/3457/09/10 28 0AP/3458/09/10 0 0AP/3459/09/10 0 0AP/3460/09/10 0 0

Blank +IS with AP/3451/09/10 0 557914

LOQ with AP/3451/09/10 4932 551115







140


standard. No significant response (≤ 20% for the analyte response and ≤ 5% of the



presented in Table 5.5




(Human K2EDTA Plasma) for Valacyclovir


LLOQArea


Interference withAnalyte(% of

LLOQ Response)

Interferencewith IS(% of IS

Response)

AP/3451/09/10 4930 551118 0 0

AP/3452/09/10 4876 568732 0 0

AP/3454/09/10 4798 563455 0 0

AP/3456/09/10 4689 558974 0 0

AP/3458/09/10 4768 557687 0 0

AP/3459/09/10 4812 568794 0 0



blank.

2. Internal standard response should be ≤ 5% of mean internal standard response

in at least 75% of the blank.

141


Table 5.5 Limit of Quantitation for analyte (Valacyclovir)


Analyte RTLLOQ

responseLLOQ S/N

RATIO

AP/3451/09/10

0 4932 17.4

0 4836 15.9

0 4860 15.2

0 4734 17.8

0 4677 17.4

0 4505 13.6

N 6 6 6Mean 0 4757 16.2

LLOQ was spiked in -AP/3451/09/10





Intra batch accuracy and precision evaluation were assessed by analyzing

1 calibration curve and 6 replicate each of the LLOQ, LQC, MQC, HQC, from


The Intra batch percentage of nominal concentrations for Valacyclovir was



Valacyclovir.


142


Table 5.6 Intra batch (Within-Batch) Accuracy and Precision for determination

of Valacyclovir levels in human plasma

AnalyticalRun ID

LLOQ0.50 ng/mL

Low QC1.50 ng/mL

Mid QC210.00 ng/mL

High QC490.00 ng/mL

Conc.found

%nominal

Conc.found

%nominal

Conc.found

%nominal

Conc.found

%nominal

P&A Batch 1

0.49 97.00 1.43 95.33 206.00 98.10 478.00 97.550.47 94.20 1.43 95.33 204.00 97.14 482.00 98.370.47 94.80 1.55 103.33 208.00 99.05 476.00 97.140.48 96.00 1.42 94.67 206.00 98.10 472.00 96.330.49 97.60 1.44 96.00 204.00 97.14 486.00 99.180.44 87.00 1.44 96.00 205.00 97.62 478.00 97.55

N 6 6 6 6Mean 0.47 1.45 205.50 478.67SD (±) 0.02 0.05 1.52 4.84CV (%) 4.09 3.36 0.74 1.01

%Accuracy94.43 96.78 97.86 97.69



2. Mean % Nominal (100 ±15% and for LLOQ 100±20%).



sets of calibration curves for Valacyclovir and 5 sets of QC samples, 6 replicates each

of the LLOQ, LQC, MQC and HQC.

The inter batch percentage of nominal concentrations for Valacyclovir was



Valacyclovir.


143


Table 5.7 Inter batch (Between-Batch) Accuracy and Precision for determinationof Valacyclovir levels in human plasma

AnalyticalRun ID

LLOQ0.50 ng/mL

Low QC1.50 ng/mL

Mid QC210.00 ng/mL

High QC490.00 ng/mL

Conc.found

%nominal

Conc.found

%nominal

Conc.found

%nominal

Conc.found

%nominal

P&ABatch 1

0.49 97.00 1.43 95.33 206.00 98.10 478.00 97.550.47 94.20 1.43 95.33 204.00 97.14 482.00 98.370.47 94.80 1.55 103.33 208.00 99.05 476.00 97.140.48 96.00 1.42 94.67 206.00 98.10 472.00 96.330.49 97.60 1.44 96.00 204.00 97.14 486.00 99.180.44 87.00 1.44 96.00 205.00 97.62 478.00 97.55

P&ABatch 2

0.47 94.40 1.40 93.33 199.00 94.76 458.00 93.470.49 97.00 1.41 94.00 185.00 88.10 426.00 86.940.47 93.60 1.45 96.67 192.00 91.43 462.00 94.290.46 92.60 1.33 88.67 202.00 96.19 452.00 92.240.47 94.20 1.37 91.33 199.00 94.76 424.00 86.530.47 93.80 1.49 99.33 203.00 96.67 428.00 87.35

P&ABatch 3

0.54 107.20 1.66 110.67 216.00 102.86 497.00 101.430.56 111.60 1.53 102.00 201.00 95.71 458.00 93.470.56 111.60 1.49 99.33 203.00 96.67 456.00 93.060.55 109.40 1.49 99.33 202.00 96.19 476.00 97.140.53 105.60 1.48 98.67 210.00 100.00 470.00 95.920.58 116.60 1.46 97.33 203.00 96.67 470.00 95.92

P&ABatch 4

0.50 100.20 1.47 98.00 189.00 90.00 429.00 87.550.49 98.60 1.38 92.00 199.00 94.76 418.00 85.310.53 105.40 1.46 97.33 202.00 96.19 457.00 93.270.53 106.40 1.55 103.33 212.00 100.95 469.00 95.710.53 106.00 1.51 100.67 212.00 100.95 482.00 98.370.52 103.40 1.51 100.67 199.00 94.76 466.00 95.10

P&ABatch 5

0.54 107.60 1.55 103.33 211.00 100.48 504.00 102.860.50 99.00 1.38 92.00 202.00 96.19 486.00 99.180.59 118.40 1.40 93.33 196.00 93.33 464.00 94.690.50 100.00 1.43 95.33 201.00 95.71 478.00 97.550.56 112.40 1.42 94.67 202.00 96.19 481.00 98.160.56 112.80 1.40 93.33 197.00 93.81 493.00 100.61

N 30 30 30 30Mean 0.51 1.46 202.33 465.87SD(±) 0.04 0.07 6.65 22.27% CV 7.80 4.68 3.29 4.78

%Nominal 102.15 97.18 96.35 95.07

Acceptance criteria: Same as presented in Table 5.6

144


Calibration Curve


Valacyclovir over 0.50 -700.00 ng/mL for calibration range. The correlation

coefficient is greater than 0.9988 for Valacyclovir. Back calculations were made from

the calibration curves to determine Valacyclovir concentrations of each calibration

standard.

Results are presented in Tables 5.8 & 5.9

Table 5.8 Summary of calibration curve parameters for Valacyclovir in human

plasma

Analytical Run ID A B CCoefficient of

regression(r2)

P&A Batch-1 -0.000001537 0.02206 0.0004753 0.9980P&A Batch-2 -0.000004956 0.02394 -0.0003083 0.9994P&A Batch-3 -0.000002361 0.02194 -0.0009788 0.9994P&A Batch-4 -0.000002326 0.02453 -0.0002957 0.9987P&A Batch-5 -0.000001149 0.02193 0.00008181 0.9989

N 5 5 5Mean -000002.4658 0.02288 -0.00021 0.9988SD (±) 000001.48562 0.001255 0.000539 0.00058CV (%) -60.24 5.48 -262.82 0.05

Regression model y = ax2 +bx+c

where:

y = peak area ratio (PAR) of valacyclovir to internal standard.

x = concentration (ng/mL) of valacyclovir in plasma.



145



for Valacyclovir in human plasma.

Analytical RunID


ng/mL1.00

ng/mL5.00

ng/mL35.00

ng/mL70.00

ng/mLP&A Batch-1 0.52 0.91 4.81 37.30 70.70P&A Batch-2 0.51 0.99 4.77 36.50 70.90P&A Batch-3 0.51 0.96 4.90 35.30 72.40P&A Batch-4 0.51 0.98 4.75 35.40 72.20P&A Batch-5 0.50 1.02 4.74 37.10 69.80

N 5 5 5 5 5Mean 0.51 0.97 4.79 36.32 71.20SD(±) 0.01 0.04 0.07 0.93 1.09CV% 1.87 4.05 1.36 2.57 1.53

%Nominal 101.72 97.26 95.88 103.77 101.71

Analytical runID

Nominal Concentration (ng/mL)CS6 CS7 CS8 CS9 CS10

140.00ng/mL

280.00ng/mL

420.00ng/mL

560.00ng/mL

700.00ng/mL


N 5 5 5 5 5Mean 141.20 280.00 414.00 559.80 702.80SD(±) 3.49 6.44 11.55 6.69 7.98CV% 2.47 2.30 2.79 1.19 1.14

%Nominal 100.86 100.00 98.57 99.96 100.40

Acceptance criteria

1. Mean % Nominal (100±15%) except lowest calibration standard.

2. Mean % Nominal (100±20%) for lowest calibration standard (CS1).


146


Recovery

The percentage recovery of Valacyclovir was determined by comparing the

mean peak area of Valacyclovir in extracted LQC, MQC, HQC samples with freshly


The mean % recovery for LQC, MQC, HQC samples of Valacyclovir were


The mean recovery of Valacyclovir across QC levels is 99.17%.

The mean recovery of % CV recovery of Valacyclovir across QC levels is

10.9%.



recovery is 110.84%.

The % CV recovery of IS Valacyclovir- D8 for extracted is 7.9%.


147


Table 5.10 Recovery of Analyte Valacyclovir and Valacyclovir-D8 from humanplasma




Low QC: 1.50 ng/mL

21804 614170 27316 72904919882 608307 23875 65580722415 708869 22084 59993524321 769709 24652 65799618076 575189 22374 62884120168 651502 22846 630215

N 6 6 6 6Mean 88.48SD (±) 9.193%CV 10.4

Medium QC: 210.00 ng/mL

3082852 666064 3005197 6198882873999 667969 2830170 5807642700670 619936 2357070 4805033164543 729545 3024239 6073382813385 623809 2504035 5067652500981 575139 2460142 505789

N 6 6 6 6Mean 105.88SD(±) 9.071%CV 8.6

High QC: 490.00 ng/mL

6632999 643479 6561725 6031866093578 637962 5900211 5356846016152 632263 5769571 5142916211171 626532 6337750 5728156070756 620561 5607332 5049145914901 604012 5636899 509386

N 6 6 6 6Mean 103.15SD(±) 4.225%CV 4.1

Drug ISMean recovery of across QC levels 99.17 110.84Mean SD(±) of across QC levels 10.77 8.742The Mean % CV across QC levels 10.9 7.9


1. The coefficient of variation for mean recovery across LQC, MQC and HQC

shall not exceed 25%.


148


Matrix EffectSamples were prepared at LQC & HQC level in triplicate in each of 6 different



No significant matrix effect found in different sources of human plasma tested

for Valacyclovir, Valacyclovir- D8.

Results are presented in Tables 5.11 and 5.12.

Table 5.11 Assessment of Matrix Effect on determination of Valacyclovirat LQC levels in human plasma




Matrix factor


N 18 18 18Grand Mean 0.013872

SD(±) 0.0013% CV 9.36

Acceptance criteria:1. Mean % Nominal 100±15% of nominal value.2. % CV ≤ 15%.

149


Table 5.12 Assessment of Matrix Effect on determination of Valacyclovirat HQC levels in human plasma



Matrix factor


N 18 18 18Grand Mean 13.55533

SD(±) 0.2438% CV 1.80



2. % CV ≤ 15%.

150


Dilution Integrity





The % accuracy of Valacyclovir nominal concentrations ranged between

90.50% and 92.43% for 1 in 2 dilutions and 1 in 4 dilutions respectively.

The % CV is 4.29% to 4.59%.


Table 5.13 Assessment of Dilution integrity for Valacyclovirat DQC Conc (ng/mL)


Nominal conc: 1125.00 ng/mL


Nominal conc: 1125.00 ng/mLConc. Found % Nominal Conc. Found %Nominal

1090.00 96.89 1090.00 96.89986.00 87.64 1050.00 93.331040.00 92.44 1040.00 92.44976.00 86.76 1050.00 93.331030.00 91.56 949.00 84.36987.00 87.73 1060.00 94.22

N 6 6Mean

%Nominal 90.50 92.43SD (±) 43.73 47.71CV (%) 4.29 4.59


1. % CV ≤ 15%.

2. Mean % Nominal (100 ±15%).

151




P & A batch-2 were kept at auto sampler temperature for approx. 26 hrs after the

initial analysis and were re-injected again after approx. 26 hrs. Concentrations were


The Accuracy of Valacyclovir QC samples in reinjection was between

100.23% and 106.84%.

The Precision (% CV) of Valacyclovir QC samples in reinjection was between

3.27 % and 5.47%.

Valacyclovir was found to be stable at autosampler temperature postextraction (in reconstitution solution) for approx. 26 hrs and reproducible afterreinjection.

Results are presented in Table 5.14Table 5.14 Assessment of Whole Batch Re-injection Reproducibility during

estimation of Valacyclovir in human plasma

AnalyticalRun ID



sample1.47 1.53 429 5161.38 1.45 418 4861.46 1.55 457 4791.55 1.43 469 4641.51 1.45 482 4881.51 1.49 466 474

N 6 6 6 6Mean 1.48 1.48 453.50 484.50SD(±) 0.06 0.05 24.83 17.71%CV 3.96 3.27 5.47 3.65

%NOM 100.23 106.84Acceptance criteria:1. % CV≤ 15% Except LLOQ for which it is ≤ 20%.2. Mean % Nominal (100 ±15% and for LLOQ 100 ±20%).3. 67% 0f the re-injected QCs at each level shall be within ± 20% of their previous

concentration.

152






The Accuracy of Valacyclovir QC samples within the range of 98.43% to116.90%.

The Precision of Valacyclovir QC samples within the range of 0.74% to4.81%.

These results indicated that the method is rugged and reproducible by differentanalyst.


Table 5.15 Ruggedness of the method for estimation of Valacyclovir Plasmalevels in human plasma with different Analyst.

AnalyticalRun ID

LLOQ0.50 ng/mL

Low QC1.50 ng/mL

Mid QC210.00 ng/mL

High QC490.00 ng/mL

AnalystID 1

AnalystID 2

AnalystID 1

AnalystID 2

AnalystID 1

AnalystID 2

AnalystID 1

AnalystID 2

P&A Batch3

0.49 0.54 1.43 1.66 206.00 216.00 478.00 497.000.47 0.56 1.43 1.53 204.00 201.00 482.00 458.000.47 0.56 1.55 1.49 208.00 203.00 476.00 456.000.48 0.55 1.42 1.49 206.00 202.00 472.00 476.000.49 0.53 1.44 1.48 204.00 210.00 486.00 470.000.44 0.58 1.44 1.46 205.00 203.00 478.00 470.00

N 6 6 6 6 6 6 6 6Mean 0.47 0.55 1.45 1.52 205.50 205.83 478.67 471.17SD (±) 0.02 0.02 0.05 0.07 1.52 5.91 4.84 14.81CV (%) 3.93 3.16 3.36 4.81 0.74 2.87 1.01 3.14

%Accuracy116.90 104.59 100.16 98.43



2. Mean % Nominal (100±15% & for LLOQ 100 ±20%).

153






The Accuracy of Valacyclovir QC samples within the range of 98.05% to

114.44%.

The Precision of Valacyclovir QC samples within the range of 0.74% to

6.65%.


analyst.


Table 5.16 Ruggedness of the method for estimation of Valacyclovir Plasmalevels in human plasma with different Analytical column

AnalyticalRun ID

LLOQ0.50 ng/mL

Low QC1.50 ng/mL

Mid QC210.00 ng/mL

High QC490.00 ng/mL

ColumnID

LC/312

ColumnID

LC/345

ColumnID

LC/312

ColumnID

LC/345

ColumnID

LC/312

ColumnID

LC/345

ColumnID

LC/312

ColumnID

LC/345

P&A Batch5

0.49 0.54 1.43 1.55 206.00 211.00 478.00 504.000.47 0.50 1.43 1.38 204.00 202.00 482.00 486.000.47 0.59 1.55 1.40 208.00 196.00 476.00 464.000.48 0.50 1.42 1.43 206.00 201.00 472.00 478.000.49 0.56 1.44 1.42 204.00 202.00 486.00 481.000.44 0.56 1.44 1.40 205.00 197.00 478.00 493.00

N 6 6 6 6 6 6 6 6

Mean 0.47 0.54 1.45 1.43 205.50 201.50 478.67 484.33

SD(±) 0.02 0.04 0.05 0.06 1.52 5.32 4.84 13.63

CV (%) 3.93 6.65 3.36 4.29 0.74 2.64 1.01 2.81

%Accuracy114.44 98.51 98.05 101.18

Acceptance criteria:1. % CV ≤ 15 % except LLOQ for which it is ≤ 20%.2. Mean % Nominal (100±15% & for LLOQ 100 ±20%).

154


Bench Top Stability (at room temp for 41.0 hrs)

Spiked LQC and HQC samples were retrieved from deep freezer and were kept at

room temperature for 41.0 hrs and were processed and analyzed along with freshly prepared

calibration standards, comparison LQC and HQC samples. Concentrations were calculated to

determine mean % change during stability period.

The mean Accuracy for LQC & HQC samples of Valacyclovir from


The plasma samples of Valacyclovir were found to be stable for

approximately 41.0 hrs min at room temperature.


Table 5.17 Assessment of stability of Analyte (Valacyclovir) in Biological matrixat Room temperature



Stability samples(41.0 hrs)



Conc.found

%nominal

Conc.found

%nominal

Conc.found

%nominal

Conc.found

%nominal

1.49 99.33 1.46 97.33 521.00 106.33 485.00 98.981.36 90.67 1.34 89.33 501.00 102.24 479.00 97.761.44 96.00 1.58 105.33 490.00 100.00 451.00 92.041.43 95.33 1.45 96.67 511.00 104.29 476.00 97.141.47 98.00 1.43 95.33 512.00 104.49 483.00 98.571.38 92.00 1.43 95.33 504.00 102.86 492.00 100.41

N 6 6 6 6Mean 1.43 1.45 506.50 477.67SD(±) 0.05 0.08 10.67 14.17

CV (%) 3.53 5.34 2.11 2.97%Accuracy 95.22 96.55 103.37 97.48Acceptance criteria:

1. % Ratio (stability/comparison) should be within 85-115 %.2. %CV ≤ 15%.3. Mean % Nominal (100 ±15%).

155



Samples were prepared at LQC and HQC levels, aliquoted and frozen at -

30±5°C six samples from each concentration were subjected to three freeze and thaw




The mean Accuracy for LQC & HQC samples of Valacyclovir from


The plasma samples of Valacyclovir were found to be stable after 3 cycles at -

30 ±5°C.


Table 5.18 Assessment of Freeze-Thaw stability of Analyte (Valacyclovir)at -30±5°C


samplesStability sample

at 4th cycleComparison


4th cycleConc.found

%nominal

Conc.found

%nominal

Conc.found

%nominal

Conc.found

%nominal

1.49 99.33 1.45 96.67 521 106.33 486 99.181.36 90.67 1.36 90.67 501 102.24 470 95.921.44 96.00 1.4 93.33 490 100.00 467 95.311.43 95.33 1.49 99.33 511 104.29 475 96.941.47 98.00 1.48 98.67 512 104.49 499 101.841.38 92.00 1.39 92.67 504 102.86 495 101.02

N 6 6 6 6Mean 1.43 1.43 506.50 482.00SD(±) 0.05 0.05 10.67 13.36

CV (%) 3.53 3.69 2.11 2.77%Accuracy

95.22 95.22 103.37 98.37

Acceptance criteria

Same as presented in Table 5.17

156







The mean Accuracy change for LQC & HQC samples of Valacyclovir from


Valacyclovir samples were stable for 79 hrs at 2-8°C in autosampler.


Table 5.19 Assessment of Autosampler stability of Analyte (Valacyclovir)

at 2-8°C



(79 hr)Comparison


(79 hr)Conc.found

%nominal

Conc.found

%nominal

Conc.found

%nominal

Conc.found

%nominal

1.49 99.33 1.47 98.00 521 106.33 517 105.511.36 90.67 1.43 95.33 501 102.24 480 97.961.44 96.00 1.47 98.00 490 100.00 463 94.491.43 95.33 1.48 98.67 511 104.29 472 96.331.47 98.00 1.46 97.33 512 104.49 487 99.391.38 92.00 1.41 94.00 504 102.86 494 100.82

N 6 6 6 6Mean 1.43 1.45 506.50 485.50SD(±) 0.05 0.03 10.67 18.90

CV (%) 3.53 1.88 2.11 3.89%Accuracy 95.22 96.89 103.37 99.08


157


Long-term stability (at -30°C temp for 55 days)





The mean Accuracy for LQC and HQC samples of Valacyclovir from


The plasma samples of Valacyclovir were found to be stable for

approximately 34 days at -30°C temp.


Table 5.20 Assessment of Long term plasma stability of Analyte(Valacyclovir) at -30°C.

