DEVELOPMENT AND VALIDATION OF ANALYTICAL METHODS FOR NEW CHEMICAL ENTITIES AND THEIR DOSAGE FORMS BY USING HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

Transcription

1 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 CELL JAWAHARLAL NEHRU TECHNOLOGICAL UNIVERSITY ANANTAPUR ANANTAPUR A.P., INDIA SEPTEMBER- 2012

2 ii DEDICATED TO MY BELOVED PARENTS

3 iii JAWAHARLAL NEHRU TECHNOLOGICAL UNIVERSITY ANANTAPUR ANANTAPUR , 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 Dr.K.B. CHANDRA SEKHAR Professor in Chemistry & Director of Evaluation J N T University Anantapur, Anantapur, A.P, INDIA Research Supervisor Dr.CH.BABU RAO Professor & Director, Don Bosco P G College of Pharmacy 5 Th Mile, PulladiGunta (Po), Guntur (D.t.) A.P, INDIA

4 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

5 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.

6 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

7 vii CONTENTS Chapter Contents Page No. List of Tables ix List of Figures xvi List of Abbrevations xix Abstract xxii 1 Introduction 1.1 Pharmaceutical Analysis Extraction Procedures for Drugs and Metabolites from Biological Samples Method development and validation Estimation of Drugs in Biological Sample by HPLC-MS 1.5 Bioavailability and Bioequivalence Studies Aim and objectives of Present Research work 25 3 Analytical method development and validation of Rasagiline by High Performance Liquid Chromatography with mass spectrometry 3.1 Introduction Experimental Investigations Method Development Method Validation Application Pharmacokinetic Studies 73 4 Analytical method development and validation of Almotriptan by High Performance Liquid Chromatography with mass spectrometry 4.1 Introduction Experimental Investigations Method Development 79

8 viii 4.4 Method Validation Application Pharmacokinetic Studies Analytical method development and validation of Valacyclovir by High Performance Liquid Chromatography with mass spectrometry 5.1 Introduction Experimental Investigations Method Development Method Validation Application Pharmacokinetic Studies Analytical method development and validation of Memantine by High Performance Liquid Chromatography with mass spectrometry 6.1 Introduction Experimental Investigations Method Development Method Validation Application Pharmacokinetic Studies Summary and Conclusion Bibliography Appendx: Research Publications

9 ix LIST OF TABLES Table No. Description Page No. 3.1 Preparation of Reagents and Solvents Preparation of Stock solutions Screening of Different batches of blank matrix (Human K 2 EDTA Plasma) for interference free Rasagiline blank plasma 3.4 Specificity of Different batches of blank matrix (Human K 2 EDTA Plasma) for Rasagiline Limit of Quantitation for analyte (Rasagiline) Intra batch (Within-Batch) Accuracy and Precision for determination of Rasagiline levels in human plasma 3.7 Inter batch (Between-Batch) Accuracy and Precision for determination of Rasagiline levels in human plasma 3.8 Summary of calibration curve parameters for Rasagiline in human Plasma 3.9 Back-calculated standard concentrations from each calibration curve for Rasagiline in human plasma Recovery of analyte Rasagiline and Rasagiline- 13 C 3 mesylate from human plasma 3.11 Assessment of Matrix Effect on determination of Rasagiline at LQC levels in human plasma 3.12 Assessment of Matrix Effect on determination of Rasagiline at HQC levels in human plasma 3.13 Assessment of Dilution integrity for Rasagiline at DQC Conc (pg/ml) 3.14 Assessment of Whole Batch Re-injection Reproducibility during estimation of Rasagiline in human plasma 3.15 Ruggedness of the method for estimation of Rasagiline Plasma levels in human plasma with different Analyst