Low QC 1.50 ng/ml High QC 490.0 ng/mlComparison


(34 days)Comparison samples

(0.0 hr)Stability samples

(34 days)Conc.found

%nominal

Conc.found

%nominal

Conc.found

%nominal

Conc.found

%nominal

1.49 99.33 1.5 100.00 521 106.33 506 103.271.36 90.67 1.38 92.00 501 102.24 496 101.221.44 96.00 1.41 94.00 490 100.00 481 98.161.43 95.33 1.38 92.00 511 104.29 498 101.631.47 98.00 1.42 94.67 512 104.49 504 102.861.38 92.00 1.33 88.67 504 102.86 500 102.04

N 6 6 6 6Mean 1.43 1.40 506.50 497.50SD(±) 0.05 0.06 10.67 8.89CV (%) 3.53 4.05 2.11 1.79%Accuracy 95.22 93.56 103.37 101.53


158


Short Term Stock Solution Stability of Valacyclovir, and Valacyclovir- D8 at

Room Temperature



Stock solutions of Valacyclovir and Valacyclovir-D8 were freshly prepared and

aliquots of stocks were kept at room temperature for 9.0 hr (stability samples).

Aqueous equivalent highest calibration standard of Valacyclovir and solution of

Valacyclovir- D8 were prepared from the stability samples and analyzed. Areas of

stability samples and freshly prepared samples were compared to determine mean %

change during stability period.

The % CV for of Valacyclovir stock solution from comparison samples was


The % CV for of Valacyclovir- D8 stock solution from comparison samples

was 1.06% and % Ratio (stability/comparison) was 100.08

The % CV for Valacyclovir- D8 working solution (Internal standard spiking


comparison) was 101.57

Valacyclovir, Valacyclovir- D8 stock solutions and Valacyclovir- D8 spiking


Results are present in Tables 5.21 and 5.22

159


Table 5.21 Assessment of Short term stock solution stability of Analyte(Valacyclovir) and Internal standard (Valacyclovir- D8) at Room temperature



(0.0 hr)

Stability stocksolution response

(9.0 hr)


(0.0 hr)


Response(9.0 hr)

1125523 1164383 247074 2426271148859 1161817 238708 2450901140661 1158323 244735 2496791157040 1136488 243203 2435941112872 1152873 240936 2438481160514 1157245 240507 243193

N 6 6 6 6Mean 1140911.50 1155188.17 242527.17 244671.83SD (±) 18604.47 9975.97 3072.58 2586.71CV (%) 1.63 0.86 1.27 1.06

%Ratio

101.25 100.88


1. % change should be ± 15 %

Table 5.22 Assessment of Short term solution stability of internal standardspiking solution (Valacyclovir- D8) at refrigerated conditions


Response (0.0 hr)


Response (9.0 hr)240177 242981238872 248870232906 236707241784 241009231518 239699238928 237213

N 6 6Mean 237364.17 241079.83SD (±) 4152.57 4478.55CV (%) 1.75 1.86

% Ratio101.57



160




stands validated and is suitable for estimation of plasma concentrations of

Valacyclovir in a single analytical run. The rugged, efficient Solid phase extraction

method provides exceptional sample clean up and constant recoveries using 200µl of

plasma. The high extraction efficiency, low limit of quantification, and wide linear

dynamic range make this a suitable method for use in clinical samples from

Pharmacokinetic studies following oral administration of Valacyclovir fixed dose

(1000/1000 mg) tablets in healthy human subjects.

161

Chapter 5 Valacyclovir - Application

5.5 Application

In order to evaluate the practical applicability of the developed method, we used it for

drug analysis throughout a project designed for bioequivalence analysis of Valacyclovir

generic product (Test tablet) with the innovator product. For this purpose, twenty healthy,

non-alcoholic, non-smoking, male volunteers were enrolled in this study. These volunteers

were, contracted in APL Research Pvt.Lt.D, Hyderabad, India. The clinical protocol was

approved by the IEC (Institutional Ethics Committee) as per ICMR (Indian council for

medical research) guidelines. The volunteers gave written informed consent after they had

received detailed instructions about the aims, restrictions and possible adverse effect, which

could be experienced as a result of taking the drug. Volunteers were healthy and had no

history of kidneys and metabolic diseases. Also they had a routine physical examination and

the routine laboratory tests found them to be normal. Subjects did not receive any medication

during the 2 weeks period prior to the start and also were not undergoing any pharmacological

treatment during the study period. The study was an open, randomized, two-period, two-

group crossover design over a 7days washout period between doses. The tablets were

administered to the volunteers in the next morning after an overnight fast, with 200 ml of

water. No other food was permitted for consumption during the sampling period. Blood

samples (2 ml) were collected via the catheter into K2EDTA anticoagulant containing tubes at

0, 0.33, 0.5, 0.67, 0.83, 1.0, 1.25, 1.5, 1.75, 2.0, 2.33, 2.67, 3.0, 3.33, 3.67, 4.0, 4.5, 5.0 and

6.0 h post-dosing. The blood samples were centrifuged at 4000 rpm for 10 min and the

plasma was separated stored at -80 °C until assayed for Valacyclovir content 40,41

The Mean Plasma concentration data for 20 volunteers is represented in Table

5.23 with respective concentration-time curve is shown in Figure 5.13.

162

Chapter 5 Valacyclovir - Application

Table 5.23. Valacyclovir Mean concentration (ng/mL) data for the subject samples obtained fromthe LC-MS/MS

Time in hoursMean Plasma Concentration dataTest Reference

0 0 00.33 73.38014 81.519860.5 104.2249 98.44586

0.67 111.6632 105.02260.83 119.4778 105.2104

1 115.1178 114.22831.25 131.4125 143.73941.5 129.6616 157.2031

1.75 115.9391 139.23792 98.02067 125.6938

2.33 77.84638 93.850572.67 40.61176 55.72605

3 22.69424 29.171573.33 14.01033 16.900433.67 10.23967 11.39776

4 7.706762 8.6464.5 5.779857 5.6915245 4.134095 4.2061436 2.475619 2.457048

Figure 5.13 Mean plasma Concentration Vs Time curve for Valacyclovir

163

Chapter 5 Valacyclovir – Pharmacokinetic Studies






concentration time curve (AUC) ratio higher than 80% as per FDA guidelines8-10.

Plasma Valacyclovir concentration-time profiles were visually inspected Cmax and

Tmax values were determined. The AUC0–t was obtained by trapezoidal method.

AUC0-∞ was calculated up to the last measureable concentration and extrapolations

were obtained using the last measureable concentration and the terminal elimination

rate constant (Kel). The terminal elimination rate constant (Kel), was estimated from

the slope of the terminal exponential phase of the plasma of Valacyclovir

concentration-time curve by means of the linear regression method. The terminal

elimination half-life, t1/2, was then calculated as 0.693/Kel. Regarding AUC0–t, AUC0-∞

and Cmax bioequivalence was assessed by means of analysis of variance (ANOVA)

and calculating the standard 90% confidence intervals (90% CIs) of the ratios



AUC0-∞ and Cmax. Pharmacokinetic data is shown in Table 5.24 and Table 5.25.

164

Chapter 5 Valacyclovir – Pharmacokinetic Studies

Table 5.24 Valacyclovir Pharmacokinetic dataValacyclovir Pharmacokinetic data

PharmacokineticParameter

Test ReferenceMean±SD Mean±SD

Cmax

(ng/mL )157.20 ± 56.09 131.41 ± 51.74

AUC 0-t

(ng h/mL)322.32 ± 10.85 286.78 ± 12.78

AUC 0-inf

(ng h/mL)325.78± 8.44 290.89± 10.21

Tmax(hr) 1.5 1.25

t1/20.9760 1.125

Table 5.25 Valacyclovir Pharmacokinetic data (Test/Reference)


Cmax AUC 0-tAUC 0-∞

Test/Reference88.97 89.29 83.59



Valacyclovir 1000 mg was bioequivalent with Valtrex Tablets (GSK, Australia) 1000

mg tablet under fasting conditions.

In vivo data was predicted by using Solid Phase Extraction procedure andconcentrations were found through Liquid Chromatography Tandem MassSpectroscopy detection. The Pharmacokinetic parameters assessed were AUC0-t,AUC0-, Cmax, Tmax, t1/2. The bioequivalence criteria are based on the 90% confidenceintervals whose acceptance range is in between 80% -125%.

The results obtained for Valacyclovir was within the acceptance range.

Therefore, it can be concluded that the two Valacyclovir formulations (reference and

test) analyzed were bioequivalent in terms of rate and extent of absorption.

CHAPTER 6

Analytical method development andvalidation of Memantine by High


165

Chapter 6 Memantine- Introduction

6.1 Introduction

Memantine (1-amino-3,5-dimethyladamantane hydrochloride) (Figure.6.1) is

the first in a novel class of Alzheimer's disease medication acting on the glutamatergic

system by blocking N-methyl-D-aspartate (NMDA) glutamate receptors62

Fig.6.1.Chemical structures of Memantine and Memantine-D6

It is used in Parkinson’s disease, movement disorders and recently it has been

demonstrated to be useful in dementia syndrome. The mode of action is thought to be

due to prevention of damage to retinal ganglion as a result of increased intraocular

pressure. The accumulation of a drug in melanin-rich tissues may have serious

physiological consequences as it could lead to potentially toxic effects. Despite

several investigations into the nature of drug melanin binding, the exact mechanism of

the interaction remains unknown. Memantine is well absorbed, with peak plasma

concentrations (Cmax) ranging from 22 to 46 ng/mL following a single dose of 20 mg.

The time to achieve maximum plasma concentration (Tmax) following single doses of

10-40 mg ranges from 3 to 8 hr. The drug is 45% bound to plasma proteins presenting

a distribution volume of approximately 9-11 L/kg, which suggests an extensive

distribution into tissues. It is poorly metabolized by the liver and 57-82% of the

administered dose is excreted unchanged in the urine with a mean terminal half-life

of 70 hr. 62.

166

Chapter 6 Memantine- Introduction

There were few methods established previously to determine Memantine in a

variety of matrices. These methods include LC-MS62-65 HPLC66-69 GC–MS70 and

Micellar electrokinetic chromatography71 Among all methods LC-MS62-65 has gained

more importance.

M.Y. Liuet.al 62 developed the method with the Linear concentration range of

0.2- 200 ng/mL, with 0.2 ng/mL sensitivity. This sensitivity was improved by

A.A.Almeida et.al 63.They developed the method with the linear concentration range

of 0.1 to 50 ng/mL, with 0.1 ng/mL sensitivity. R.N.Pan et.al 64 developed the method

with the linear concentration range of 0.1 to 25 ng/mL, They used 0.5 mL plasma

usage to get 0.1 ng/mL of sensitivity. M.J.Koeberle et.al65 developed the method in

different melanins.

The reported methods does not show the usage of deuterated internal standard

comparision with analyte which is most important in bioanalytical method

development. All the reported methods develop the method with long run time and

more amount of plasma sample for extraction of drug and Internal Standard (IS).

The purpose of this investigation was to develop a rapid, simple, sensitive and

selective LC-MS/MS method for the quantitative estimation of Memantine in less

volume of human plasma using deuterated internal standard. It is also expected that

this method would provide an efficient solution for pharmacokinetic, bioavailability

or bioequivalence studies of memantine.

.

167

Chapter 6 Memantine - Experimental

6.2 Experimental Investigations


Memantine hydrochloride was obtained from Varda biotech Pvt.Ltd. Andheri,

Mumbai, India Memantine -D6 hydrochloride obtained from Toronto Research

Chemicals, Toronto, Canada. Human plasma (K2EDTA), obtained from Navjeevan

blood bank, Hyderabad. HPLC-grade methanol and acetonitrile were purchased from

Jt.Baker, USA. Diethyl ether, n-hexane were purchased from Lab Scan, Asia Co. Ltd,

Bangkok, Thailand. Formic acid and sodium hydroxide were purchased from Merck

Mumbai, India. Ultra pure water obtained from Milli-Q System.


Refer Chapter - 3.2.2

6.2.3 Preparation of Reagents and SolventsTable 6.1 Preparation of Reagents and Solvents


0.1% Formic acid Dilute 1mL of formic acid to 1L with water.

10mM NaOH Dissolve 0.4 g of sodium hydroxide in 1000 mL of water.

Extraction Solvent Mix 700 mL of diethyl ether with 300 mL of n-hexane.


Reconstitution solution Mix 350 mL of 0.1% formic acid with 650 mL of acetonitrile.

Autosampler wash Mix 800 mL of methanol with 200 mL of water.

Mobile phase0.1% Formic acid: Acetonitrile in the ratio of 35:65 and


168

Chapter 6 Memantine - Experimental



Name of thesolutions

Concentration Volume (mL) Diluent

Memantine stocksolution

100.0 g/mL 50 mL Methanol

Memantine -D6

stock solution100.0 g/mL 50 mL Methanol


Standard stock solutions of Memantine (100.00µg/mL) and Memantine -D6

(100.00µg/mL) were prepared in methanol. The spiking solution for Memantine -D6

was prepared in 50% methanol from respective standard stock solution. Standard

stock solutions and IS spiking solutions were stored in refrigerator conditions (2-8 °C)

until analysis. Standard stock solutions were added to drug-free human plasma to

obtain Memantine concentration levels of 50.00, 100.00, 500.00, 1000.00, 5000.00,

10000.00, 20000.00, 30000.00, 40000.00 and 50000.00 pg/mL for Analytical

standards and 50.0, 150.0, 15000.0, 35000.0 pg/mL for Quality control standards

and stored in a -30°C set point freezer until analysis . The Aqueous standards were

prepared in reconstitution solution (0.1% Formic acid: Acetonitrile 35:65,v/v) for

validation excercises until analysis.

169

Chapter 6 Memantine – Method Development


HPLC-MS has been used as one of the most powerful analytical tools in

clinical pharmacokinetics for its selectivity, sensitivity and reproducibility. The goal

of this work is to develop and validate a simple, sensitive, rapid method for

quantitative estimation of Memantine from plasma samples.

Mass spectrometry parameters, fragmentation pattern and mode of ionization

are the main task in mass spectrometry tuning to obtain respective fragmented ions

and response for both Memantine and Memantine -D6 which were shown in Figures

6.2, 6.3, 6.4 and 6.5. MRM technique was chosen for the assay development. The

MRM parameters were optimized to maximize the response for the analyte. The

instrumental parameters for mass spectroscopy were optimized. The source

temperature was 600°C, The gas pressures of nebulizer, heater, curtain and CAD were

40, 30, 20 and 4 psi respectively. The ion spray voltage, entrance potential,

declustering potential, collision energy and collision cell exit potential were optimized

at 5500, 10, 50, 32 and 12 V respectively. The dwell time 400milli seconds for both

Memantine and Memantine-D6.

170


Figure 6.2 Parent ion mass spectra (Q1) of Memantine

171


Figure 6.3 Product ion mass spectra (Q3) of Memantine

172


Figure 6.4 Parent ion mass spectra (Q1) Memantine-D6

173


Figure 6.5 Product ion mass spectra (Q3) of Memantine-D6

174


Chromatographic conditions optimization

The chromatographic conditions, particularly the composition of mobile phase,

flow-rate of mobile phase, choosing of suitable column, injection volume, column

oven temperature, auto sampler temperature, splitting of sample in to ion source, as

well as a short run time were optimized through several trials to achieve good

resolution and symmetric peak shapes for the Memantine and Memantine- D6. It was

found that a mixture of 0.1% formic acid: acetonitrile (35:65 v/v) could achieve this

purpose and this was finally adopted as the mobile phase. The formic acid was found

to be necessary in order to lower the pH to protonate the Memantine and thus deliver

good peak shape. The percentage of formic acid was optimized to maintain this peak

shape while being consistent with good ionization and fragmentation in the mass

spectrometer. A good separation and elution were achieved using Zorbax SB-C18

(4.6 x 75 mm,3.5m) was selected as the analytical column. The mobile phase

composition was 0.1% Formic acid:acetonitrile (35:65 v/v) at a flow rate of 0.6

mL/min and 10L injection volume was used. Column temperature was set at 30°C.

Memantine -D6 was found to be appropriate internal standard. Retention time of

Memantine and Memantine- D6 were found to be 1.45 ± 0.2 min, with overall runtime

of 3.5 min.

Extraction optimization

Initially we tried with several extraction techniques like SPE, Precipitation,

Liquid-liquid extraction (LLE). Finally Liquid-liquid extraction was used for the

175


sample preparation in this work. LLE can be helpful to clean the samples. Clean

samples are essential for minimizing ion suppression and matrix effect in LC-MS/ MS

analyses. Several organic solvents and their mixtures in different combinations and

ratios were evaluated. Finally, diethyl ether/n-hexane (70:30) were found to be

optimal, which produced a clean chromatogram for a blank plasma sample and

yielded the highest recovery for the Memantine and Memantine-D6 from the plasma.

Memantine-D6 hydrochloride was used as internal standard for the present purpose.

Clean chromatograms were obtained and no significant direct interferences in the

MRM channels at the relevant retention times were observed.

Sample preparation

Liquid-liquid extraction method was used to isolate Memantine and

Memantine-D6 from human plasma. For this purpose,50 µL of Memantine-D6

(25 ng/mL) and 100 µl of plasma sample and 100 µL of 10mM NaOH were added

into labeled 5 mL ria vials and vortexed briefly. This was followed by addition of

3 ml extraction solvent (diethyl ether: n-hexane 70:30 v/v) and vortexed for 10 min.

Then samples were centrifuged at 4000 rpm for 5 min at ambient temperature. The

supernatant from each sample was transferred into labelled vials by using the dry-ice

acetone flash freeze technique. The supernatant of each sample was evaporated to

dryness under nitrogen stream at 40°C. The dried residue was reconstituted with 400

µl of 0.1% of formic acid: acetonitrile (35:65 v/v) mixture and vortexed until

dissolved. A 20 µL of each sample was transferred into auto sampler vials and

injected into HPLC connected with mass spectrometer.

176



The analytical curves were constructed using values ranging from 50.0 to

50000.0 pg/mL of Memantine in human plasma. Calibration curves were obtained by

weighted 1/Conc2 quadratic regression analysis

y = ax2+bx+c

where, x = concentration (pg/mL) of memantine in plasma.

y = peak area ratio (PAR) of memantine to internal standard.

The ratio of memantine peak area to Memantine- D6 peak area was plotted

against the ratio of Memantine concentration in pg/ mL. Calibration curve standard

samples and quality control samples were prepared in replicates (n=6) for analysis.

Accuracy and precision for the back calculated concentrations of the calibration

points should be within ≤ 15 and ± 15% of their nominal values. However, for LLOQ,

the precision and accuracy should be within ≤ 20 and ± 20%.


The developed method is suitable for estimation of Memantine concentrations

in plasma as a single analytical run, in clinical samples from Pharmacokinetic studies.

This was followed by method validation.

177

Chapter 6 Memantine – Method Validation


The objective of the work is to validate specific HPLC-MS method for the

determination of Memantine in human plasma for clinical / Pharmacokinetic study.

Chromatography


LLOQC, LQC, MQC, HQC, Calibration curve are shown in Figure 6.6 to 6.14.


178



179


Figure 6.8 Chromatogram of LOQ Sample (Memantine & IS)

180


Figure 6.9 Chromatogram of ULOQ Sample (Memantine & IS)

181


Figure 6.10 Chromatogram of LLOQ Sample (Memantine & IS)

182


Figure 6.11 Chromatogram of LQC Sample (Memantine & IS)

183


Figure 6.12 Chromatogram of MQC Sample (Memantine & IS)

184


Figure 6.13 Chromatogram of HQC Sample (Memantine & IS)

185


Figure 6.14 Calibration Curve of Memantine

186







Table 6.3 Screening of Different batches of blank matrix (Human K2EDTAPlasma) for interference free Memantine blank plasma


Blank plasma Area

Analyte (Memantine) RTInternalstandard

RT

AP/2203463 A 4690

AP/5519618 A 012

AP/5519599 A 00

AP/5519615 A 0213

AP/5519602 A 0321

AP/5519602 A 1210

AP/5519616 A 2265

AP/5519617 A 460

AP/5519618 A 00

AP/5519619A 570

Blank+IS with AP/2203463 A 01326218

LOQ with AP/2203463 A 3912 1321436

187








standard. No significant response (≤ 20% for the analyte response and ≤ 5% of the



presented in Table 6.4.



188


Table 6.4 Specificity of Different batches of blank matrix(Human K2EDTA Plasma) for Memantine


BlankArea

LLOQResponse)


Interferencewith Analyte(% of LLOQ

Response)

Interferencewith IS(% ofIS Response)

AP/2203463 A 469 39121336014

11.990.03

AP/5519618 A 0 36181320435

0.00 0.00

AP/5519599 A 0 39301220421

0.00 0.00

AP/5519615 A 0 37121120568

0.00 0.00

AP/5519602 A 0 38361321243

0.00 0.00

AP/5519602 A 121 38921226542

3.110.01

AP/5519616 A 22 41271228763

0.530.01

AP/5519617 A 46 38301326345

1.200.01

AP/5519618 A 0 40871221343

0.00 0.00

AP/5519619A 57 413212287564

1.380.00



blank.

2. Internal standard response should be ≤5% of mean internal standard response in

at least 75% of the blank.

189


Table 6.5 Limit of Quantitation for analyte (Memantine)


Analyte RTLLOQ

responseLLOQ S/N

RATIO

AP/2203463 A

469.00 4165.00 11.80121.00 3830.00 11.50111.00 3812.00 13.7023.00 3063.00 12.1034.00 3100.00 10.30124.00 3199.00 12.20

N 6 6 6Mean 147.00 3528.17 11.93

LLOQ was spiked in AP/2203463 A





Intra batch accuracy and precision evaluation were assessed by analyzing

1 calibration curve and 6 replicate each of the LLOQ, LQC, MQC, HQC, from


The Intra batch percentage of nominal concentrations for Memantine was



Memantine.


190


Table 6.6 Intra batch (Within-Batch) Accuracy and Precision for determinationMemantine levels in human plasma

AnalyticalRun ID

LLOQ50.00 pg/mL

Low QC150.00 pg/mL

Mid QC15000.00 pg/mL

High QC35000.00 pg/mL

Conc.found

%nominal

Conc.found

%nominal

Conc.found

%nominal

Conc.found

%nominal

P&A Batch 1

52.2 104.4 145.2 96.8 15084.9 100.6 35686.7 102.047.3 94.6 145 96.7 15156.3 101.0 35509.8 101.547.8 95.6 145.9 97.3 14893.4 99.3 35167.2 100.548.2 96.4 140.9 93.9 14446.2 96.3 34563.7 98.848.8 97.6 145.3 96.9 14471.1 96.5 33679.7 96.249.3 98.6 138.1 92.1 14426.3 96.2 35003.9 100.0

N 6 6 6 6Mean 48.9 143.4 14746.4 34935.2SD (±) 1.8 3.2 338.4 730.4% CV 3.7 2.2 2.3 2.1

%Accuracy 87.8 95.6 98.3 99.8



2. Mean % Nominal (100 ±15% and for LLOQ 100 ±20%).



sets of calibration curves for Memantine and 5 sets of QC samples, 6 replicates each

of the LLOQ, LQC, MQC and HQC.

The inter batch percentage of nominal concentrations for Memantine was



Memantine.


191


Table 6.7 Inter batch (Between-Batch) Accuracy and Precision for determinationMemantine levels in human plasma

AnalyticalRun ID

LLOQ50.00 pg/mL

Low QC150.00 pg/mL



Conc.found

%nominal

Conc.found

%nominal

Conc.found

%nominal

Conc.found

%nominal

P&ABatch 1

52.20 104.4 145.20 96.8 15084.90 100.6 35686.70 102.047.30 94.6 145.00 96.7 15156.30 101.0 35509.80 101.547.80 95.6 145.90 97.3 14893.40 99.3 35167.20 100.548.20 96.4 140.90 93.9 14446.20 96.3 34563.70 98.848.80 97.6 145.30 96.9 14471.10 96.5 33679.70 96.249.30 98.6 138.10 92.1 14426.30 96.2 35003.90 100.0

P&ABatch 2

45.30 90.6 141.90 94.6 14455.80 96.4 34275.30 97.946.20 92.4 139.80 93.2 14975.30 99.8 34108.90 97.550.80 101.6 145.20 96.8 14724.90 98.2 34746.70 99.364.30 108.6 146.50 97.7 14837.40 98.9 34570.00 98.845.80 91.6 144.10 96.1 15046.60 100.3 34594.60 98.846.70 93.4 155.40 103.6 14894.70 99.3 34844.90 99.6

P&ABatch 3

51.50 103 140.40 93.6 14890.50 99.3 34512.00 98.649.70 99.4 146.10 97.4 14896.10 99.3 34375.30 98.249.80 99.6 142.10 94.7 14837.70 98.9 35008.70 100.050.10 100.2 147.30 98.2 14885.80 99.2 34752.50 99.352.50 105 145.50 97.0 14688.60 97.9 34359.90 98.249.80 99.6 146.30 97.5 14732.20 98.2 34283.80 98.0

P&ABatch 4

51.80 103.6 141.80 94.5 14966.90 99.8 35031.40 100.148.10 96.2 147.50 98.3 15030.90 100.2 35447.80 101.347.40 94.8 143.80 95.9 14641.20 97.6 35198.20 100.649.20 98.4 144.70 96.5 14475.20 96.5 35137.40 100.450.70 101.4 146.30 97.5 14729.10 98.2 34871.50 99.652.60 105.2 145.60 97.1 14713.20 98.1 35274.10 100.8

P&ABatch 5

46.50 93 139.30 92.9 14587.90 97.3 34265.80 97.944.40 88.8 136.50 91.0 14385.60 95.9 34730.00 99.246.80 93.6 137.60 91.7 14339.30 95.6 34546.50 98.743.20 86.4 1390 92.7 14553.60 97.0 34292.10 98.046.20 92.4 138.40 92.3 14624.00 97.5 33593.40 96.048.20 96.4 142.40 94.9 14183.70 94.6 34543.10 98.7

N 30 30 30 30Mean 49.00 143.50 14719.10 34699.20SD(±) 3.80 3.90 248.30 498.40% CV 7.80 2.70 1.70 1.40

%Nominal98.00 95.70 98.10 99.10


192


Calibration Curve


Memantine over 50.00 - 50000.00 pg/mL for calibration range. The correlation

coefficient is greater than 0.9986 for Memantine. Back calculations were made from

the calibration curves to determine Memantine concentrations of each calibration

standard.


Table 6.8 Summary of calibration curve parameters for Memantine in humanplasma

AnalyticalRun ID A B C

Coefficientof

regression(r2)

P&A Batch-1 -0.00000000009204 0.00008698 0.0003389 0.9999P&A Batch-2 -0.00000000006902 0.00009203 0.0003816 0.9997P&A Batch-3 -0.0000000001003 0.00009350 0.0001899 0.9998P&A Batch-4 -0.0000000001074 0.00009355 0.0001643 0.9999P&A Batch-5 -0.0000000001026 0.00009364 0.0004198 0.9997

N 5 5 5Mean -0.00000000009427 0.00009194 0.0002989 0.9998SD (±) -0.00000000001517 0.000002852 0.0001152 0.0001CV (%) -16.1 3.1 38.5 0.0

Regression Footnote(s): Resp. = A * (Conc.2) + B * Conc. + C



193



for Memantine in human plasma.

Analytical RunID


pg/mL100.00pg/mL

500.00pg/mL

1000.00pg/mL

5000.00pg/mL


N 5 5 5 5 5Mean 49.90 100.20 502.70 1000.50 5005.00SD(±) 0.40 1.90 6.80 11.50 38.70% CV 0.80 1.90 1.40 1.10 0.80

%Nominal 99.80 100.20 100.50 100.10 100.10

Analytical runID

Nominal Concentration (ng/mL)CS6 CS7 CS8 CS9 CS10

10000.00pg/mL

20000.00pg/mL

30000.00pg/mL

40000.00pg/mL

50000.00pg/mL


N 5 5 5 5 4Mean 9978.90 19871.00 29759.80 40310.80 50084.80SD(±) 160.50 303.40 508.40 123.80 266.70CV% 1.60 1.50 1.70 0.30 0.50

%Nominal 99.80 99.40 99.20 100.80 100.20Acceptance criteria

1. Mean % Nominal (100 ±15%) except lowest calibration standard.

2. Mean % Nominal (100 ±20%) for lowest calibration standard (CS1).


194


Recovery

The percentage recovery of Memantine was determined by comparing the

mean peak area of Memantine in extracted LQC, MQC, HQC samples with freshly


The mean % recovery for LQC, MQC, HQC samples of Memantine were


The mean recovery of Memantine across QC levels is 86.07%.

The mean recovery of % CV recovery of Memantine across QC levels is

19.6%.



recovery is 80.31%.

The %CV recovery of IS Memantine -D6 for extracted is 19.60%.


195


Table 6.10 Recovery of Analyte (Memantine) and Memantine –D6 from human plasma




Low QC: 150.00 pg/mL

16666 1240218 17262 136124716879 1274261 17689 142883817207 1252007 18675 147644010984 792193 17456 1422982

6164 451731 18606 148673017975 1224717 18413 1451081

N 6 6 6 6% Recovery 79.45

SD (±) 26.203% CV 33.0

Medium QC: 15000.00 pg/mL

1544005 1172972 1711122 13420541506570 1105276 1784561 14065721604998 1197278 1820986 14394491712284 1267735 1844421 14328291754830 1281384 1819181 14104391735332 1279914 1823941 1444836

N 6 6 6 6% Recovery 91.25

SD(±) 5.879% CV 6.4

High QC: 35000.00 pg/mL

3640155 1184317 3935392 13490423506538 1146264 4051762 13966133811506 1223688 4097227 14010812557367 825131 4102019 14006373969619 1279905 4085863 13864923794514 1214894 4039585 1383573

N 6 6 6 6% Recovery 87.52

SD(±) 12.591% CV 14.4

Drug ISMean recovery of across QC levels 86.07 80.31Mean SD(±) of across QC levels 16.865 15.704The Mean % CV across QC levels 19.60 19.60


1. The coefficient of variation for mean recovery across LQC, MQC and HQCshall not exceed 25%.


196


Matrix Effect

Samples were prepared at LQC & HQC level in triplicate in each of 6 different



No significant matrix effect found in different sources of human plasma testedfor Memantine, Memantine-D6.

Results are presented in Tables 6.11 and 6.12.Table 6.11 Assessment of Matrix Effect on determination of Memantine at LQC

levels in human plasmaIdentification of

matrixDrug responsein Matrix atLQC Level


Matrix factor

AP/2203463 A 3937 890913 0.004419AP/2203463 A 3705 852767 0.004345AP/2203463 A 4054 901294 0.004498AP/5519618 A 4106 887716 0.004625AP/5519618 A 3844 889182 0.004323AP/5519618 A 3889 875217 0.004443AP/5519599 A 3668 852101 0.004305AP/5519599 A 3799 853610 0.004451AP/5519599 A 3794 867318 0.004374AP/5519615 A 3615 842646 0.00429AP/5519615 A 3757 858802 0.004375AP/5519615 A 3948 873326 0.004521AP/5519602 A 3898 837676 0.004653AP/5519602 A 3525 849627 0.004149AP/5519602 A 3746 874224 0.004285AP/5519616 A 3728 845726 0.004408AP/5519616 A 3443 828837 0.004154AP/5519616 A 3584 841163 0.004261

N 18 18 18Grand Mean 0.004382

SD(±) 0.0001CV (%) 3.16


1. Mean % Nominal 100 ± 15% of nominal value.2. %CV ≤ 15%.

197


Table 6.12 Assessment of Matrix Effect on determination of Memantine at HQClevels in human plasma



Matrix factor

AP/2203463 A 3151645 841230 3.746472AP/2203463 A 3211633 856335 3.75044AP/2203463 A 3105236 828311 3.748877AP/5519618 A 3082605 814024 3.786872AP/5519618 A 3191090 849578 3.756088AP/5519618 A 3182366 855932 3.718013AP/5519599 A 3139352 834447 3.762195AP/5519599 A 3212771 858024 3.744384AP/5519599 A 3065968 821738 3.731077AP/5519615 A 3076849 827108 3.720009AP/5519615 A 3109595 836216 3.71865AP/5519615 A 3094915 829896 3.729281AP/5519602 A 3110384 827635 3.758159AP/5519602 A 3106450 824714 3.7667AP/5519602 A 3041956 809277 3.758856AP/5519616 A 3032206 805064 3.766416AP/5519616 A 3109184 842222 3.691644AP/5519616 A 3099056 831666 3.726323

N 18 18 18Grand Mean 3.743359

SD(±) 0.0232% CV 0.62

Acceptance criteria:1. Mean % Nominal 100 ±15% of nominal value.2. % CV ≤ 15%.

Matrix Factor = Peak response ratio (analyte/IS) in the presence of extracted matrixPeak response ratio (analyte/IS) in the absence of extracted matrix

198


Dilution Integrity





The % accuracy of Memantine nominal concentrations ranged between

98.96 % and 98.72 % for 1 in 2 dilutions and 1 in 4 dilutions respectively.

The % CV is 0.66% to 0.63%.


Table 6.13 Assessment of Dilution integrity for Memantineat DQC Conc (Pg/mL)


Nominal conc: 75000.0 pg/mL


Nominal conc: 75000.0 Pg/mLConc. Found % Nominal Conc. Found %Nominal73700 98.27 73600 98.174300 99.07 73700 98.374300 99.07 73700 98.373600 98.13 74800 99.774500 99.33 74000 98.774900 99.87 74400 99.2

N 6 6Mean

%Nominal 98.96 98.72SD (±) 0.66 0.62CV (%) 0.66 0.63


1. % CV ≤ 15%.

2. Mean % Nominal (100 ±15%).

199




P & A batch-2 were kept at auto sampler temperature for approx 27 hrs after the

initial analysis and were re-injected again after approx. 27 hrs. Concentrations were


The Accuracy of Memantine QC samples in reinjection was between 96.44%

and 98.71%.

The Precision (% CV) of Memantine QC samples in reinjection was between

0.76 % and 3.60%.

Memantine was found to be stable at autosampler temperature post extraction

(in reconstitution solution) for approx. 27 hrs and reproducible after

reinjection.


Table 6.14 Assessment of Whole Batch Re-injection Reproducibility during

estimation of Memantine in human plasma

AnalyticalRun ID

Low QC 150.00 pg/mL High QC 35000.00 pg/mL


sample142 145.00 34300 34800.00140 146.00 34100 34400.00145 142.00 34700 34700.00147 150.00 34600 34200.00144 142.00 34600 34100.00155 143.00 34800 35100.00

N 6 6 6 6Mean 145.50 144.67 34516.67 34550.00SD(±) 5.24 3.08 263.94 383.41%CV 3.60 2.13 0.76 1.11

%NOM 97.00 96.44 98.62 98.71Acceptance criteria:

1. % CV≤ 15% Except LLOQ for which it is ≤ 20%.

2. Mean % Nominal (100±15% and for LLOQ 100 ± 20%).3. 67% 0f the re-injected QCs at each level shall be within ±20% of their previous

concentration.

200






The Accuracy of Memantine QC samples within the range of 95.60% to

101.13%.

The Precision of Memantine QC samples within the range of 0.61% to 3.58%.


analyst.


Table 6.15 Ruggedness of the method for estimation of Memantine Plasma levelsin human plasma with different analyst

AnalyticalRun ID

LLOQ50.00 pg/mL

Low QC150.00 pg/mL



AnalystID 1

AnalystID 2

AnalystID 1

AnalystID 2

AnalystID 1

AnalystID 2

AnalystID 1

AnalystID 2

P&ABatch 3

52.2 51.5 145.2 140.4 15084.9 14890.5 34512 35686.747.3 49.7 145 146.1 15156.3 14896.1 34375.3 35509.847.8 49.8 145.9 142.1 14893.4 14837.7 35008.7 35167.248.2 50.1 140.9 147.3 14446.2 14885.8 34752.5 34563.748.8 52.5 145.3 145.5 14471.1 14688.6 34359.9 33679.749.3 49.8 138.1 146.3 14426.3 14732.2 34283.8 35003.9

N 6 6 6 6 6 6 6 6Mean 48.93 50.57 143.40 144.62 14746.37 14821.82 34548.70 34935.17SD (±) 1.75 1.16 3.16 2.73 338.40 89.84 279.42 730.45CV (%) 3.58 2.30 2.21 1.88 2.29 0.61 0.81 2.09%NOM 97.87 101.13 95.60 96.41 98.31 98.81 98.71 99.81



2. Mean % Nominal (100±15% & for LLOQ 100 ± 20%).

201






The Accuracy of Memantine QC samples within the range of 91.77% to

98.71%.

The Precision of Memantine QC samples within the range of 0.81% to

3.91%.


analyst.


Table 6.16 Ruggedness of the method for estimation of Memantine Plasma levelsin human plasma with different Analytical column

AnalyticalRun ID

LLOQ5.00 ng/mL

Low QC15.00 ng/mL

Mid QC700.00 ng/mL

High QC1400.00 ng/mL

ColumnID

LC/221

ColumnID

LC/245

ColumnID

LC/221

ColumnID

LC/245

ColumnID

LC/221

ColumnID

LC/245

ColumnID

LC/221

ColumnID

LC/245

P&ABatch 5

52.2 46.5 145.2 139.3 15084.9 14587.9 34512 34265.847.3 44.4 145 136.5 15156.3 14385.6 34375.3 3473047.8 46.8 145.9 137.6 14893.4 14339.3 35008.7 34546.548.2 43.2 140.9 139 14446.2 14553.6 34752.5 34292.148.8 46.2 145.3 138.4 14471.1 14624 34359.9 33593.449.3 48.2 138.1 142.4 14426.3 14183.7 34283.8 34543.1

N 6 6 6 6 6 6 6 6Mean 48.93 45.88 143.40 138.87 14746.37 14445.68 34548.70 34328.48SD(±) 1.75 1.79 3.16 2.01 338.40 171.60 279.42 400.10

CV (%) 3.58 3.91 2.21 1.44 2.29 1.19 0.81 1.17%NOM 97.87 91.77 95.60 92.58 98.31 96.30 98.71 98.08

Acceptance criteria:1. % CV ≤ 15 % except LLOQ for which it is ≤ 20%.2. Mean % Nominal (100 ± 15% & for LLOQ 100 ± 20%).

202


Bench Top Stability (at room temp for 26.0 hrs)


kept at room temperature for 26 hrs and were processed and analyzed along with



The mean Accuracy for LQC & HQC samples of Memantine from comparison

samples were 92.67% and 98.00% respectively.

The plasma samples of Memantine were found to be stable for approximately

26.0 hrs min at room temperature.


Table 6.17 Assessment of stability of Analyte (Memantine) in Biological matrix atRoom temperature


samples(0.00 hr)




Conc.found

%nominal

Conc.found

%nominal

Conc.found

%nominal

Conc.found

%nominal

135 90.00 143 95.33 34800 99.43 34200 97.71140 93.33 139 92.67 34400 98.29 34600 98.86140 93.33 141 94.00 33800 96.57 34100 97.43139 92.67 140 93.33 34200 97.71 33900 96.86142 94.67 140 93.33 34200 97.71 34500 98.57138 92.00 139 92.67 34400 98.29 34000 97.14

NMean 139.00 140.33 34300.00 34216.67SD(±) 2.37 1.51 328.63 278.69

CV (%) 1.70 1.07 0.96 0.81

%NOM92.67 93.56 98.00 97.76


85-115 %.2. % CV ≤ 15%.3. Mean % Nominal (100 ± 15%).

203




-30±5°C six samples from each concentration were subjected to three freeze and thaw




The mean Accuracy for LQC & HQC samples of Memantine from comparison

samples were 94.56% and 97.52% respectively.

The plasma samples of Memantine were found to be stable after 3 cycles at-30±5°C.

Results are present in Table 6.18.Table 6.18 Assessment of Freeze-Thaw stability of Analyte (Memantine) at

-30±5°CLow QC 150.00 pg/mL High QC 35000.00 pg/mL

Comparisonsamples

Stability sampleat 4th cycle

Comparisonsamples

Stability sample at4th cycle

Conc.found

%nominal

Conc.found

%nominal

Conc.found

%nominal

Conc.found

%nominal

135 90.00 143 95.33 34800 99.43 34500 98.57140 93.33 141 94.00 34400 98.29 34200 97.71140 93.33 147 98.00 33800 96.57 34500 98.57139 92.67 142 94.67 34200 97.71 34000 97.14142 94.67 142 94.67 34200 97.71 33900 96.86138 92.00 136 90.67 34400 98.29 33700 96.29

N 6 6 6 6Mean 139.00 141.83 34300.00 34133.33SD(±) 2.37 3.54 328.63 326.60

CV (%) 1.70 2.50 0.96 0.96%Accuracy 92.67 94.56 98.00 97.52

Acceptance criteria

1. % change should be ± 15 or % Ratio (stability/comparison) should be within85-115 %.

2. %CV ≤ 15%.3. Mean % Nominal (100±15%)

204







The mean Accuracy change for LQC & HQC samples of Memantine from


Memantine samples were stable for 79 hrs at 2-8°C in autosampler.