10 x Table No. Description Page No Ruggedness of the method for estimation of Rasagiline Plasma levels in human plasma with different Analytical column 3.17 Assessment of stability of Analyte (Rasagiline) in Biological matrix at Room temperature 3.18 Assessment of Freeze-Thaw stability of Analyte(Rasagiline ) at -30 ±5 C 3.19 Assessment of Auto sampler stability of Analyte (Rasagiline) at 2-8 C 3.20 Assessment of Long term plasma stability of Analyte (Rasagiline) at -30 C 3.21 Assessment of Short term stock solution stability of Analyte (Rasagiline) and Internal standard (Rasagiline- 13 C 3 mesylate) at Room temperature 3.22 Assessment of Short term solution stability of Internal standard Spiking solution (Rasagiline- 13 C 3 mesylate) at refrigerated conditions 3.23 Rasagiline mean concentration (pg/ml) data for the subject samples obtained from the LC-MS/MS Rasagiline Pharmacokinetic data Rasagiline Pharmacokinetic data (Test/Reference) Preparation of Reagents and Solvents Preparation of Stock solutions Screening of Different batches of blank matrix (Human K 2 EDTA Plasma) for interference free Almotriptan blank plasma 4.4 Specificity of Different batches of blank matrix (Human K 2 EDTA Plasma) for Almotriptan Limit of Quantitation for analyte (Almotriptan) Intra batch (Within-Batch) Accuracy and Precision for determination of Almotriptan levels in human plasma 98

11 xi Table No. Description Page No. 4.7 Inter batch (Between-Batch) Accuracy and Precision for determination of Almotriptan levels in human plasma 4.8 Summary of calibration curve parameters for Almotriptan in human Plasma 4.9 Back-calculated standard concentrations from each calibration curve for Almotriptan in human plasma 4.10 Recovery of analyte Almotriptan and Almotriptan- D 6 from human plasma 4.11 Assessment of Matrix Effect on determination of Almotriptan at LQC levels in human plasma 4.12 Assessment of Matrix Effect on determination of Almotriptan at HQC levels in human plasma 4.13 Assessment of Dilution integrity for Almotriptan at DQC Conc (ng/ml) 4.14 Assessment of Whole Batch Re-injection Reproducibility during estimation of Almotriptan in human plasma 4.15 Ruggedness of the method for estimation of Almotriptan Plasma levels in human plasma with different Analyst 4.16 Ruggedness of the method for estimation of Almotriptan Plasma levels in human plasma with different Analytical column Assessment of stability of Analyte (Almotriptan) in Biological matrix at Room temperature Assessment of Freeze-Thaw stability of Analyte (Almotriptan) at -30 ±5 C 4.19 Assessment of Auto sampler stability of Analyte (Almotriptan) at 2-8 C 4.20 Assessment of Long term plasma stability of Analyte (Almotriptan) at -30 C 4.21 Assessment of Short term stock solution stability of Analyte (Almotriptan) and Internal standard (Almotriptan- D 6 ) at Room temperature

12 xii Table No. Description Page No Assessment of Short term solution stability of Internal standard Spiking solution (Almotriptan- D 6 ) at refrigerated conditions 4.23 Almotriptan mean concentration (ng/ml) data for the subject samples obtained from the LC-MS/MS Almotriptan Pharmacokinetic data Almotriptan Pharmacokinetic data (Test/Reference) Preparation of Reagents and Solvents Preparation of Stock solutions Screening of Different batches of blank matrix (Human K 2 EDTA Plasma) for interference free Valacyclovir blank plasma 5.4 Specificity of Different batches of blank matrix (Human K 2 EDTA Plasma) for Valacyclovir Limit of Quantitation for analyte (Valacyclovir) Intra batch (Within-Batch) Accuracy and Precision for determination of Valacyclovir levels in human plasma 5.7 Inter batch (Between-Batch) Accuracy and Precision for determination of Valacyclovir levels in human plasma 5.8 Summary of calibration curve parameters for Valacyclovir in human Plasma 5.9 Back-calculated standard concentrations from each calibration curve for Valacyclovir in human plasma Recovery of analyte Valacyclovir and Valacyclovir- D 8 from human plasma 5.11 Assessment of Matrix Effect on determination of Valacyclovir at LQC levels in human plasma 5.12 Assessment of Matrix Effect on determination of Valacyclovir at HQC levels in human plasma