Table 6.19 Assessment of Autosampler stability of Analyte (Memantine) at 2-8°C



(79 hr)Comparison


(79 hr)Conc.found

%nominal

Conc.found

%nominal

Conc.found

%nominal

Conc.found

%nominal

135 90.00 141 94.00 34800 99.43 34500 98.57140 93.33 142 94.67 34400 98.29 34600 98.86140 93.33 143 95.33 33800 96.57 34000 97.14139 92.67 142 94.67 34200 97.71 34600 98.86142 94.67 146 97.33 34200 97.71 35100 100.29138 92.00 145 96.67 34400 98.29 34500 98.57

N 6 6 6 6Mean 139.00 143.17 34300.00 34550.00SD(±) 2.37 1.94 328.63 350.71

CV (%) 1.70 1.36 0.96 1.02

%NOM92.67 95.45 98.00 98.72

Acceptance criteria:1. % change should be ± 15 or % ratio (stability/comparison) should be within 85-

115 %.2. %CV ≤ 15%.3. Mean % Nominal (100 ±15%).

205


Longterm stability (at -30°C temp for 76 days)

Spiked LQC and HQC samples were retrieved from deep freezer after 76days




The mean Accuracy for LQC and HQC samples of Memantine from


The plasma samples of Memantine were found to be stable for approximately

76 days at -30°C temp.


Table 6.20 Assessment of Long term plasma stability of Analyte (Memantine) at -30°C.



(76 days)Comparison


(76 days)Conc.found

%nominal

Conc.found

%nominal

Conc.found

%nominal

Conc.found

%nominal

135 90.00 143 95.33 34800 99.43 34800 99.43140 93.33 138 92.00 34400 98.29 33500 95.71140 93.33 138 92.00 33800 96.57 34500 98.57139 92.67 140 93.33 34200 97.71 34700 99.14142 94.67 146 97.33 34200 97.71 33700 96.29138 92.00 140 93.33 34400 98.29 33900 96.86

N 6 6 6 6Mean 139.00 140.83 34300.00 34183.33SD(±) 2.37 3.13 328.63 552.87CV (%) 1.70 2.22 0.96 1.62

%Accuracy 92.67 93.89 98.00 97.67


85-115 %.2. %CV ≤ 15%.3. Mean % Nominal (100±15%).

206


Short Term Stock Solution Stability of Memantine and Memantine D6 at Room

Temperature



Stock solutions of Memantine and Memantine-D6 were freshly prepared and aliquots

of stocks were kept at room temperature for 9.0 hr (stability samples). Aqueous

equivalent highest calibration standard of Memantine and solution of Memantine D6

were prepared from the stability samples and analyzed. Areas of stability samples and

freshly prepared samples were compared to determine mean % change during stability

period.

The % CV for of Memantine stock solution from comparison samples was


The % CV for of Memantine- D6 stock solution from comparison samples was

1.00% and % Ratio (stability/comparison) was 100.15.

The % CV for Memantine- D6 working solution (Internal standard spiking


comparison) was 99.81.

Memantine, Memantine- D6 stock solutions and Memantine D6 working


Results are present in Table 6.21 and 6.22.

207


Table 6.21 Assessment of Short term stock solution stability of Analyte(Memantine) and Internal standard (Memantine -D6) at Room temperature



(0.0 hr)

Stability stocksolution response

(9.0 hr)


(0.0 hr)


Response(9.0 hr)

1917103 1885033 1584157 15797851879449 1948746 1553139 15901501961019 1912343 1569834 15658121905164 1897432 1572770 15718241880625 1896795 1561019 15619702007600 1992436 1559263 1544637

N 6 6 6 6Mean 1925160 1922131 1566697 1569030

SD (±) 50239.11 40927.18 11161.52 15650.87

CV (%) 2.61 2.13 0.71 1.00

%Ratio 99.84 100.15

Acceptance criteria:1. % change should be ± 5 %

Table 6.22 Assessment of short term solution stability of internal standardSpiking solution (Memantine -D6) at refrigerated conditons


Response (0.0 hr)


Response (9.0 hr)1535237 15650851531198 15516471539471 15337411557196 15560201568876 15100881556709 1554489

N 6 6Mean 1548115 1545178SD (±) 14927.07 20022.45CV (%) 0.96 1.30% Ratio 99.81



208




stands validated and is suitable for estimation of plasma Memantine concentrations in

a single analytical run. The rugged, efficient Liquid-liquid extraction method provides

exceptional sample clean up and constant recoveries using 100µL of plasma. The high

extraction efficiency, low limit of quantification, and wide linear dynamic range make

this a suitable method for use in clinical samples from Pharmacokinetic studies

following oral administration of Memantine fixed dose (10/10 mg) tablets in 20

healthy human subjects.

209

Chapter 6 Memantine - Application

6.5 Application

The above described analytical method was applied to determine plasma

concentrations of Memantine following oral administration in healthy human

volunteers. These volunteers have informed consent before participation of study

and study protocol was approved by IEC (Institutional Ethics Committee) as per

DCGI (Drug control general of India) guidelines. Each volunteer was

administered 10 mg dose (one 10 mg tablet) in 20 healthy human volunteers by

oral administration with 240 mL of drinking water. The reference product

Namenda tablets 10 mg, Forest laboratories, Ireland 10 mg, and test product

Memantine tablet 10 mg (Test tablet) was used. Blood samples were collected

as a pre-dose (0 h) 5 min prior to dosing followed by further samples at 1, 2, 3, 4,

4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 12, 24, 48 and 72 hours. After dosing 5 mL blood was

collected each time in vacutainer containing K2EDTA. A total of 34 (17 time

points from test and reference respectively) time points were collected by using

centrifugation at 3200 rpm, 10°C, 10 min and stored below -30 °C until sample

analysis. Test and reference was administered to same human volunteers under

fasting conditions separately with a gap of 18 days washing period as per

approved protocol.

The Mean Plasma concentration vs. time curve is shown in Table 6.23and

Figure 6.15.

210


Table 6.23 Memantine Mean concentration (pg/mL) data for the subjectsamples obtained from the LC-MS/MS

Time in hoursMean Concentration data

Period 1 Period 20 0 0

1 4516.8 3218.905

2 9300.885 8740.68

3 11410.29 11072.07

4 11792.31 12114.35

4.5 12498.97 12857.6

5 12765.46 13896.71

5.5 13558.31 13748.34

6 13653.12 14251.17

6.5 14054.96 14166.9

7 13996.62 14368.57

7.5 14328.28 13991.82

8 14302.7 14033.61

12 12773.3 12735.65

24 10607.12 10344.13

48 8155.335 7783.835

72 6003.395 5610.035

211


Figure 6.15 Mean plasma concentration Vs time curve for Memantine

212

Chapter 6 Memantine – Pharmacokinetic Studies






concentration time curve (AUC) ratio higher than 80% as per FDA guideline39-41

Plasma Memantine concentration-time profiles were visually inspected Cmax and Tmax

values were determined. The AUC0–t was obtained by trapezoidal method. AUC0-∞

was calculated up to the last measureable concentration and extrapolations were

obtained using the last measureable concentration and the terminal elimination rate

constant (Kel). The terminal elimination rate constant (Kel), was estimated from the

slope of the terminal exponential phase of the plasma of Memantine concentration–

time curve by means of the linear regression method. The terminal elimination half-

life, t1/2, was then calculated as 0.693/Kel. Regarding AUC0–t , AUC0-∞ and Cmax

bioequivalence was assessed by means of analysis of variance (ANOVA) and

calculating the standard 90% confidence intervals (90% CIs) of the ratios



AUC0-∞ and Cmax. Pharmacokinetic data is shown in Table 6.24 and Table 6.25.

213

Chapter 6 Memantine – Pharmacokinetic Studies

Table 6.24 Memantine Pharmacokinetic data

Memantine Pharmacokinetic dataPharmacokinetic

ParameterTest Reference

Mean±SD Mean±SD

Cmax(Pg/ml)14368.57 ± 4044.16 14328 ± 4324.76

AUC 0-t

(Pg hr/ml)654545.5 ± 70423.12 674564.4 ± 67858.99

AUC 0-∞(Pg hr/ml)

1053469.0 ± 77690.79 1136607 ± 74862.04

Tmax(h) 7.0 7.5t1/2 49.29 53.35

Table 6.25 Memantine Pharmacokinetic data (Test/Reference)


Cmax AUC 0-t AUC 0-∞

Test/Reference 99.72 103.06 107.89

Pharmacokinetic Conclusion


Memantine 10 mg was not bioequivalent with Namenda tablet (Forest Laboratories,

Ireland) 10 mg tablet under fasting conditions.

In vivo data was predicted by using Liquid-liquid Extraction procedure and

concentrations were found through Liquid Chromatography Tandem Mass

Spectroscopy detection. The Pharmacokinetic parameters assessed were AUC0-t,

AUC0-, Cmax, Tmax, t1/2. The bioequivalence criteria are based on the 90% confidence


The results obtained for Memantine was within the acceptance range.

Therefore, it can be concluded that the two Memantine formulations (reference and

test) analyzed were Bioequivalent terms of rate and extent of absorption.

CHAPTER 7

Summary & Conclusion

214

Chapter 7 Summary and Conclusion

7. SUMMARY AND CONCLUSION

The present work aimed to assess the applicability of High Performance

Liquid chromatography with mass spectrometry (HPLC-MS) for analysis of different

class of drugs in healthy human volunteers. The Dissertation described the research

work is composed of 8 chapters.

In Chapter 1, a general introduction and background on the current research

is given. HPLC has been suggested as an alternative but the lack of selective detection

has limited its capabilities for a long time.

Today this has changed with the introduction of High Performance Liquid

chromatography with mass spectrometry (HPLC-MS). The tremendous evolution in

interface and instrument design over the last decade has resulted in the creation of

state-of-the-art instrumentation for target analysis in complex mixtures. In recent

years, HPLC-MS/MS has been applied in numerous scientific fields, including

Toxicology. Evaluating the application of HPLC-MS for analysis of selected drugs

offered an interesting research challenges and was the basis for the present work.

Simultaneously we have discussed about pharmaceutical analysis, different extraction

procedures, method development, method validation parameters and pharmacokinetic

studies.

In Chapter 2 we have discussed about the Aim and Objectives of the present

research work for the selected drugs namely Rasagiline, Almotriptan,Valacyclovir

and Memantine in human plasma by using HPLC-MS detection. Simultaneously we

proved the validation parameters like Selectivity, Specificity, Sensitivity, Intra &Inter

215


Assay Precision and Accuracy, Recovery, Stability parameters like Short time

stability, Long time stability, Auto sampler stability, Bench Top Stability and Freeze-

thaw stability.

In Chapter 3 we have developed and validated the simple, highly sensitive,

selective, rugged and reproducible bioanalytical method for Rasagiline within the

concentration range of 5.0 – 12000.0 pg/mL using a simple liquid-liquid extraction

technique for drug and internal standard within 3minutes of analysis time in biological

fluids. Rasagiline- 13C3 mesylate was used as an internal standard. Simultaneously it

was successfully employed in the analysis of Rasagiline in human plasma samples by

oral administration of Rasagiline (1 mg) in 22 healthy human subjects.

In Chapter 4 we have developed simple, sensitive, rapid, good, linear,

reproducible analytical method for Almotriptan and validated over a concentration

range of 0.5 – 150.00 ng/mL using a Liquid-Liquid Extraction technique. Deuterated

compound Almotriptan- D6 was used as an internal standard. Simultaneously it was

successfully employed in the analysis of Almotriptan in human plasma samples by

oral administration of Almotriptan (12.5 mg) in 18 healthy human subjects.

In Chapter 5 we have developed and validated highly sensitive, selective,

rapid, rugged and reproducible highly efficient bio-analytical method for the

determination Valacyclovir and Valacyclovir-D8, in plasma samples over a

216


concentration range of 0.5 –700.0 ng/mL using a Solid Phase Extraction Technique

for drug and internal standard, using HPLC-MS/MS. This method was fully validated

as per FDA guidelines and was successfully employed in 20 healthy human subjects

by oral administration of Valacyclovir (1000mg) tablet and evaluated

pharmacokinetic parameters.

In Chapter 6 we have developed and validated simple, sensitive method for

Memantine over a concentration range of 50.0 - 50000.0 pg/mL for by a simple

liquid-liquid extraction technique for drug and internal standard. Deuterated

compound Memantine- D6 was used as an internal standard. Simultaneously it was

successfully employed in the analysis of human plasma samples by oral

administration of Memantine (10 mg) tablet in 20 healthy human subjects.

The above validated methods were successfully employed in analysis,

followed by pharmacokinetic study by non-compartmental statistics model using

Win-Non-Lin 5.0 software. The Cmax, Tmax, AUC0-t and AUC0-∞ were within the

acceptance criteria for selected drugs. The overall pharmacokinetic parameters were

within the range of 80-125% therefore it can be concluded that the two formulations

(test and reference) for Rasagiline, Almotriptan, Valacyclovir and Memantine

analyzed were bioequivalent in terms of rate and extent of absorption.

217


CONCLUSION

The present work compiled with our initial research objectives and

successfully demonstrated the applicability of HPLC-MS/MS for biopharmaceutical

analysis of different class of drugs namely Rasagiline, Almotriptan,Valacyclovir and

Memantine in human plasma.

The developed and validated methods shown high degree of sensitivity,

selectivity, reproducibility and high recovery, stability with less matrix effects when

compared with previously reported methods. Moreover it is proved by publishing the

methods in reputed journals.

This research has contributions in 2 important scientific fields. From an

analytical point of view, the extensive study of this novel instrumentation has resulted

in innovative methodology for selected drugs in human plasma.

From a bioequivalence and pharmacokinetic point of view, application of the

new HPLC-MS/MS procedures and usage of Non-compartmental statistics model

using WinNon-Lin 5.0 software broadened our knowledge, concentration-time

profiles and in-vivo studies calculations in human plasma.

The tremendous potential of HPLC-MS/MS for clinical and bioanalysis is

evident and will unquestionably expand future research capabilities in terms of shorter

runtimes, high rugged and reproducible methods with less precision and high

accuracy.

CHAPTER 8

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67) C.Shuangjin, F.Fang, L.Han, M.Ming, J. Pharm. Biomed Anal. 44(2007)1100-5.

68) Y.Higashi, S.Nakamura, H.Matsumura, Y.Fujii, Biomed Chromatogr.20(5)

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69) T.H.Duh, H.L.Wu, C.W.Pan, H.S.Kou, J. Chromatography A. 1088 (2005) 175

70) HJ Leis, G.Faule, W.Windischhofer, J. mass spectrometry. 37 (2002) 477.

71) H.H.YehY.H.Yang,S.Hwei, Electrophoresis.31(2010)1903.DOI:10.1002/elps.20

1000001.

Appendix

Appendix – Research Publications

LIST OF PUBLICATIONS - RELAVENT TO RESEARCH WORK

Publicationtype

Title Status

Full LengthArticle

Bio-analytical method development andvalidation of Rasagiline by High performanceLiquid chromatography tandem massspectrometry detection and its application toPharmacokinetic study.

Ravi kumar Konda, Babu Rao Chandu,

B.R.Challa, Chandrasekhar.K.B,

Journal ofPharmaceutical

Analysis

Elsevierpublication.

- Published

http://dx.doi.org/10.1016/j.jpha.

2012.04.001

Full LengthArticle

Method Development and Validation ofAlmotriptan in Human Plasma by HPLCTandem Mass spectrometry: Application toPharmacokinetic Study

Ravi kumar Konda, Babu.Rao Chandu,


ScientiaPharmaceuticaPeer reviewed-Austria Journal.

- Publishedhttp://dx.doi.org/10.3797/sciphar

m.1112-01

Full LengthArticle

Development and Validation of a sensitive LC-MS/MS method for determination ofValacyclovir in Human Plasma: Application toa Bioequivalence Study.

Ravi kumar Konda, Babu Rao Chandu,


Acta ChromatoGraphica

InternationalPeer reviewed

journal-- Accepted

Full LengthArticle

Bioanalytical Method Development andValidation of Memantine in Human Plasma byHigh Performance Liquid chromatography withTandem Mass spectrometry: Application toBioequivalence Study

Ravi kumar Konda, B.R.Challa,

Babu Rao Chandu, Chandrasekhar.K.B,

Journal ofAnalyticalMethods inChemistry

Hindawipublishing

corporation.

-Published

doi:10.1155/

2012/101249

ORIGINAL ARTICLE

Bio-analytical method development and validation of

Rasagiline by high performance liquid chromatography

tandem mass spectrometry detection and its application

to pharmacokinetic study

Ravi Kumar Kondaa,d, Babu Rao Chandub, B.R. Challac,n,

Chandrasekhar B. Kothapallid

aHindu College of Pharmacy, Amaravathi Road, Guntur, Andhra Pradesh 522002, IndiabDon Bosco College of Pharmacy, Pulladigunta, Guntur, Andhra Pradesh 522001, IndiacNirmala College of Pharmacy, Madras Road, Kadapa, Andhra Pradesh 516002, IndiadJawaharlal Nehru Technological University, Anantapur, Andhra Pradesh 515002, India

Received 27 November 2011; accepted 6 April 2012

KEYWORDS

High performance liquid

chromatography;

Mass spectrometry;

Rasagiline;

Liquid–liquid extraction

Abstract The most suitable bio-analytical method based on liquid–liquid extraction has beendeveloped and validated for quantification of Rasagiline in human plasma. Rasagiline-13C3

mesylate was used as an internal standard for Rasagiline. Zorbax Eclipse Plus C18 (2.1 mm� 50mm, 3.5 mm) column provided chromatographic separation of analyte followed by detection withmass spectrometry. The method involved simple isocratic chromatographic condition and massspectrometric detection in the positive ionization mode using an API-4000 system. The total runtime was 3.0 min. The proposed method has been validated with the linear range of 5–12000 pg/mLfor Rasagiline. The intra-run and inter-run precision values were within 1.3%–2.9% and 1.6%–2.2% respectively for Rasagiline. The overall recovery for Rasagiline and Rasagiline-13C3 mesylateanalog was 96.9% and 96.7% respectively. This validated method was successfully applied to thebioequivalence and pharmacokinetic study of human volunteers under fasting condition.

& 2012 Xi’an Jiaotong University. Production and hosting by Elsevier B.V. All rights reserved.

1. Introduction

Rasagiline ((1R)-N-prop-2-ynyl-2,3-dihydro-1H-inden-1-amine) is

used as a monotherapy in early Parkinson’s disease or as an

adjunct therapy in more advanced cases [1–3]. The empirical

formula is C12H13N with its molecular weight 171.24 (Fig. 1).

Rasagiline is rapidly absorbed, reaching peak plasma concentra-

tion (Cmax) in approximately 1 h. The absolute bioavailability of

Rasagiline is about 36%. Food does not affect the tmax of

Rasagiline, although Cmax and exposure (AUC) decreased by

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Journal of Pharmaceutical Analysis

2095-1779 & 2012 Xi’an Jiaotong University. Production and hosting

by Elsevier B.V. All rights reserved.

Peer review under responsibility of Xi’an Jiaotong University.

http://dx.doi.org/10.1016/j.jpha.2012.04.001

nCorresponding author. Tel.: þ91 8088259567.

E-mail address: [email protected] (B.R. Challa)

Journal of Pharmaceutical Analysis ]]]];](]):]]]–]]]

Please cite this article as: R.K. Konda, et al., Bio-analytical method development and validation of Rasagiline by high performance liquid

chromatography tandem mass spectrometry detection and its..., J. Pharm. Anal. (2012), http://dx.doi.org/10.1016/j.jpha.2012.04.001

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approximately 60% and 20%, respectively, when the drug is taken

with a high fat meal. Rasagiline’s pharmacokinetics is linear with

doses over the range of 1–10 mg. Its mean steady-state half-life is

3 h but there is no correlation of pharmacokinetics with its

pharmacological effect. Plasma protein binding ranges from

88%–94% with a mean extent of binding of 61%–63% to human

albumin over the concentration range of 1–100 ng/mL. Rasagiline

is almost metabolized in the liver and undergoes urinary excretion.

Half-life of Rasagiline is about 38–45 min [4].

Literature survey reveals that only a few methods were

reported for quantification of Rasagiline in human plasma and

pharmaceutical analysis [5–9]. These include HPLC [5,6],

crystallographic analysis [7], and LC-MS/MS [8,9]. Only two

methods were reported for quantification of Rasagiline in

human plasma with LC-MS/MS [8,9].

Song et al. [8] developed a method in a concentration range

0.01–40 ng/mL in human plasma and 0.025–40 ng/mL in urine

with runtime 5.5 min. Papavarin was used as an internal

standard and the pharmacokinetic study was conducted in

30 human volunteers. The main drawbacks of this method are

longer runtime and unsuitable internal standard.