13 xiii Table No. Description Page No Assessment of Dilution integrity for Valacyclovir at DQC Conc (ng/ml) 5.14 Assessment of Whole Batch Re-injection Reproducibility during estimation of Valacyclovir in human plasma 5.15 Ruggedness of the method for estimation of Valacyclovir Plasma levels in human plasma with different Analyst 5.16 Ruggedness of the method for estimation of Valacyclovir Plasma levels in human plasma with different Analytical column Assessment of stability of Analyte (Valacyclovir) in Biological matrix at Room temperature Assessment of Freeze-Thaw stability of Analyte (Valacyclovir) at -30 ±5 C 5.19 Assessment of Auto sampler stability of Analyte (Valacyclovir) at 2-8 C 5.20 Assessment of Long term plasma stability of Analyte (Valacyclovir) at -30 C 5.21 Assessment of Short term stock solution stability of Analyte (Valacyclovir) and Internal standard (Valacyclovir- D 8 ) at Room temperature 5.22 Assessment of Short term solution stability of Internal standard Spiking solution (Valacyclovir- D 8 ) at refrigerated conditions 5.23 Valacyclovir mean concentration (ng/ml) data for the subject samples obtained from the LC-MS/MS bioanalytical method Valacyclovir Pharmacokinetic data Valacyclovir Pharmacokinetic data (Test/Reference) Preparation of Reagents and Solvents Preparation of Stock solutions 168

14 xiv Table No. Description Page No. 6.3 Screening of Different batches of blank matrix (Human K 2 EDTA Plasma) for interference free Memantine blank plasma 6.4 Specificity of Different batches of blank matrix (Human K 2 EDTA Plasma) for Memantine Limit of Quantitation for analyte (Memantine) Intra batch (Within-Batch) Accuracy and Precision for determination of Memantine levels in human plasma 6.7 Inter batch (Between-Batch) Accuracy and Precision for determination of Memantine levels in human plasma 6.8 Summary of calibration curve parameters for Memantine in human Plasma 6.9 Back-calculated standard concentrations from each calibration curve for Memantine in human plasma Recovery of analyte Memantine and Memantine- D 6 from human plasma 6.11 Assessment of Matrix Effect on determination of Memantine at LQC levels in human plasma 6.12 Assessment of Matrix Effect on determination of Memantine at HQC levels in human plasma 6.13 Assessment of Dilution integrity for Memantine at DQC Conc (pg/ml) 6.14 Assessment of Whole Batch Re-injection Reproducibility during estimation of Memantine in human plasma 6.15 Ruggedness of the method for estimation of Memantine Plasma levels in human plasma with different Analyst 6.16 Ruggedness of the method for estimation of Memantine Plasma levels in human plasma with different Analytical column Assessment of stability of Analyte (Memantine) in Biological matrix at Room temperature

15 xv Table No. Description Page No Assessment of Freeze-Thaw stability of Analyte (Memantine) at -30 ±5 C 6.19 Assessment of Auto sampler stability of Analyte (Memantine) at 2-8 C 6.20 Assessment of Long term plasma stability of Analyte (Memantine) at -30 C 6.21 Assessment of Short term stock solution stability of Analyte (Memantine) and Internal standard (Memantine- D 6 ) at Room temperature 6.22 Assessment of Short term solution stability of Internal standard Spiking solution (Memantine- D 6 ) at refrigerated conditions 6.23 Memantine mean concentration (pg/ml) data for the subject samples obtained from the LC-MS/MS Memantine Pharmacokinetic data Memantine Pharmacokinetic data (Test/Reference) 213