The drawbacks of Song et al. are overcome by Ma et al. [9]

with shorter runtime of 3.5 min for each sample in a

concentration range of 0.02–50 ng/mL, Pseudoephedrine was

used as an internal standard and the pharmacokinetic study

was conducted in 12 human volunteers. The main drawbacks

of Ma et al. [9] method are sensitivity which is not achieved

when compared with Song et al. [8] and suitable internal

standard like deuterated or analogs of Rasagiline is not used.

The purpose of the present investigation is to explore rapid run

analysis time (3 min), more sensitive method (5 pg/mL), with the

small amount of plasma sample (100 mL plasma) utilization during

sample processing, simple extraction and analyte comparison with

isotope labeled internal standard (Rasagiline-13C3 mesylate).

2. Experimental

Rasagiline and Rasagiline-13C3 mesylate were obtained from TLC

PharmaChem, Canada. LC grade methanol, methyl t-butyl ether

and dichloromethane were purchased from J.T. Baker Inc.

(Phillipsburg, NJ, USA). Analytical reagent grade formic acid

and Na2CO3 were procured from Merck (Mumbai, India).

Human plasma (K2EDTA) was obtained from Doctors Patholo-

gical Lab, Hyderabad. The AZILECTs tablets, containing 1 mg

Rasagiline per tablet, were obtained from Teva Pharma (USA).

2.1. Instrumentation

An HPLC system (1200 series model, Agilent Technologies,

Waldbronn, Germany) connected with mass spectrometer API

4000 triple quadrupole instrument (ABI-SCIEX, Toronto,

Canada) was used. Data processing was performed with

Analyst 1.4.1 software package (SCIEX).

2.2. Detection

The mass spectrometer was operated in the multiple reaction

monitoring (MRM) modes. Sample introduction and ionization

were electrospray ionization in the positive ion mode. Sources

dependent parameters optimized were as follows: nebulizer gas

flow, 30 psi; curtain gas flow, 25 psi; ion spray voltage, 2000 V;

temperature (TEM), 375 1C. The compound dependent para-

meters such as the declustering potential (DP), focusing potential

(FP), entrance potential (EP), collision energy (CE), cell exit

potential (CXP) were optimized during tuning as 40, 35, 10, 12,

8 eV for Rasagiline and Rasagiline-13C3 mesylate, respectively.

The collision activated dissociation (CAD) gas was set at 4 psi

using nitrogen gas. Quadrupole 1 and quadrupole 3 were both

maintained at a unit resolution and dwell time was set at 300 ms

for Rasagiline and Rasagiline-13C3 mesylate. The mass transitions

were selected as m/z 172.1-117.1 for Rasagiline and m/z 175.1-117.1 for Rasagiline-13C3 mesylate. The data acquisition was

ascertained by Analyst 1.5.1 software.

2.3. Chromatography

Zorbax Eclipse Plus C18 (2.1 mm� 50 mm, 3.5 mm) was

selected as the analytical column. Column temperature was

set at 45 1C. Mobile phase composition was 0.1% formic

acid:methanol (80:20, v/v). Source flow rate was 300 mL/min

without split with injection volume of 10 mL. Rasagiline and

Rasagiline-13C3 mesylate were eluted at 1.270.2 min, with a

total run time of 3.0 min for each sample.

2.4. Calibration curve and quality control samples

Two separate stock solutions of Rasagiline were prepared for bulk

spiking of calibration curve and quality control samples for the

method validation exercise as well as the subject sample analysis.

The stock solutions of Rasagiline and Rasagiline-13C3 mesylate

were prepared in methanol at free base concentration of 50 mg/mL. Primary dilutions and working standard solutions were

prepared from stock solutions using water:methanol (50:50, v/v)

solvent mixture. These working standard solutions were used to

prepare the calibration curve and quality control samples. Blank

human plasma was screened prior to spiking to ensure it was free

of endogenous interference at retention times of Rasagiline and

internal standard Rasagiline-13C3 mesylate. Ten point standard

curve and four quality control samples were prepared by spiking

the blank plasma with an appropriate amount of Rasagiline.

Calibration samples were made at concentrations of 5.0, 10.0,

100.0, 600.0, 1200.0, 2400.0, 4800.0, 7200.0, 9600.0 and

12000.0 pg/mL and quality control samples were made at con-

centrations of 5.0, 15.0, 4500.0 and 9000.0 pg/mL for Rasagiline.

2.5. Sample preparation

For sample preparation, 100 mL of plasma sample or Rasagi-

line spiked standard or quality control plasma sample was

added to 5 mL ria vial tubes. 50 mL of internal standard and

200 mL of 1 M Na2CO3 solution were added and vortexed

Figure 1 Chemical structures of Rasagiline (A) and Rasagili-

ne-13C3 mesylate (B).

R.K. Konda et al.2



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briefly. Then liquid–liquid extraction with 3 mL of extraction

solvent (Methyl tertiary butyl ether (MTBE):Dichloromethane

(DCM) (3:1, v/v)) was added to each tube and vortexed for

10 min. After centrifugation at 4000 rpm for approximately

10 min at 20 1C, the supernatant was transferred to respective

ria vial tubes and evaporated to dryness under nitrogen at

25 1C. Finally, the residue was redissolved in 200 mL of

reconstitution solution (MeOH:0.1% formic acid(1:4)).

Further, samples were centrifuged at 4000 rpm for approxi-

mately 2 min at 20 1C and the supernatant was transferred to

auto sampler vials with caps and 10 mL of sample was injected

into the LC-MS/MS system.

2.6. Selectivity

Selectivity was performed by analyzing the human blank

plasma samples from six different sources (donors) with an

additional hemolyzed group and lipedimic group to test for

interference at the retention time of analytes.

2.7. Matrix effect

Matrix effect for Rasagiline and internal standard was

evaluated by comparing the peak area ratio in the post-

extracted plasma sample from 6 different drug-free blank

plasma samples and neat reconstitution samples. Experiments

were performed at MQC levels in triplicate with six different

plasma lots with the acceptable precision (% CV) of r15%.

2.8. Precision and accuracy

It was determined by replicate analysis of quality control

samples (n¼6) at a lower limit of quantification (LLOQ), low

quality control(LQC), medium quality control (MQC), high

quality control (HQC) levels. The % CV should be less than

15%, and accuracy should be within 15% except LLOQ where

it should be within 20%.

2.9. Recovery

The extraction efficiencies of Rasagiline and Rasagiline-13C3

mesylate were determined by analysis of six replicates at each

quality control concentration level for Rasagiline and at one

concentration for Rasagiline-13C3 mesylate. The percentage

recovery was evaluated by comparing the peak areas of

extracted standards to the peak areas of nonextracted stan-

dards (spiked into mobile phase).

2.10. Stability

Stock solution stability was performed by comparing the area

response of analyte and internal standard in the stability

sample, with the area response of sample prepared from fresh

stock solution. Stability studies in plasma were performed at

the LQC and HQC concentration levels using six replicates at

each level. Analyte was considered stable if the change is less

than 15% as per US FDA guidelines [10]. The stability of

spiked human plasma samples stored at room temperature

(bench top stability) was evaluated for 24 h. The stability of

spiked human plasma samples stored at 2–8 1C in autosampler

(autosampler stability) was evaluated for 55 h. The

autosampler sample stability was evaluated by comparing

the extracted plasma samples that were injected immediately

(time 0 h), with the samples that were reinjected after storing

in the autosampler at 2–8 1C for 26 h. The reinjection

reproducibility was evaluated by comparing the extracted

plasma samples that were injected immediately (time 0 h),

with the samples that were re-injected after storing in the

autosampler at 2–8 1C for 26 h. The freeze–thaw stability was

conducted by comparing the stability samples that had been

frozen at �30 1C and thawed three times, with freshly spiked

quality control samples. Six aliquots each of LQC and HQC

concentration levels were used for the freeze–thaw stability

evaluation. For long-term stability evaluation the concentra-

tions obtained after 78 days were compared with initial

concentrations.

2.11. Application of method

The validated method has been successfully used to analyze

Rasagiline concentrations in 22 human volunteers under

fasting conditions after administration of a single tablet

containing 1 mg (1� 1 mg) Rasagiline as an oral dose. The

study design was a randomized, two-period, two-sequence,

two-treatment single dose, open label, bioequivalence study

using AZILECTs manufactured by Teve Pharma, USA as the

reference formulation. The test formulation was conducted for

APL Research Pvt. Ltd., India. The study was conducted

according to current GCP guidelines and after obtaining

signed consent of the volunteers. Before conducting the study

it was also approved by an authorized ethics committee. There

were a total of 19 blood collection time points including the

predose sample, per period. The blood samples were collected

at time intervals (0, 0.083, 0.167, 0.25, 0.333, 0.417, 0.5, 0.667,

0.833, 1, 1.25, 1.5, 2, 2.5, 3, 3.75, 4.5, 5.5 and 6.5 h) in separate

vacutainers containing K2EDTA as an anticoagulant. The

plasma from these samples was separated by centrifugation at

4000 rpm within the range of 10 1C. The plasma samples thus

obtained were stored at �30 1C until analysis. The pharma-

cokinetic parameters were computed using Win-Nonlins

software version 5.2 and 90% confidence interval was com-

puted using SASs software version 9.2.

3. Results and discussion

3.1. Method development

During method development, different options were evaluated

to optimize mass spectrometry detection parameters, chroma-

tography and sample extraction.

3.1.1. Mass spectrometry detection parameters optimization

Electrospray ionization (ESI) provided a maximum response

over atmospheric pressure chemical ionization (APCI) mode,

and was chosen for this method. The instrument was opti-

mized to obtain sensitivity and signal stability during infusion

of the analyte in the continuous flow of mobile phase to

electrospray ion source operated at both polarities at a flow

rate of 10 mL/min. Rasagiline gave more response in positive

ion mode as compare to the negative ion mode. The pre-

dominant peaks in the primary ESI spectra of Rasagiline

and Rasagiline-13C3 mesylate correspond to the MHþ ions at

Bio-analytical method development and validation of Rasagiline 3



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m/z 172.1 and 175.1 respectively (Fig. 2A and C). Product

ions of Rasagiline and Rasagiline-13C3 mesylate scanned in

quadrupole 3 after a collision with nitrogen in quadrupole 2

had an m/z of 117.1 and 117.2 respectively (Fig. 2B and D).

3.1.2. Chromatography optimization

Initially, a mobile phase consisting of ammonium acetate and

acetonitrile in varying combinations was tried, but a low

response was observed. The mobile phase containing acetic

acid:acetonitrile (20:80, v/v) and acetic acid:methanol (20:80,

v/v) gave the better response, but poor peak shape was

observed. A mobile phase of 0.1% formic acid in water in

combination with methanol and acetonitrile with varying

combinations was tried. The best signal along with a marked

improvement in the peak shape was observed for Rasagiline

and Rasagiline-13C3 mesylate using a mobile phase containing

0.1% formic acid in water in combination with methanol

(20:80, v/v). Short length columns, such as Symmetry

Shield RP18 (50 mm� 2.1 mm, 3.5 mm), Inertsil ODS-2V

(50 mm� 4.6 mm, 5 mm), Hypurity C18 (50 mm� 4.6 mm,

5 mm) and Hypurity Advance (50 mm� 4.0 mm, 5 mm),

YMC basic (50 mm� 2 mm, 5 mm), Zorbax Eclipse Plus C18

(2.1 mm� 50 mm, 3.5 mm), were tried during the method

development. The best signal and good peak shape was

obtained using the Zorbax Eclipse Plus C18 (2.1 mm� 50 mm,

3.5 mm) column. It gave satisfactory peak shapes for both

Rasagiline and Rasagiline-13C3 mesylate. Flow rate of 0.3 mL/

min without splitter was used and reduced the run time to

3.0 min. Both the drug and internal standard were eluted in

shorter time at 2.0 min. For an LC-MS/MS analysis, utiliza-

tion of stable isotope-labeled or suitable analog drugs as an

internal standard proves helpful when a significant matrix

effect is possible. In our case, Rasagiline-13C3 mesylate was

found to be best for the present purpose. The column oven

temperature was kept at a constant temperature of about

45 1C. Injection volume of 10 mL sample is adjusted for better

ionization and chromatography.

3.1.3. Extraction optimization

Prior to load the sample for LC injection, the co-extracted

proteins should be removed from the prepared solution. For

this purpose, initially we tested with different extraction

procedures like Protein precipitation(PPT), Liquid–liquid extra-

ction(LLE) and Solid phase extraction(SPE). We found ion

suppression effect in protein precipitation method for the drug

and internal standard. Further, we tried with SPE and LLE.

Out of all, we observed LLE is suitable for extraction of the

drug and internal standard. We tried with several organic

solvents (ethyl acetate, chloroform, n-hexane, dichloromethane

and methyl tertiary butyl ether) individually as well with

Figure 2 Mass spectra (A) Rasagiline Parent ion, (B) Rasagiline Product ion, (C) Rasagiline-13C3 mesylate Parent ion, and

(D) Rasagiline-13C3 mesylate Product ion.

R.K. Konda et al.4



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combinations in LLE to extract analyte from the plasma

sample. In our case methyl tertiary butyl ether:dichloromethane

(75:25) combination served as good extraction solvent. Auto

sampler wash is optimized as 80% methanol. Several com-

pounds were investigated to find a suitable internal standard,

and finally Rasagiline-13C3 mesylate was found to be the most

appropriate internal standard for the present purpose. There

was no significant effect of IS on analyte recovery, sensitivity or

ion suppression. High recovery and selectivity was observed in

the liquid–liquid extraction method. These optimized detection

parameters, chromatographic conditions and extraction proce-

dure resulted in reduced analysis time with accurate and precise

detection of Rasagiline in human plasma.

3.2. Method validation

A thorough and complete method validation of Rasagiline in

human plasma was done following US FDA guidelines [10].

The method was validated for selectivity, sensitivity, matrix

effect, linearity, precision and accuracy, recovery, reinjection

reproducibility and stability.

3.2.1. Selectivity and sensitivity

Representative chromatograms obtained from blank

plasma and plasma spiked with a lower limit of quantification

(LOQ) sample are shown in Figs. 3 and 4 for Rasagiline and

Rasagiline-13C3 mesylate. The mean % interference observed

at the retention time of analytes between six different lots of

human plasma, including hemolyzed and lipedemic plasma

containing K2EDTA as an anti-coagulant was 0.00% and

0.00% for Rasagiline and Rasagiline-13C3 mesylate respec-

tively, which was within acceptance criteria. Six replicates of

extracted samples at the LLOQ level in one of the plasma

sample having least interference at the retention time of

Rasagiline were prepared and analyzed. The % CV of the

area ratios of these six replicates of samples was 1.1% for

Rasagiline, confirming that interference does not affect the

quantification at the LLOQ level. The LLOQ for Rasagiline

was 5 pg/mL. All the values obtained below 5 pg/mL for

Rasagiline were excluded from statistical analysis as they were

below the LLOQ values validated for Rasagiline.

3.2.2. Matrix effect

The % CV of ion suppression/enhancement in the signal was

found to be 1.0% at MQC level for Rasagiline, indicating that

the matrix effect on the ionization of analyte is within the

acceptable range under these conditions.

3.2.3. Linearity

The peak area ratios of calibration standards were propor-

tional to the concentration of Rasagiline in each assay over the

nominal concentration range of 5–12000 pg/mL. The calibra-

tion curves appeared linear and were well described by least-

squares linear regression lines. As compared to the 1/x

Figure 3 Blank plasma chromatograms of Rasagiline and Rasagiline-13C3 mesylate in human plasma.




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weighing factor, a weighing factor of 1/x2 achieved the best

result and was chosen to achieve homogeneity of variance. The

correlation coefficient was Z0.9991 for Rasagiline. The

observed mean back-calculated concentration with accuracy

and precision (% CV) of five linearity’s analyzed during

method validation is given in Table 1. The deviations of the

back calculated values from the nominal standard concentra-

tions were less than 15%. This validated linearity range

justifies the concentration observed during real sample

analysis.

3.2.4. Precision and accuracy

The inter-run precision and accuracy were determined by

pooling all individual assay results of replicate (n¼6) quality

control over five separate batch runs analyzed on four

different days. The inter-run, intra-run precision (% CV)

was r5% and inter-run, intra-run accuracy was in between

85 and 115 for Rasagiline. All these data presented in Table 2

indicate that the method is precise and accurate.

3.2.5. Recovery

Six aqueous replicates (samples spiked in reconstitution solu-

tion) at low, medium and high quality control concentration

levels for Rasagiline were prepared for recovery determina-

tion, and the areas obtained were compared with the areas

obtained for extracted samples of the same concentration

levels from a precision and accuracy batch run on the same

day. The mean recovery for Rasagiline was 96.9% with a

precision of 2.4%, and the mean recovery for Rasagiline-13C3

mesylate was 96.7% with a precision of 2.1%. This indicates

that the extraction efficiency for Rasagiline as well as Rasa-

giline-13C3 mesylate was consistent and reproducible.

3.2.6. Reinjection reproducibility

Reinjection reproducibility exercise was performed to check

whether the instrument performance remains unchanged after

hardware deactivation due to any instrument failure during

real subject sample analysis. The change was less than 2.5% at

LQC and HQC concentration levels; hence batch can be

reinjected in the case of instrument failure during real subject

sample analysis. Furthermore, samples were prepared to be

reinjected after 27 h, which shows % change less than 2.8% at

LQC and HQC concentration levels; hence batch can be

reinjected after 27 h in the case of instrument failure during

real subject sample analysis.

3.2.7. Stabilities

Stock solution stability was performed to check stability of

Rasagiline and Rasagiline-13C3 in stock solutions prepared in

methanol and stored at 2–8 1C in a refrigerator. The freshly

prepared stock solutions were compared with stock solutions

prepared before 28 days. The % change for Rasagiline and

Rasagiline-13C3 mesylate was �0.01% and 0.02% respectively,

which indicates that stock solutions were stable at least for 28

days. Bench top and autosampler stability for Rasagiline was

investigated at LQC and HQC levels. The results revealed that

Figure 4 LLOQ chromatograms of Rasagiline and Rasagiline-13C3 mesylate in human plasma.

Table 1 Calibration curve details.

Concentration

(pg/mL)

Mean

(pg/mL)

SD CV

(%)

Accuracy

5.0 4.8 0.0 0.4 96.0

10.0 9.8 0.2 1.7 98.0

100.0 100.5 2.6 2.6 100.5

600.0 595.2 16.7 2.8 99.2

1200.0 1180.6 22.4 1.9 98.4

2400.0 2496.1 69.9 2.8 104.0

4800.0 4505.4 108.1 2.4 93.9

7200.0 7268.4 247.1 3.4 101.0

9600.0 9468.2 236.7 2.5 98.6

12000.0 11864.5 178.0 1.5 98.9

SD: Standard deviation.

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Rasagiline was stable in plasma for at least 24 h at room

temperature, and 55 h in an auto sampler at 20 1C. It was

confirmed that repeated freezing and thawing (three cycles) of

plasma samples spiked with Rasagiline at LQC and HQC levels

did not affect their stability. The long-term stability results also

indicated that Rasagiline was stable in a matrix up to 78 days at

a storage temperature of �30 1C. The results obtained from all

these stability studies are tabulated in Table 3.

3.3. Application

The validated method has been successfully used to quantify

Rasagiline concentrations in 22 human volunteers, under fasting

conditions after administration of 1 mg (1� 1 mg) tablet con-

taining Rasagiline as an oral dose. The study was carried out

after obtaining signed consent from the volunteers. These

volunteers were contracted in APL Research Centre, Hyderabad,

India. The study protocol was approved from an IEC (indepen-

dent ethics committee) as per DCGI (Drug Control General of

India) guidelines. The pharmacokinetic parameters evaluated

were Cmax (maximum observed drug concentration during the

study), AUC0–6.5 (area under the plasma concentration–time

curve measured 6.5 h, using the trapezoidal rule), tmax (time to

observe maximum drug concentration), Kel (apparent first order

terminal rate constant calculated from a semi-log plot of the

plasma concentration versus time curve, using the method of the

least square regression) and t1/2 (terminal half-life as determined

by the quotient 0.693/Kel, Table 4).

The Test/Reference ratios for Cmax, AUC0–6.5, and AUC0–

N were 80.22, 90.86 and 90.70 respectively, and they were

within the acceptance range of 80%–125% demonstrating the

bioequivalence of the two formulations of Rasagiline [11–12].

The mean concentration versus time profile of Rasagiline in

human plasma from 22 subjects that are receiving 1� 1 mg

oral dose of Rasagiline tablet as test and reference is shown in

Figure 5.