16 xvi LIST OF FIGURES Figure No. Description Page No. 3.1 Chemical structures of Rasagiline, Rasagiline- 13 C 3 mesylate Parent ion mass spectra (Q 1 ) of Rasagiline Product ion mass spectra (Q 3 ) of Rasagiline Parent ion mass spectra (Q 1 ) of Rasagiline- 13 C 3 mesylate Product ion mass spectra (Q 3 ) of Rasagiline- 13 C 3 mesylate MRM Chromatogram of Blank Human Plasma Sample Chromatogram of Blank + IS Chromatogram of LOQ sample (Rasagiline & IS) Chromatogram of ULOQ Sample (Rasagiline & IS) Chromatogram of LLOQ Sample (Rasagiline & IS) Chromatogram of LQC Sample (Rasagiline & IS ) Chromatogram of MQC Sample (Rasagiline & IS) Chromatogram of HQC Sample (Rasagiline& IS) Calibration Curve of Rasagiline Mean plasma concentration Vs time curve for Rasagiline Chemical structures of Almotriptan, Almotriptan- D 6 malate Parent ion mass spectra (Q 1 ) of Almotriptan Product ion mass spectra (Q 3 ) of Almotriptan Parent ion mass spectra (Q 1 ) of Almotriptan- D 6 malate Product ion mass spectra (Q 3 ) of Almotriptan- D 6 malate MRM Chromatogram of Blank Human Plasma Sample Chromatogram of Blank + IS Chromatogram of LOQ sample (Almotriptan & IS) 88

17 xvii Figure No. Description Page No. 4.9 Chromatogram of ULOQ Sample (Almotriptan & IS ) Chromatogram of LLOQ Sample (Almotriptan & IS) Chromatogram of LQC Sample (Almotriptan & IS ) Chromatogram of MQC Sample (Almotriptan & IS) Chromatogram of HQC Sample (Almotriptan & IS) Calibration Curve of Almotriptan Mean plasma concentration Vs time curve for Almotriptan Chemical structures of Valacyclovir, Valacyclovir- D Parent and Product ion mass spectra of Valacyclovir Parent and Product ion mass spectra of Valacyclovir- D MRM Chromatogram of Blank Human Plasma Sample Chromatogram of Blank + IS Chromatogram of LOQ sample (Valacyclovir & IS) Chromatogram of ULOQ sample (Valacyclovir & IS) Chromatogram of LLOQ Sample (Valacyclovir & IS) Chromatogram of LQC Sample (Valacyclovir & IS) Chromatogram of MQC Sample (Valacyclovir & IS) Chromatogram of HQC Sample (Valacyclovir & IS) Calibration Curve of Valacyclovir Mean plasma concentration Vs time curve for Valacyclovir Chemical structures of Memantine, Memantine- D Parent ion mass spectra (Q 1 ) of Memantine Product ion mass spectra (Q 3 ) of Memantine Parent ion mass spectra (Q 1 ) of Memantine -D 6 172

18 xviii Figure No. Description Page No. 6.5 Product ion mass spectra (Q 3 ) of Memantine- D MRM Chromatogram of Blank Human Plasma Sample Chromatogram of Blank + IS Chromatogram of LOQ sample (Memantine & IS) Chromatogram of ULOQ sample (Memantine & IS ) Chromatogram of LLOQ Sample (Memantine & IS) Chromatogram of LQC Sample (Memantine & IS) Chromatogram of MQC Sample (Memantine & IS) Chromatogram of HQC Sample (Memantine & IS) Calibration Curve of Memantine Mean plasma concentration Vs time curve for Memantine 211

19 xix LIST OF ABBREVATIONS RSG RSG-IS ALM : Rasagiline : Rasagiline - 13 C 3 mesylate : Almotriptan ALM- D 6 : Almotriptan- D 6 VL : Valacyclovir VL- D 8 : Valacyclovir- D 8 ME : Memantine ME-D 6 : Memantine- D 6 IS MP SP TP LC-MS/MS HPLC LLOQ LLOQC LQC DQC MQC HQC ULOQ LOD : Internal Standard : Mobile Phase : Stationary Phase : Theoretical Plates : Liquid Chromatography - Tandem Mass Spectrometry : High Performance Liquid Chromatography : Lower Limit of Quantitation : Lower Limit of Quality Control : Low Quality Control : Dilution Quality Control : Medium Quality Control : High Quality Control : Upper Limit of Quantitation : Limit of Detection

20 xx PPT LLE SPE CS QC mg : Precipitation : Liquid-liquid extraction : Solid phase extraction : Calibration Standards : Quality Control : Milli Grams µg : Micro Grams µl : Micro Litre ng pg ml Hrs Min Sec rpm RT Temp Conc DCM SD CV Max. : Nano Grams : Pico Grams : Milli Litre : Hours : Minutes : Seconds : Rotations per Minute : Retention Time : Temperature : Concentration : Dichloro methane : Standard Deviation : Coefficient of Variation : Maximum