4. Conclusion

The proposed bio-analytical method is simple, highly sensitive,

selective, rugged and reproducible. The major advantage of

this method is rapid analysis time (3 min), less plasma volume

(0.1 mL) usage for analysis, suitable internal standard usage.

This method was successfully applied in bioequivalence study

to evaluate the plasma concentrations of Rasagiline in healthy

human volunteers.

Table 2 Within-run and between-run precision and accuracy.

Nominal added

concentration

(pg/mL)

Within-run (n¼6) Between-run (n¼36)

Mean

(pg/mL)

SD Precision

(CV, %)

Accuracy Mean

(pg/mL)

SD Precision

(CV, %)

Accuracy

5.0 4.9 0.1 1.3 98.0 5.1 0.1 1.6 102.0

15.0 15.2 0.2 1.3 101.3 15.2 0.2 1.6 101.3

4500.0 4485.6 130.1 2.9 99.7 4465.2 98.2 2.2 99.2

9000.0 8965.3 215.2 2.4 99.6 8965.2 251.0 2.8 99.6

SD: Standard deviation, CV¼Coefficient of variation.

Table 3 Stability of the samples.

Stability experiments Spiked plasma concentration

(n¼6, pg/mL, mean7SD)

Concentration measured

(n¼6, pg/mL, mean7SD)

CV

(%, n¼6)

Room temperature stability (24 h) 15.0 14.970.2 1.3

9000.0 8799.37211.2 2.4

Autosampler stability (55 h) 15.0 15.270.2 1.3

9000.0 8896.47213.5 2.4

Long-term stability (78 days) 15.0 14.670.2 1.3

9000.0 8897.57213.5 2.4

Freeze–thaw stability (cycle 3, 48 h) 15.0 14.970.2 1.3

9000.0 8897.57213.5 2.4

Table 4 Mean pharmacokinetic parameters of Rasagiline

in 22 healthy volunteers after oral administration of 1 mg

(1� 1 mg) test and reference products.

Pharmacokinetic parameter Rasagiline

Test Reference

AUC0–t (pg h/mL) 4005.11 4407.78

Cmax (pg/mL) 4529.87 5647.15

AUC0–N (pg h/mL) 4022.33 4434.56

Kel 0.64892 0.59415

t1/2 1.07 1.17

tmax(h) 0.417 0.417

AUC0–N: area under the curve extrapolated to infinity; AUC0–t:

area under the curve up to the last sampling time; Cmax: the

maximum plasma concentration; tmax: the time to reach peak

concentration; Kel: the apparent elimination rate constant. t1/2:

0.693/Kel




dx.doi.org/10.1016/j.jpha.2012.04.001

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Acknowledgments

The authors are grateful to the Indian Institute of Chemical

Technology, Hyderabad for literature survey and Manipal

Accunova, Manipal, India for their Lab facility for this

research work.

References

[1] O. Weinreb, T. Amit, O. Bar-Am, et al., Rasagiline: a novel anti-

parkinsonian monoamine oxidase-b inhibitor with neuroprotec-

tive activity, Prog. Neurobiol. 92 (3) (2010) 330–344.

[2] J. Leegwater-Kim, E. Bortan, The role of rasagiline in the treatment

of Parkinson’s disease, Clin. Interv. Aging 5 (2010) 149–156.

[3] M.F. Lew, R.A. Hauser, H.I. Hurtig, et al., Long-term efficacy of

rasagiline in early Parkinson’s disease, Int. J. Neurosci. 20 (6)

(2010) 404–408.

[4] S. Won Hyuk, K.S. Suslick, S. Yoo-Hum, Therapeutic agents for

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Agents 5 (2005) 259–269.

[5] M. Fernandez, E. Barcia, S. Negro, Development and validation

of a reverse phase liquid chromatography method for the

quantification of rasagiline mesylate in biodegradable

PLGA microspheres, J. Pharm. Biomed. Anal. 49 (5) (2009)

1185–1191.

[6] D. Haberle, K. Magyar, E. Szoko, Determination of the nor-

epinephrine level by high-performance liquid chromatography to

assess the protective effect of MAO-B inhibitors against DSP-4

toxicity, J. Chromatogr. Sci. 40 (9) (2002) 495–499.

[7] C. Binda, F. Hubalek, M. Li, et al., Binding of rasagiline-related

inhibitors to human monoamine oxidases: a kinetic and crystal-

lographic analysis, J. Med. Chem. 48 (26) (2005) 8148–8154.

[8] M. Song, L. Wang, H. Zhao, et al., Rapid and sensitive liquid

chromatography-tandem mass spectrometry: assay development,

validation and application to a human pharmacokinetic study, J.

Chromatogr. B Analyt. Technol. Biomed. Life Sci. 875 (2) (2008)

515–521.

[9] J. Ma, X. Chen, X. Duan, et al., Validated LC-MS/MS method

for quantitative determination of rasagiline in human plasma and

its application to a pharmacokinetic study, J. Chromatogr. B

Analyt. Technol. Biomed. Life Sci. 873 (2) (2008) 203–208.

[10] Guidance for Industry: Bioanalytical Method Validation, U.S.

Department of Health and Human Services, Food and Drug

Administration, Center for Drug Evaluation and Research

(CDER), Center for Biologics Evaluation and Research (CBER),

May 2001.

[11] Guidance for Industry: Food-Effect Bioavailability and Fed

Bioequivalence Studies. U.S. Department of Health and Human

Services, Food and Drug Administration, Center for Drug

Evaluation and Research (CDER), December 2002.

[12] Guidance for Industry: Bioavailability and Fed Bioequivalence

Studies for Orally Administered Drug Products—General Con-

siderations, U.S. Department of Health and Human Services,

Food and Drug Administration, Center for Drug Evaluation and

Research (CDER), March 2003.

Figure 5 Mean plasma concentrations of test versus reference

after 1 mg dose (1� 1 mg tablet) in 22 healthy volunteers.

R.K. Konda et al.8



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Sci Pharm www.scipharm.at

Research article Open Access Method Development and Validation of

Almotriptan in Human Plasma by HPLC Tandem Mass Spectrometry:

Application to Pharmacokinetic Study Konda RAVIKUMAR 1,2, Babu Rao CHANDU 3,

Balasekhara Reddy CHALLA * 4, Kottapalli B. CHANDRASEKHAR 2

1 Hindu college of Pharmacy, Amaravathi Road, Guntur, Andhrapradesh, 522002, India. 2 Jawaharlal Nehru Technological University, Anantapur, 515002, India. 3 Donbosco college of Pharmacy, Pulladigunta, Guntur, 522201, India. 4 Nirmala college of Pharmacy, Madras road, Kadapa, Andhrapradesh, 516002, India.

* Corresponding author. E-mail: [email protected] (B. R. Challa)

Sci Pharm. 2012; 80: 367–378 doi:10.3797/scipharm.1112-01

Published: February 27th 2012 Received: December 1st 2011 Accepted: February 27th 2012

This article is available from: http://dx.doi.org/10.3797/scipharm.1112-01

© Ravikumar et al.; licensee Österreichische Apotheker-Verlagsgesellschaft m. b. H., Vienna, Austria.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract A simple, sensitive and selective method has been developed for quantification of Almotriptan (AL) in human plasma using Almotriptan-d6 (ALD6) as an internal standard. Almotriptan and Almotriptan-d6 were detected with proton adducts at m/z 336.1→201.1 and 342.2→207.2 in multiple reaction monitoring (MRM) positive mode, respectively. The method was linear over a concentration range of 0.5–150.0 ng/mL. The limit of detection (LOD) and limit of quantification (LOQ) for Almotriptan were 0.2 pg/mL and 0.5 ng/mL, respectively. Liquid-liquid extraction was used followed by MS/MS (ion spray). The method was shown to be precise with an average within-run and between-run variation of 0.68 to 2.78% and 0.57 to 0.86%, respectively. The average within-run and between-run accuracy of the method throughout its linear range was 98.94 to 102.64% and 99.43 to 101.44%, respectively. The mean recovery of drug and internal standard from human plasma was 92.12 ± 4.32% and 89.62 ± 6.32%. It can be applied for clinical and pharmacokinetic studies.

Keywords HPLC • MS/MS • Almotriptan • Human plasma • Pharmacokinetic study • LLE

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mailto:[email protected]

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368 K. Ravikumar et al.:

Sci Pharm. 2012; 80: 367–378

Introduction Almotriptan, N,N-dimethyl-2-{5-[(pyrrolidin-1-ylsulfonyl)methyl]-1H-indol-3-yl}ethanamine, is a novel 5-HT1B/1D receptor agonist used for the treatment of symptomatic relief of migraines (Fig. 1) [1]. Almotriptan is absorbed well orally, with an absolute bioavailability of around 70%. The drug shows a dose linear pharmacokinetics and a mean elimination half-life of 1.4 to 3.8 h. Approximately 40 to 50 % of the dose is recovered unchanged in the urine; renal elimination probably occurs via active tubular secretion. The balance of the dose is eliminated unchanged in faecus (approximately 5%) or is metabolised [2, 3].

NH

N

SN

O

O

OH

O

O

OH

OH

NH

ND3C

SN

CD3

O

O

OH

O

O

OH

OH

Almotriptan malate Almotriptan-d6 malate

Fig. 1. Chemical structure of Almotriptan malate and Almotriptan-d6 malate

To our knowledge, several methods for the determination of Almotriptan in biological matrixes [1, 4, 5], pharmaceutical formulations [6–9] by LC–MS/MS [4], HPLC [6, 7] HPTLC [8], fluorimetric and calorimetry [9] have been reported.

However, Fleischhacker et al. [4] concentrated more on pharmacokinetics rather than method development and validation. The authors have not explained briefly extraction procedure, stability aspects, matrix factor effect, and recovery for determination of Almotriptan by LC-MS/MS. The purpose of this study was to develop and validate a novel sensitive LC-MS/MS method to quantify Almotriptan in human plasma.

Material and methods Standards and chemicals Almotriptan malate was obtained from USP and Almotriptan malate-d6 was obtained from clear synth Labs (P) Ltd, Mumbai, India. All other chemicals (Ammonium formate, formic acid, sodium carbonate, acetonitrile, methanol) and solvents were purchased from s. d. fine chemical's Mumbai. Human plasma was obtained from Navjeevan blood bank, hyderabad, India.

Instrumentation Almotriptan was analyzed using HPLC system (1200 Series Agilent Technologies, Germany). MS/MS (ABI-SCIEX, Toronto, Canada) using MRM. A turbo electrospray interface in positive ionization mode was used. Data processing was performed on Analyst 1.4.1 software package (SCIEX).

Method Development and Validation of Almotriptan in Human Plasma by HPLC Tandam Mass … 369

Sci Pharm. 2012; 80: 367–378

Detection Turbo Ion Spray (API) positive mode with Unit Resolution, MRM was used for the detection. For Almotriptan, the MH+ (m/z: 336.1) was monitored as the precursor ion and a fragment at m/z: 201.3 was chosen as the product ion (Fig. 2). For internal standard, the MH+ (m/z: 342.2) was monitored as the precursor ion and a fragment at m/z: 207.2 was monitored as the product ion (Fig. 3). Mass parameters were optimised as Source temperature 500 °C, Ion source gas 1 (GS1) 25 (nitrogen) psi, Ion source gas 1 (GS2) 35 (nitrogen) psi, Curtain gas 25 (nitrogen) psi, CAD gas 8 (nitrogen) psi, Ion Spray (IS) voltage 4000 volts, Source flow rate 500 µl/min without split, Entrance potential 10 V, Declustering potential 40 V for both analyte and IS, Collision energy 22 V for both Analyte and IS, Collision cell exit potential, 12 V for both Analyte and IS.

Chromatographic conditions Chromatographic separation was carried out on a reversed phase Zorbax, SB C18, 4.6 x 75mm, 3.5 μm column using a mixture of 10 mM ammonium formate buffer (pH 4.5) and acetonitrile (50:50 v/v) as mobile phase with a flow-rate of 0.5 mL/min. The column temperature was set to 40°C. Retention time of Almotriptan and Almotriptan-d6 was found at 1.5 ± 0.2 min approximately with a total runtime of 3 min.

Preparation of standards and quality control (QC) Samples To prepare stock standard solution (100 µg/mL) of Almotriptan, accurate volume of Almotriptan was dissolved in methanol in 20 ml volumetric flask. The stock solution was then further diluted with blank plasma to obtain the different working solutions ranging from 50, 150 and 1000 ng/mL, from which analytical standards were prepared at concentration levels of 0.5, 1.0, 5.0, 15.0, 30.0, 45.0, 60.0, 90.0, 120.0 and 150.0 ng/mL by appropriate dilution with blank plasma. Quality control (QC) samples were prepared at Lower limit of quality control (LLOQ) (0.5 ng/mL), Low quality control (LQC) (1.5 ng/mL), medium quality control (MQC) (75.0 ng/mL) and high quality control (HQC) (105.0 ng/mL) concentrations in the same way as the plasma samples for calibration. All samples were stored in a −30°C freezer until analysis.

Sample preparation Liquid-Liquid extraction procedure was used in this study to isolate Almotriptan from the plasma samples. For this purpose, 100 µL of Almotriptan-d6 (80 ng/mL) and 200 µL plasma (respective concentration of plasma sample) was added into riavials then vortexed for 30 sec and then 100 µl of 0.5 N sodium carbonate solution was added and vortexed for 10 min. Then samples were centrifuged at 4000 rpm for approximately 5 min at ambient temperature and the supernatant from each sample was transferred into respective ria vials, evaporated to dryness and reconstituted with 10mM ammonium formate (pH:4.5) acetonitrile (50:50v/v) and vortexed briefly. The sample was transferred into auto sampler vials to inject into LC-MS/MS.

Linearity Linearity was evaluated by using bulk spiked calibration curve standards and quality control standards. The calibration curve was constructed by using 10 non-zero calibration curve standard points spanning the range of 0.5–150.0 ng/mL, (0.5, 1.0, 5.0, 15.0, 30.0, 45.0, 60.0, 90.0, 120.0 and 150.0 ng/mL), four non-zero quality control standards (0.5, 1.5,


Sci Pharm. 2012; 80: 367–378

75.0 and 105.0 ng/mL), and, in addition, a blank sample (spiked only with blank plasma), blank + IS sample (spiked only with blank plasma and IS sample). Calibration curves were obtained by weighted 1/x2 linear regression model (y = mx + c). The ratio of Almotriptan peak area to Almotriptan-d6 peak area was plotted against the concentration of Almotriptan in ng/mL. The suitability of the calibration curve was confirmed by back-calculating the concentrations of the calibration standards.

Precision and Accuracy For determination of within-run and between-run precision and accuracy, four different series of samples at concentrations of 0.5, 1.5, 75.0 and 105.0 ng/mL of Almotriptan were analyzed within a single instrument run and in different runs. The accuracy was calculated from the ratio of measured concentration, based on the standard curve, to the nominal added concentration. Precision was evaluated by calculating the within-run and between-run coefficients of variations of the measured concentrations at each level (CV%).

Recovery The extraction recovery of Almotriptan and Almotriptan-d6 from human plasma was determined by analyzing quality control samples. Recovery at three concentrations (1.5, 75.0 and 105.0 ng/mL) was determined by comparing peak areas obtained from the plasma sample, and the standard solution was spiked with the blank plasma residue. A recovery of more than 50 % was considered adequate to obtain required sensitivity.

Stability Low quality control (1.50 ng/mL) and high quality control (105.0 ng/mL) samples (n=6) were retrieved from the deep freezer after three freeze-thaw cycles according to the clinical protocols. Samples were stored at −30°C in three cycles of 24, 36 and 48 h. In addition, the long-term stability of Almotriptan in quality control samples was also evaluated by analysis after 65 days of storage at −30°C. Autosampler stability was studied following a 57-h storage period in the autosampler tray with control concentrations. Bench top stability was studied for a 26-h period with control concentrations. Stability samples were processed and extracted along with the freshly spiked calibration curve standards. The precision and accuracy for the stability samples must be within ≤15 and ± 15 %, respectively, of their nominal concentrations [10].

Application of method The validated method has been successfully used to analyze Almotriptan concentrations in 18 human volunteers under fasting conditions after oral administration of a single tablet containing 12.5mg (1x12.5mg) Almotriptan. The study design was a randomized, two-period, two-sequence, two-treatment single dose, open label, bioequivalence study using AXERT® (manufactured by Ortho-McNeil-Janssen Pharmaceuticals, Inc., USA) as the reference formulation. The test formulation was conducted for APL Research Pvt. Ltd, India. The study was conducted according to current GCP guidelines and after signed consent of the volunteers. Before conducting the study it was also approved by an authorized ethics committee. There was a total of 13 blood collection time points, including the predose sample, per period. The blood samples were collected at time intervals (0, 0.5, 1.0, 1.5, 2, 2.5, 3, 4, 6, 8, 12, 16 and 24 h) in separate vacutainers containing K2EDTA as an anticoagulant. The plasma from these samples was separated by centrifugation at


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4000 rpm at 10°C. The plasma samples thus obtained were stored at −30°C until analysis. Post analysis, the pharmacokinetic parameters were computed using win nonlin® software version 5.2 and 90% confidence interval was computed using SAS® software version 9.2.

Results and Discussion Method Development The goal of this work was to develop and validate a simple, rapid and sensitive assay method for the quantitative determination of Almotriptan from human plasma samples by LC-MS/MS detection. We tested a wide spectrum of organic solvents from different physicochemical categories with different volume fractions as well as combinations. In terms of the analysis condition, various mobile phases, in different proportions, buffered and non-buffered at various pH, were attempted to provide the best peak shape and less retention time. Also, we tried different column packing, even from normal phase. The MS optimization was performed by direct infusion of solutions of both Almotriptan and Almotriptan-d6 into the ESI source of the mass spectrometer. The critical parameters in the ESI source include the needle (ESI) voltage. Other parameters, such as the nebulizer and the desolvation gases, were optimized to obtain a better spray shape, resulting in better ionization. A CAD product ion spectrum for Almotriptan and Almotriptan-d6 yielded high-abundance fragment ions at m/z 336.1→201.1 and 342.2→207.2 in multiple reaction monitoring (MRM) positive mode, respectively. After the MRM channels were tuned, the mobile phase was changed from an aqueous phase to a more organic phase with acid dopant to obtain a fast and selective LC method. The most accurate extraction method for analyte was selected as Liquid-Liquid extraction. A good separation and elution were achieved using 10 mM ammonium formate (pH 4.5.): acetonitrile (50:50 v/v) as the mobile phase, at a flow-rate of 0.5 mL/min and injection volume of 10 µL. The developed method was found to be the most sensitive and accurate one compared with known methods.

Fig. 2. Mass spectra of the Almotriptan Q1, Almotriptan Q3


Sci Pharm. 2012; 80: 367–378

Fig. 3. Mass-spectra of Almotriptan-d6 (Q1), Almotriptan-d6 (Q3)

Method Validation Linearity and Range The method produced highly linear responses within the wide concentration range of 0.5–150.0 ng/mL, which is desirable for the majority of PK studies on the drug (Table 1).

Specificity and Selectivity To investigate specificity, a series of blank (drug-free) human plasma (total 6 plasma samples) in addition to the different concentrations spiked were screened, and no endogenous interference was observed at the retention time of Almotriptan and internal standard (Fig. 4 & 5).

Tab. 1. Calibration curves details

Spiked plasma concentration (ng/mL)

Concentration measured (mean ± SD)

(ng/mL)

CV (%) (n = 5)

Accuracy (%)

0.50 0.49 ± 0.01 2.73 97.84 1.00 0.97 ± 0.01 1.37 96.84 5.00 4.89 ± 0.14 2.81 97.88 15.00 14.56 ± 0.22 1.52 97.09 30.00 29.22 ± 0.57 1.94 97.41 45.00 43.76 ± 0.74 1.70 97.24 60.00 58.49 ± 1.21 2.07 97.48 90.00 87.73 ± 1.81 2.07 97.48 120.00 117.25 ± 2.90 2.48 97.71 150.00 146.27 ± 3.10 2.12 97.51


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Fig. 4. Chromatogram of Blank Plasma sample

Fig. 5. Blank human plasma spiked with 0.5 ng/mL Almotriptan and human plasma

spiked with 100 ng/mL Almotriptan-d6 (LOQ)

Precision and Accuracy In Table 2, the CV% values of the measurements made by the method at different levels have been shown along with the corresponding accuracies. As shown, all the values of variations and accuracies are within the generally acceptable ranges (Precission (cv%) ≤ 15%, accuracy ± 15% for all concentrations, for LOQ accuracy ± 20%). This in turn assures obtaining accurate and precise results from the method.


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Tab. 2. Precision and accuracy (analysis with spiked plasma samples at four different concentrations)

Spiked plasma concentration (ng/mL)

Within-run Concentration measured

(n=6) (ng/mL) (mean ± SD) % CV % Accuracy

0.50 0.51 ± 0.01 2.05 102.64 1.50 1.52 ± 0.04 2.78 101.00 75.00 74.20 ± 0.50 0.68 98.94 105.00 104.52 ± 0.49 0.47 99.54 Spiked plasma concentration (ng/mL)

Between-run Concentration measured

(n=30) (ng/mL) (mean ± SD) % CV % Accuracy

0.50 0.51 ± 0.01 0.86 101.44 1.50 1.50 ± 0.01 0.61 99.91 75.00 74.57 ± 0.42 0.57 99.43 105.00 104.21 ± 0.49 0.47 99.24

Recovery A variety of extraction procedures were tested, as described, and the best recovery was achieved with Liquid-Liquid extraction. The mean recoveries of Almotriptan and Almotriptan-d6 were found to be 92.12 ± 4.32 % and 89.62 ± 6.32 %. These data indicate an acceptable degree of drug recovery by the extraction method within the whole concentration range tested.

Tab. 3. Stability of the samples Spiked plasma concentration (ng/mL)

Room Temperature stability Processed sample stability

26.0 h 57 h Concentration

measured (n=6)

(ng/mL) (mean ±SD)

% CV (n=6)


(n=6) (ng/mL)

(mean ± SD)

% CV (n=6)

1.50 1.49 ± 0.13 2.6 1.51 ± 0.14 2.2 105.00 104.85 ± 1.20 3.2 104.37 ± 24.78 1.4 Spiked plasma concentration (ng/mL)

Long term stability Freeze and thaw stability

65 days Cycle 3 (48 h)


(n=6) (ng/mL) (mean ± SD)

% CV (n=6)


(n=6) (ng/mL)

(mean ± SD)

% CV (n=6)

1.50 1.46 ± 0.04 0.7 1.49 ± 0.4 2.3 105.00 103.92 ± 6.13 1.2 104.81 ± 5.99 3.4


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LOD and LOQ The LOD and LOQ of the method for Almotriptan were 0.02 pg/mL and 0.50 ng/mL, respectively. These results confirm the significant sensitivity of the method for drug analysis (Fig. 6).

Stability Quantification of the Almotriptan in plasma subjected to 3 freeze-thaw (−30°C to room temperature) cycles showed the stability of the analyte. No significant degradation of the Almotriptan was observed even after the 57-h storage period in the autosampler tray. In addition, the long-term stability of Almotriptan in QC samples after 65 days of storage at −30°C was also evaluated. These results confirmed the stability of Almotriptan in human plasma for at least 65 days at −30°C (Table 3).

Application The validated method has been successfully used to quantify Almotriptan concentrations in 18 human volunteers, under fasting conditions after oral administration of 12.5 mg (1x12.5mg) tablet containing Almotriptan. The study was carried out after obtaining signed consent from the volunteers. These volunteers were contracted in APL Research centre, Hyderabad, India. The study protocol was approved from an IEC (institutional ethics committee) as per DCGI (Drug control general of India) guidelines. The pharmacokinetic parameters evaluated were Cmax (maximum observed drug concentration during the study), AUC0–24 (area under the plasma concentration–time curve measured 24 h, using the trapezoidal rule), Tmax (time to observe maximum drug concentration), Kel (apparent first-order terminal rate constant calculated from a semi-log plot of the plasma concentration versus time curve, using the method of the least square regression) and T1/2 (terminal half-life as determined by the quotient 0.693/Kel) (Table 4).

Tab. 4. Mean Pharmacokinetic Parameters of Almotriptan in 18 Healthy Volunteers after Oral Administration of 12.5 mg (1x12.5 mg) Test and Reference Product

Pharmacokinetic Parameter Almotriptan Test Reference

AUC0–t (ng h/mL) 293.55 272.24 Cmax (ng/mL) 50.76 49.75 AUC0–∞ (ng h/mL) 293.55 272.24 Kel 0.34467 0.33982 Tmax (h) 2.5 2.5 AUC0–∞: area under the curve extrapolated to infinity; AUC0–t: area under the curve up to the last sampling time; Cmax: the maximum plasma concentration; Tmax: the time to reach peak concentration; Kel: the apparent elimination rate constant.

The 90% confidence intervals of the ratios of means Cmax, AUC0-24 within the acceptance range of 80–125%, (Table 5) demonstrate the bioequivalence of the two formulations of Almotriptan [11, 12]. The mean concentration versus time profile of Almotriptan in human plasma from 18 subjects that are receiving 1x12.5mg oral dose of Almotriptan tablet as test and reference is shown in Fig. 6.


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Tab. 5. Test/Reference values for Log-Transformed Pharmacokinetic parameters of Almotriptan after Administration of 12.5 mg (1x12.5 mg) of Test and Reference products in 18 healthy male volunteers

Pharmacokinetic parameters Cmax AUC0-t AUC0–∞ Test/Ref 102.02 107.83 107.82

Fig. 6. Mean Pharmacokinetic graph of Almotriptan in 18 human volunteers

Conclusion A simple, sensitive, rapid LC-MS/MS method with Liquid-Liquid extraction method was developed and validated as per FDA guidelines for quantification of Almotriptan in human plasma over a concentration range of 0.5–150.0 ng/mL. Almotriptan-d6 (ALD6) was used as an internal standard and 200 µL of plasma was used for extraction of drug and internal standard.

Acknowledgment The authors wish to thank the IICT (Indian Institute of Chemical Technology) Hyderabad, India, for providing literature survey, Jawaharlal Nehru Technological University, Anantapur, India and APL Research centre, India

Authors’ Statements Competing Interests The authors declare no conflict of interest.


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Informed Consent, Ethical Approvals Research followed the ethical standard formulated in the Helsinki declaration of 1964, revised in 2000, and was approved by the institutional human experimentation committee and IEC followed by ICMR guidelines. See the experimentation part for details.

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[4] Fleishaker JC, Ryan KK, Jansat JM, Carel BJ, Bell DJ, Burke MT, Azie NE.. Effect of MAO-A inhibition on the pharmacokinetics of almotriptan, an antimigraine agent in humans. Br J Clin Pharmacol. 2001; 51: 437–441. http://dx.doi.org/10.1046/j.1365-2125.2001.01367.x

[5] Salva M, Jansat JM, Martinez-Tobed A, Palacios JM. Identification of the human liver enzymes involved in the metabolism of the antimigraine agent almotriptan. Drug Metab Dispos. 2003; 31: 404–411. http://dx.doi.org/10.1124/dmd.31.4.404

[6] Kumar AP, Ganesh VR, Rao DV, Anil C, Rao BV, Hariharakrishnan VS, Suneetha A, Sundar BS. A validated reversed phase HPLC method for the determination of process-related impurities in almotriptan malate API. J Pharm Biomed Anal. 2008; 46: 792–798. http://dx.doi.org/10.1016/j.jpba.2007.11.029

[7] Suneetha A, Syama Sundar B. A Validated RP HPLC Method for Estimation of Almotriptan Malate in Pharmaceutical Dosage Form. J Chin Chem Soc. 2010; 57: 1067–1070.

[8] Suneetha A, Syamasundar B. Fluorimetric and colorimetric methods for the determination of some antimigraine drugs. Indian J Pharm Sci. 2010; 72: 629–632. http://dx.doi.org/10.4103/0250-474X.78534

[9] El-Bagary RI, Mohammed NG, Nasr HA. Fluorimetric and colorimetric methods for the determination of some antimigraine drugs. J Chem Pharm Res. 2011; 3: 304–314.

[10] Guidance for industry: bioanalytical method validation. U. S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER), Center for Biologics Evaluation and Research (CBER), May 2001.

[11] Guidance for industry Food- effect bio availability and Fed Bio equivalence studies. U. S. Department of Health and Human services Food and Drug Administration Centre for Drug Evaluation and research (CDER) December 2002.

http://dx.doi.org/10.1177/0091270002042012006

http://www.ncbi.nlm.nih.gov/pubmed/15320857

http://dx.doi.org/10.2165/00003088-200140030-00004

http://dx.doi.org/10.1046/j.1365-2125.2001.01367.x

http://dx.doi.org/10.1124/dmd.31.4.404

http://dx.doi.org/10.1016/j.jpba.2007.11.029

http://dx.doi.org/10.4103/0250-474X.78534


Sci Pharm. 2012; 80: 367–378

[12] Guidance for Industry Bio availability and Fed Bio equivalence Studies for Orally Administered Drug Products. General considerations U. S. Department of Health and Human services Food and Drug Administration Centre for Drug Evaluation and research (CDER).

On Sun, 4/15/12, [email protected] <[email protected]> wrote:

From: [email protected] <[email protected]>Subject: Final decisionTo: [email protected]: Sunday, April 15, 2012, 1:51 PM

Dear R.K.Konda,

Your revised re-submission was repeatedly evaluated for its quality andnow I am very pleased to let you know that it finally conforms withscientific and formal demands of our journal.I remain in full agreement with the Reviewers’ recommendations andtherefore my decision is to accept your manuscript for publication in ActaChromatographica.

Your accepted manuscript is scheduled for the Acta Chromatographica issueno. 4 / 2013. However, beginning from January, 2012, the electronic ONLINEPREVIEW of the manuscripts accepted to our journal in the DOI systemstarts (http://www.akademiai.com/content/1233-2356), so that your acceptedpaper will appear in the Internet much earlier and owing to the DOI ID, itwill immediately obtain the status of a published paper.

Kind regards,

Danica AgbabaEditor, Acta Chromatographica

DEVELOPMENT AND VALIDATION OF A SENSITIVE LC-MS/MS METHOD FOR DETERMINATION OF

VALACYCLOVIR IN HUMAN PLASMA: APPLICATION TOA BIOEQUIVALENCE STUDY

Ravi Kumar.Konda1,4*, Babu.Rao.Chandu2 , B.R.Challa3,Chandrasekhar.K.B4,

1Department of pharmaceutical chemistry,Hindu college of Pharmacy, Amaravathi Road,

Guntur,Andhrapradesh, India - 522002

2 Department of pharmaceutical Sciences, Donbosco college of Pharmacy, pulladigunta,

Guntur, India – 522201

3Department of pharmaceutical Analysis, Nirmala college of Pharmacy, Madras road,

Kadapa, Andhrapradesh, India - 516002.

4 Department of chemistry, Jawaharlal Nehru Technological University, Anantapur, India -

515002.

*Correspondence to:

Ravi Kumar.Konda, Department of pharmaceutical chemistry Hindu college of Pharmacy,

Amaravathi Road, Guntur,Andhrapradesh, India - 522002

Email: [email protected] & [email protected]

#Tel# +91-8088259567

Abstract:

A simple, sensitive, and highly specific method has been developed for determination of

Valacyclovir (VL) in human plasma. The analytical procedure involves a Solid-Phase

extraction method using Valacyclovir-D8 (VLD8) as an internal standard. Chromatographic

separation was carried out on a reversed phase Zorbax, SB C18, 4.6 x 75mm, 3.5 m

column. Valacyclovir and Valacyclovir-D8 were detected with proton adducts at m/z

325.2152.0 and 333.3152.0 in multiple reaction monitoring (MRM) positive mode

respectively. The method was linear over the concentration range of 0.5 - 700.0 ng/ mL. The

limit of detection (LOD) and limit of quantification (LOQ) for Valacyclovir were 0.2pg/ mL

and 0.5 ng/ mL respectively. The method was shown to be precise with the average within-

run and between-run variations of 0.7 to 3.5 % and 3.1 to 4.7 %, respectively. The average

within-run and between-run accuracy of the method throughout its linear range was 96.7 to

97.9 and 94.7 to 97.3 % respectively. The mean recovery of Valacyclovir and Valacyclovir-

D8 from human plasma by the developed method was 99.17 ± 10.78 % and 110.84 ± 8.74 %

respectively. The method was successfully applied in bioequivalence study with 20 healthy

male volunteers under fasting condition.

Keywords Valacyclovir; LC-MS/MS; Solid-Phase extraction; bioequivalence

Hindawi Publishing CorporationJournal of Analytical Methods in ChemistryVolume 2012, Article ID 101249, 8 pagesdoi:10.1155/2012/101249

Research Article

Bioanalytical Method Development and Validation ofMemantine in Human Plasma by High Performance LiquidChromatography with Tandem Mass Spectrometry:Application to Bioequivalence Study

Ravi Kumar Konda,1, 2 B. R. Challa,3 Babu Rao Chandu,4

and Kothapalli B. Chandrasekhar2

1 Department of Pharmaceutical Chemistry, Hindu College of Pharmacy, Amaravathi Road, Guntur, Andhrapradesh 522002, India2 Department of Chemistry, Jawaharlal Nehru Technological University, Anantapur 515002, India3 Department of Pharmaceutical Analysis, Nirmala College of Pharmacy, Madras Road, Kadapa, Andhrapradesh 516002, India4 Department of Pharmaceutical Sciences, Donbosco College of Pharmacy, Pulladigunta, Guntur 522201, India

Correspondence should be addressed to Ravi Kumar Konda, [email protected]

Received 3 November 2011; Revised 3 January 2012; Accepted 9 January 2012

Academic Editor: Antonio Ruiz-Medina

Copyright © 2012 Ravi Kumar Konda et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

A simple, sensitive, and rapid HPLC-MS/MS method was developed and validated for quantitative estimation of memantine inhuman plasma. Chromatography was performed on Zorbax SB-C18 (4.6×75 mm, 3.5 µm) column. Memantine (ME) and internalstandard Memantine-d6(MED6) were extracted by using liquid-liquid extraction and analyzed by LC-ESI-MS/MS using multiple-reaction monitoring (MRM) mode. The assay exhibited a linear dynamic range of 50.00–50000.00 pg/ml for ME in human plasma.This method demonstrated an intra- and interday precision within the range of 2.1–3.7 and 1.4–7.8%, respectively. Further intra-and interday accuracy was within the range of 95.6–99.8 and 95.7–99.1% correspondingly. The mean recovery of ME and MED6was 86.07± 6.87 and 80.31± 5.70%, respectively. The described method was successfully employed in bioequivalence study of MEin Indian male healthy human volunteers under fasting conditions.

1. Introduction

Memantine (1-amino-3,5-dimethyladamantane hydrochlo-ride) (Figure 1) acting on the glutamatergic system by block-ing N-methyl-D-aspartate (NMDA) glutamate receptors [1].Memantine (ME) is used in Parkinson’s disease and move-ment disorders, and recently it has been demonstrated to beuseful in dementia syndrome. The mode of action is thoughtto be due to prevention of damage to retinal ganglion as aresult of increased intraocular pressure. The accumulation ofa drug in melanin-rich tissues may have serious physiologicalconsequences as it could lead to potentially toxic effects.Despite several investigations into the nature of drug melaninbinding, the exact mechanism of the interaction remains

unknown. ME is well absorbed, with peak plasma concen-trations (Cmax) ranging from 22 to 46 ng/mL following asingle dose of 20 mg. The time to achieve maximum plasmaconcentration (Tmax) following single doses of 10–40 mgranges from 3 to 8 hr. The drug is 45% bound to plasmaproteins presenting a distribution volume of approximately9–11 L/kg, which suggests an extensive distribution intotissues. It is poorly metabolized by the liver, and 57–82%of the administered dose is excreted unchanged in the urinewith a mean terminal half-life of 70 hr [1].

There were few methods established previously to deter-mine ME in a variety of matrices with different instruments.These methods include LC-MS [1–4], HPLC [5–8], GC-MS [9], and Micellar electrokinetic chromatography [10].

2 Journal of Analytical Methods in Chemistry

NH2

H3C

CH3

HCl

(a)

NH2

DD

D

D

DD

HCl

(b)

Figure 1: Chemical structures of (a) Memantine hydrochloride and (b) Memantine-D6HCL.

Among all methods LC-MS [1–4] has gained more impor-tance.

Liu et al. [1] developed the method with the linear con-centration range of 0.2–200 ng/mL, with 0.2 ng/mL sensitiv-ity. This sensitivity was improved by Almeida et al. [2]. Theydeveloped the method with the linear concentration rangeof 0.1 to 50 ng/mL, with 0.1 ng/mL sensitivity. Pan et al. [3]developed the method with the linear concentration rangeof 0.1 to 25 ng/mL. They used 0.5 mL plasma usage to get0.1 ng/mL of sensitivity. Koeberle et al. [4] developed themethod in different melanins.

The reported methods do not show the usage of deuter-ated internal standard comparision with analyte which ismost important in bioanalytical method development. Allthe reported methods develop the method with long runtime and more amount of plasma sample for extraction.

The purpose of this investigation was to develop a rapid,simple, sensitive, and selective LC-MS/MS method for thequantitative estimation of ME in less volume of humanplasma using deuterated internal standard. It is also expect-ed that this method would provide an efficient solutionfor pharmacokinetic, bioavailability, and/or bioequivalencestudies of ME.

2. Materials and Methods

2.1. Chemicals. ME (99.9%) was obtained from Vardabiotech Pvt. Ltd. Andheri, Mumbai, India. MED6 (99.0%)was obtained from the Toronto Research Chemicals, Toronto,Canada. Blank plasma lots were purchased from Navjeevanblood bank, Hyderabad. HPLC-grade methanol and ace-tonitrile were purchased from Jt. Baker, USA. Diethyl etherand n-hexane were purchased from Lab Scan, Asia Co. Ltd,Bangkok, Thailand. Formic acid and sodium hydroxide werepurchased from Merck Mumbai, India. HPLC-grade waterfrom Milli-Q System was used. All other chemicals used wereanalytical grade.

2.2. Instrumentation and Chromatographic Conditions.HPLC system (1200 series, Agilent Technologies, Germany)is connected with API 4000 triple quadrupole mass spec-trometer (ABI-SCIEX, Toronto, Canada) using multiple re-action monitoring (MRM). A turbo electrospray interfacein positive ionization mode was used. Data processing was

performed on Analyst 1.4.1 software package (SCIEX).The chromatography was performed on a Zorbax SB-C18

(4.6 × 75 mm, 3.5 µm) (Agilent technologies,Germany) at40◦C temperature. The mobile phase composition was amixture of 0.1% formic acid : acetonitrile (35 : 65 v/v) whichwas pumped at a flow-rate of 0.6 mL/min without split.

2.3. Preparation of Calibration Standards and Quality ControlSamples. Standard stock solutions of ME (100.00 µg/mL)and MED6 (100.00 µg/mL) were separately prepared inmethanol. MED6 dilution (25.00 ng/mL) was made fromMED6 standard stock solution with diluent (methanol: water50 : 50 v/v). Standard stock solution of ME was added todrug-free human plasma to obtain ME calibration stan-dards of 50.00, 100.00, 500.00, 1000.00, 5000.00, 10000.00,20000.00, 30000.00, 40000.00, and 50000.00 pg/mL. Qualitycontrol (QC) samples were also prepared as a bulk on anindependent weighing of standard drug at concentrationsof 50.00 (LLOQ), 150.00 (LQC), 15000.00 (MQC), and35000.00 pg/mL (HQC) from standard stock solutions ofME. The calibration standards and quality control sampleswere divided into aliquots in 5 mL Ria vials and stored in thefreezer at below −30◦C until analysis.

2.4. Sample Preparation. 50 µL of MED6 (25 ng/mL), 100 µLof plasma sample, and 100 µL of 10 mM NaOH were addedinto 5 mL Ria vials and vortexed briefly. This was followed byaddition of 3 mL extraction solvent (diethyl ether : n-hexane70 : 30 v/v) and vortexed for 10 min. Then samples werecentrifuged at 4000 rpm for 5 min at ambient temperatureconditions. Then, the supernatant from each sample wastransferred into labelled vials by using the dry-ice acetoneflash freeze technique and evaporated to dryness undernitrogen stream at 40◦C. The dried residue was reconstitutedwith 400 µL of 0.1% of formic acid: acetonitrile (35 : 65 v/v)mixture and vortexed until dissolved. Finally, a 20 µL of eachsample was transferred into auto sampler vials and injectedinto HPLC connected with mass spectrometer.

2.5. Recovery. Recovery of ME was evaluated by comparingthe mean peak area of six extracted low, medium, andhigh (150.00, 1500.00, and 35000.00 pg/mL) quality controlsamples to the mean peak area of six aqueous standards

Journal of Analytical Methods in Chemistry 3

with the same concentrations of low, medium, and high MEquality control samples.

Similarly the recovery of MED6 was evaluated by com-paring the mean peak area of extracted quality control sam-ples to the mean peak area of MED6 in aqueous standardssamples with the same concentrations of MED6.

2.6. Selectivity. The selectivity of the method was determinedby blank human plasma samples from six different healthyhuman volunteers to test the potential interferences of endo-genous compounds coeluted with ME and MED6. The Chro-matographic peaks of ME and MED6 were identified on thebasis of their retention times and MRM responses. The meanpeak area of LOQ for ME and MED6 at corresponding reten-tion time in blank samples should not be more than 20 and5%, respectively.

2.7. Limit of Quantification (LOQ). The LOQ was estimatedin accordance with the baseline noise method at a signal-to-noise ratio (S/N) of 5. It was experimentally determined byinjecting six samples with ME at the LLOQ concentration.The acceptance criterion for S/N was ≥5 and calculated byselecting the noise region as close as possible to the signalpeak, which was at least 8 times of the signal peak width athalf height.

2.8. Analytical Curves. The analytical curves of ME wereconstructed in the concentrations ranging from 50.00 to50000.00 pg/mL in human plasma. The calibration curvewas constructed by using instrument response (ratio of MEpeak area to MED6 peak area) against the ME concentra-tion (pg/mL) for four consecutive days by weighted 1/x2

quadratic regression model. The fitness of calibration curvewas confirmed by back-calculating the concentrations ofcalibration standards.

2.9. Calibration Curve Standards, Regression Model, Precision,and Accuracy Batches. Calibration curve standard samplesand QC samples were prepared in replicates (n = 6)for analysis. Correlation coefficients (r2) were obtained byusing quadratic regression model in whole range of testedconcentrations. The accuracy and precision for the backcalculated concentrations of the calibration points shouldbe within ±15% whereas those of LLOQ should be within±20% of their nominal values.

2.10. Stability. Low and high QC samples (n = 6) wereretrieved from the deep freezer; samples were processed forthree freeze/thaw cycles according to the clinical protocols.The samples were stored at −10◦C to −30◦C in three cyclesof 24, 36, and 48 hr. In addition, the long-term stability of MEin QC samples was also evaluated after 76 days of storage at−10 to −30◦C. The stability at refrigerated temperature wasstudied following 79 hr storage period in the autosamplertray. Bench top stability was studied for 26-hour period.Stability samples were processed and extracted along withthe freshly spiked calibration curve standards. Stability of thestock solutions was proved for 24 days. The precision and

accuracy for the stability samples were maintained within 15and ±15%, respectively, of their nominal concentrations.

2.11. Matrix Effect. The matrix effect due to plasma matrixwas used to evaluate ion suppression/enhancement in asignal by comparing the absolute response of QC samplesafter pretreatment (liquid-liquid extraction) with that ofreconstituted samples extracted blank plasma sample spikedwith analyte. Experiments were performed at low and highconcentration levels in triplicate. The acceptable precision(%CV) should be ≤15%.

2.12. Analysis of Human Plasma Samples. The bioanalyticalmethod described previously was applied to determine MEconcentrations in plasma following oral administration tohealthy adult human male volunteers below 25 years ofage. The volunteers were contracted by Micro TherapeuticsResearch Labs Pvt Ltd., Chennai, India. They were screenedbefore participation in the study and an informed consentwas taken from them. These volunteers, were not undergoneany other medication before conducting this study. To eachof the 20 volunteers a tablet containing 10 mg of ME wasorally administered along with a 240 mL of drinking water.Proper diet was provided to each volunteer as per theprotocol. The reference product (Namenda tablets 10 mg,Forest laboratories, Ireland) and test product (Memantinetablets 10 mg) were used in the study. The study protocol wasapproved by IEC (Institutional Ethical Committee) and byICMR (Indian Council of Medical Research). Blood sampleswere collected as predose (0 hr) 5 minutes prior to dosingfollowed by further samples at 1, 2, 3, 4, 4.5, 5, 5.5, 6, 6.5,7, 7.5, 8, 12, 24, 48, and 72 hr. After dosing, a 5 mL bloodsample was collected each preestablished time in vacutainerscontaining K2EDTA. A total of 34 samples (17 time pointseach for reference and test) were collected and centrifugedat 3200 rpm and10◦C for 10 min. Then they were storedat −30◦C until further analysis. Test and reference wereadministered to the same human volunteers under fastingconditions separately after a washing period of 18 days as perprotocol approved by IEC.

2.13. Pharmacokinetics and Statistical Analysis. Pharmacoki-netics parameters were calculated from plasma levels apply-ing a noncompartmental statistics model using WinNon-Lin5.0 software (Pharsight, USA). Following Food and DrugAdministration (F.D.A) guideline [11, 12], blood sampleswere drawn up to a period of three to five times the terminalelimination half-life (t1/2) and it was considered as the areaunder the concentration time curve (AUC) ratio higher than80%. The Cmax and Tmax values were determined by visualinspection of the plasma ME concentration-time profiles.The area under the concentration-time curve (AUC0−t) wasobtained by the trapezoidal method. The total area under thecurve (AUC0−∞) was calculated up to the last measureableconcentration, and extrapolations were obtained by the lastmeasureable concentration and the terminal eliminationrate constant (Ke). The Ke was estimated from the slopeof the terminal exponential phase of the plasma of ME


2.4e62.3e62.2e62.1e6

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Figure 2: (a) Mass spectra of Memantine parent ion (Q1). (b) Mass spectra of Memantine product ion (Q3). (c) Mass spectra of Memantine-D6 parent ion. (d) Mass spectra of Memantine-D6 product ion (Q3).

concentration-time curve using linear regression method.The t1/2 was then calculated as 0.693/Ke. The AUC0−t ,AUC0−∞, and Cmax bioequivalence were assessed by analysisof variance (ANOVA), and the standard 90% confidenceintervals (90% CIs) of the ratios test/reference were cal-culated after transforming the data logarithmically. Thebioequivalence was considered when the ratio of averagesof log transformed data was within 80–125% for AUC0−t ,AUC0−∞, and Cmax [11, 12].

3. Results and Discussion

3.1. Method Development and Validation. Mass spectrometryparameters, fragmentation pattern, and mode of ionizationare the main task in mass spectrometry tuning to obtainrespective fragmented ions and response for both MEand MED6 which were shown in Figures 2(a), 2(b), 2(c),and 2(d). ESI-LC-MS/MS is a very powerful techniquefor pharmacokinetic studies since it provides sensitivity


and selectivity requirements for analytical methods. MRMtechnique was chosen for the assay development. The MRMparameters were optimized to maximize the response for theanalyte.

The instrumental parameters for mass spectroscopy wereoptimized. The source temperature was 600◦C. The gas pres-sures of nebulizer, heater, curtain, and CAD were 40, 30, 20,and 4 psi, respectively. The ion spray voltage, entrance poten-tial, declustering potential, collision energy, and collision cellexit potential were optimized at 5500, 10, 50, 32, and 12 V,respectively. The dwell time was 400 milliseconds for bothME and MED6.

The product ion (Q3) mass spectra of ME and theMED6 are shown in Figures 2(b) and 2(d). [M + H]+ wasthe predominant ion in the Q1 spectrum. The Q1 for MEand MED6 was 180.2 and 186.1, respectively, and were usedas the precursor ion to obtain product ion spectra. Thecollisionally associated dissociation (CAD) mass spectrumof ME shows formation of characteristic product ions atm/z 161.8, 163.2, and 165.1. The major product ion atm/z 163.2 for ME could be explained by the splitting of1-amino-3-,5-dimethyladamantane hydrochloride from theprotonated precursor molecule. The CAD mass spectrum ofMED6 shows formation of characteristic product ions at m/z169.2. The major product ion at m/z 169.2 arose from 3,5-Dimethyl-d6-tricyclo-[3,3,1,13,7]decan-1-amine,3,5-Dim-ethyl-d6-1-adamantanamine from the protonated precursormolecule. The most sensitive mass transitions were from m/z180.2 to 1163.2 for ME and m/z 186.1 to m/z 169.2 for theMED6. The proposed fragmentation pattern is Figure 2(a)→Figure 2(b), Figure 2(c)→Figure 2(d). The inherent selecti-vity of MS-MS detection was also expected to be beneficialin developing a selective and sensitive method.

The chromatographic conditions particularly the com-position of mobile phase, flow-rate of mobile phase, choos-ing of suitable column, injection volume, column oven tem-perature, autosampler temperature, splitting of sample in toion source, as well as a short run time were optimizedthrough several trials to achieve good resolution and sym-metric peak shapes for the ME and MED6. It was foundthat a mixture of 0.1% formic acid:acetonitrile (35 : 65 v/v)could achieve this purpose and this was finally adopted asthe mobile phase. The formic acid was found to be necessaryin order to lower the pH to protonate the ME and thusdeliver good peak shape. The percentage of formic acid wasoptimized to maintain this peak shape while being con-sistent with good ionization and fragmentation in the massspectrometer. The high proportion of organic solvent elutedboth the ME and the MED6 at retention time 1.45± 0.2 minat a flow rate of 0.6 mL/min, produced good peak shapes, andpermitted a run time of 3.5 min.

Liquid-liquid extraction (LLE) was used for the samplepreparation in this work. LLE can be helpful to clean thesamples. Clean samples are essential for minimizing ionsuppression and matrix effect in LC-MS/MS analyses. Severalorganic solvents and their mixtures in different combinationsand ratios were evaluated. Finally, diethyl ether/n-hexane(70 : 30) was found to be optimal, which produced a cleanchromatogram for a blank plasma sample and yielded the

Table 1: Concentration data form validation.

Spiked plasmaconcentration(pg/ml)

Concentrationmeasured (pg/ml)

Mean ± Sdn = 5

Precision(% CV)

Accuracy%

50.00 49.92 ± 0.44 0.80 99.23

100.00 100.21± 1.91 1.90 98.14

500.00 502.73± 6.83 1.40 98.65

1000.00 1000.54± 11.53 1.10 98.92

5000.00 5005.06± 38.75 0.80 99.28

10000.00 9978.95± 160.56 1.60 98.45

20000.00 19871.04± 303.46 1.50 98.53

30000.00 29759.82± 508.47 1.70 98.37

40000.00 40310.88± 123.85 0.30 99.74

50000.00 50084.85± 266.72 0.50 99.55

highest recovery for the ME and MED6 from the plasma.Memantine-D6 hydrochloride was used as internal stan-dard for the present purpose. Clean chromatograms wereobtained, and no significant direct interferences in the MRMchannels at the relevant retention times were observed.

3.2. Selectivity. The selectivity of the method was examinedby analyzing blank human plasma extracts (n = 6). Theresult of one blank (Figure 3(a)) plasma is shown and thelack of interference is similar to other samples which werestudied which shows no significant direct interference in theblank plasma traces as observed from endogenous substancesin drug-free human plasma at the retention time of theanalyte.

3.3. Limit of Quantification (LOQ). The LOQ signal-to-noise(S/N) value found for 6 injections of ME at LOQ concen-tration was 11.93. Figure 3(b) shows a representative ion-chromatogram for the LOQ (50 pg/mL) with 20 µL injectionvolume.

3.4. Linearity, Precision, and Accuracy. The ten-point calibra-tion curve was linear over the concentration range 50.00–50000.00 pg/mL. The calibration model was selected basedon the analysis of the data by quadratic regression withintercepts and weighting factor 1/x2. The best quadraticregression for the calibration curve was achieved with a1/x2 weighing factor, giving a mean quadratic regressionequation for the calibration curve of y = −9.427× 10−11x2 +9.194 × 10−5x + 2.989 × 10−4(y = ax2 + bx + c) wherey is the peak-area ratio of the ME to the MED6 and xis the concentration of the ME in plasma (Table 1). Forthe between-batch experiments, the precision and accuracyranged from 1.4 to 2.7% and 95.7 to 99.1%, respectively(Table 2). Further, in within-batch experiments the precisionand accuracy ranged from 2.1 to 2.3% and 95.6 to 99.8%correspondingly.


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Figure 3: (a) MRM chromatogram for blank plasma. (b) Chromatogram of LOQ.

Table 2: Precision and accuracy (analysis with spiked plasma samples at three different concentrations).

Spiked plasmaconcentration(pg/ml)

Within-run (n = 6) Between-run (n = 30)

Concentrationmeasured (n = 6)

(pg/ml) (mean ± Sd.)Precision (%CV) Accuracy %

Concentrationmeasured (n = 30)

(pg/ml) (mean ± Sd.)Precision (%CV) Accuracy %

150.00 143.40± 3.20 2.20 95.60 143.50± 3.90 2.70 95.70

15000.00 14746.40± 338.40 2.30 98.30 14719.10± 248.30 1.70 98.10

35000.00 34935.20± 730.40 2.10 99.80 34699.20± 498.40 1.40 99.10


0

2000

4000

6000

8000

10000

12000

14000

16000

0 20 40 60 80

TestRef

Time (h)

Mem

anti

ne

(pg/

mL

)

Mean plasma concentration of Memantine

Figure 4: Mean plasma concentrations of test versus reference aftera 10 mg dose (one 10 mg tablet) single oral dose (20 healthy volun-teers).

Table 3: Stability of Memantine in human plasma samples.

Spiked plasmaconcentration(pg/ml n = 6)

Concentrationmeasured(pg/ml)

Precision(%CV)

Accuracy (%)

Room temperature stability for 26 hr in plasma

150.00 151.23 1.00 100.82

35000.00 34926.50 0.80 99.79

Three freeze-thaw cycles

150.00 153.02 2.30 102.01

35000.00 34818.00 0.90 99.48

Auto sampler stability for 79 hr

150.00 154.59 1.40 103.06

35000.00 35213.50 0.90 100.61

Stability for 76 days −30◦C

150.00 151.53 3.20 101.02

35000.00 35080.50 1.40 100.23

3.5. Recovery. The recoveries for ME at low (150.00 pg/mL),medium (15000.00 pg/mL) and high (35000.00 pg/mL) plas-ma concentrations with six replicate injections each showed79.45± 6.20%, 91.25± 5.9%, and 87.52± 2.59%. The overallrecovery of ME was found to be 86.07% ± 6.87%. Similarlyextraction recovery of MED6 (25.00 ng/mL) was determinedas 80.31%± 5.70%. Recoveries of ME and MED6 were high,precise, and reproducible. Therefore, the assay has proved tobe robust in high-throughput bioanalysis.

3.6. Stability Studies. Quantification of the ME in plasmathat was subjected to 3 freeze-thaw cycles (−30◦C to roomtemperature) showed the stability of the analyte. The con-centrations ranged from 98.00 to 104.00% for ME. No signif-icant degradation was observed even after a 79-hour storage

Table 4: Mean pharmacokinetic parameters of Memantine in 20healthy human volunteers after oral administration of 10 mg testand reference products.

Pharmacokinetic details of Memantine in human plasma


Reference Test

Mean ± SD Mean ± SD

Cmax (pg/ml) 14368.57± 4044.16 14328± 4324.76

AUC0−t (pg·hr/ml) 654545.5± 70423.12 674564.4± 67858.99

AUC0−∞ (pg·hr/ml) 1053469.0±77690.79 1136607± 74862.04

Tmax (hr) 7.0 7.5

t1/2 49.29 53.35

AUC0−∞: Area under the curve extrapolated to infinity.AUC0−t : Area under the curve up to the last sampling time.Cmax: The maximum plasma concentration.Tmax: The time to reach peak concentration.

Table 5: Pharmacokinetic parameters of memantine after adminis-tration of 10 mg of test and reference products in 20 healthy humanvolunteers.

Pharmacokineticparameters

Cmax (T/R) AUC0−t (T/R) AUC0−∞ (T/R)

Test/Ref 99.72 103.06 107.89

period in the autosampler tray, and the final concentrationsof ME were found between 100.00 and 105.00% The roomtemperature stability of ME in QC samples after 26 hr wasalso evaluated. The concentrations were ranged between99.00 and 102.00% for ME. In addition, the long-termstability in low and high QC samples after 76 days of storageat −30◦C was also evaluated, and the concentrations rangedfrom 98.00 to 103.00% for ME. These results confirmed thestability of ME in human plasma for at least 76 days at−30◦C. (Table 3).

3.7. Application to Biological Samples. The proposed methodwas applied to the determination of ME in plasma samplesfor the purpose of establishing the bioequivalence of asingle dose (10 mg tablet) in 20 healthy human volunteers.Typical plasma concentrations versus time profiles wereshown in Figure 4. Plasma concentrations of ME were in thestandard curve range and retained above LLOQ for the entiresampling period. The pharmacokinetic parameters for testand reference products were shown in Tables 4 and 5. Themean ratio of AUC0−t/AUC0−∞ was higher than 90% whichfollowed the Food and Drug Administration BioequivalenceGuideline [11, 12]. The ratio of test/reference (T/R) and 90%confidence intervals (90 CIs) for overall analysis were com-prised within the previously stipulated range (80–125%).Therefore, it can be concluded that the two ME formulations(reference and test) analyzed are bioequivalent in terms ofrate and extent of absorption at fasting conditions.


4. Conclusion

A simple, high sensitive, specific, rugged, and reproducibleLC-MS/MS method for the determination of memantine inhuman plasma was developed and validated as per FDAguidelines. This method was successfully applied in bioe-quivalence study to evaluate the plasma concentrations ofME in healthy human volunteers.

Acknowledgments

The authors wish to acknowledge the support received fromIndian Institute of Chemical Technology (IICT), Hyderabad,India, for providing literature survey and Micro TherapeuticsResearch Labs Pvt Ltd., Chennai, India, to carry out thisresearch work.

References

[1] M.-Y. Liu, S.-N. Meng, H.-Z. Wu, S. Wang, and M.-J. Wei,“Pharmacokinetics of single-dose and multiple-dose meman-tine in healthy chinese volunteers using an analytic method ofliquid chromatography-tandem mass spectrometry,” ClinicalTherapeutics, vol. 30, no. 4, pp. 641–653, 2008.

[2] A. A. Almeida, D. R. Campos, G. Bernasconi et al., “Determi-nation of memantine in human plasma by liquid chromatog-raphy-electrospray tandem mass spectrometry: application toa bioequivalence study,” Journal of Chromatography B, vol. 848,no. 2, pp. 311–316, 2007.

[3] R.-N. Pan, T.-Y. Chian, B. P.-C. Kuo, and L.-H. Pao, “Deter-mination of memantine in human plasma by LC-MS-MS:application to a pharmacokinetic study,” Chromatographia,vol. 70, no. 5-6, pp. 783–788, 2009.

[4] M. J. Koeberle, P. M. Hughes, C. G. Wilson, and G. G. Skellern,“Development of a liquid chromatography-mass spectromet-ric method for measuring the binding of memantine todifferent melanins,” Journal of Chromatography B, vol. 787, no.2, pp. 313–322, 2003.

[5] R. F. Suckow, M. F. Zhang, E. D. Collins, M. W. Fischman, andT. B. Cooper, “Sensitive and selective liquid chromatographicassay of memantine in plasma with fluorescence detectionafter pre-column derivatization,” Journal of ChromatographyB, vol. 729, no. 1-2, pp. 217–224, 1999.

[6] C. Shuangjin, F. Fang, L. Han, and M. Ming, “New methodfor high-performance liquid chromatographic determinationof amantadine and its analogues in rat plasma,” Journal ofPharmaceutical and Biomedical Analysis, vol. 44, no. 5, pp.1100–1105, 2007.

[7] Y. Higashi, S. Nakamura, H. Matsumura, and Y. Fujii, “Simul-taneous liquid chromatographic assay of amantadine and itsfour related compounds in phosphate-buffered saline using 4-fluoro-7-nitro-2,1,3-benzoxadiazole as a fluorescent derivati-zation reagent,” Biomedical Chromatography, vol. 20, no. 5, pp.423–428, 2006.

[8] T. H. Duh, H. L. Wu, C. W. Pan, and H. S. Kou, “Fluorimetricliquid chromatographic analysis of amantadine in urine andpharmaceutical formulation,” Journal of Chromatography A,vol. 1088, no. 1-2, pp. 175–181, 2005.

[9] H. J. Leis, G. Fauler, and W. Windischhofer, “Quantitativeanalysis of memantine in human plasma by gas chromatog-raphy/negative ion chemical ionization/mass spectrometry,”Journal of Mass Spectrometry, vol. 37, no. 5, pp. 477–480, 2002.

[10] H. H. Yeh, Y. H. Yang, and S. H. Chen, “Simultaneous deter-mination of memantine and amantadine in human plasma asfluorescein derivatives by micellar electrokinetic chromatogra-phy with laser-induced fluorescence detection and its clinicalapplication,” Electrophoresis, vol. 31, no. 11, pp. 1903–1911,2010.

[11] Guidance for industry Food-effect bio availability and fed bioequivalence studies, U.S. Department of health and humanservices food and drug administration centre for drug eval-uation and research (CDER), December 2002.

[12] Guidance for industry bio availability and fed bio equivalenceStudies for Orally Administered Drug Products-General con-siderations, U.S. Department of health and human servicesfood and drug administration centre for drug evaluation andresearch (CDER), March 2003.

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