21 0 C : Degrees Celsius xxi r API ACN MEOH USFDA ICH NDA EP FP NNC PK BA BE IEC MRM ANOVA DP CE CAD CXP MBTE : Correlation Coefficient : Active Pharmaceutical Ingredient : Acetonitrile : Methanol : United States Food and Drug Administration : International Conference on Harmonization : New Drug Application : Entrance Potential : Focussing Potential : invitro-invivo correlation : Pharmacokinetic : Bioavailability : Bioequivalence : Institutional Ethics committee : Multiple Reaction Monitoring : Analysis of Variance : Declustering Potential : Collision Energy : Collisionally Activated Dissociation : Collision exist Potential : Methyl Tertiary Butyl Ether

22 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

23 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

24 xxiv In chapter 5 discussed and developed the analytical method namely Analytical method development and validation of Valacyclovir by High Performance Liquid Chromatography with mass spectrometry In chapter 6 discussed and developed the analytical method namely Analytical method development and validation of Memantine by High Performance Liquid Chromatography with mass spectrometry In chapter 7 Summarized the overall thesis and conclusion was drawn In chapter 8 Summarized all the Bibliography.

25 CHAPTER 1 Introduction

26 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 analysis 1-3. 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 reproducible 3. 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 data 2.The main analytical phases comprise method development, method validation and sample analysis (method application) Need for pharmaceutical Analysis New Drug Development. Method Validation as for ICH Guidelines Research in Pharmaceutical Sciences Clinical Pharmacokinetic Studies Chapter 1 Introduction

27 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 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 metabolites 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. Chapter 1 Introduction

28 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 fluid 6-8. Different extraction procedures include protein precipitation or denaturation, liquid-liquid extraction, solid phase extraction and dehydration methods 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. Chapter 1 Introduction

29 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 Chapter 1 Introduction

30 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 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. Chapter 1 Introduction

31 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 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 Method development Method development 3,4-16 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. Chapter 1 Introduction

32 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 samples 12, 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, Chapter 1 Introduction

33 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 ex ceed 20% of the CV. Precision is further subdivided into: Chapter 1 Introduction

34 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) curve 3,4,26-29 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 Chapter 1 Introduction

35 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 %. 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. Chapter 1 Introduction

36 11 Stability in a Biological Fluid Drug stability in a biological fluid 3,4,10,14,24-27 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 Chapter 1 Introduction

37 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. Chapter 1 Introduction

38 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 Method Validation Method Validation 3,4,10,12,21-23,28,29 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 Chapter 1 Introduction

39 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 Chapter 1 Introduction

40 15 conducted at each site or laboratory to establish inter laboratory reliability. Crossvalidation 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 Chapter 1 Introduction

41 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 Chapter 1 Introduction

42 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 Application of Validated Method to Routine DrugAnalysis Assay and analysis 3,4,13,26-29 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. Chapter 1 Introduction

43 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. Chapter 1 Introduction

44 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 backcalculated (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: Chapter 1 Introduction

45 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 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 procedures 19.Some of the advantages are: Speed (analysis can be accomplished in 10 minutes or less) Chapter 1 Introduction

46 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 o o o o o Normal phase mode Reverse phase mode Reverse phase ion pair chromatography Ion-Exchange chromatography Affinity chromatography Size Exclusion chromatography (gel permeation and gel filtration chromatography) 1.5. Bioavailability and Bioequivalence studies 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 Chapter 1 Introduction

47 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 action 20. 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 product 20. 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 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 study 20. 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 Chapter 1 Introduction

48 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 useful 20. 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., C max, T max, mean absorption time, mean residence time, C max 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 Chapter 1 Introduction

49 24 early exposure, peak exposure, and total exposure portions of the plasma, serum, or blood concentration time profile 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 C max that are reflective of systemic exposure Chapter 1 Introduction

50 CHAPTER 2 Aim and Objectives of the Present Research work

51 25 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. Chapter 2 Aim & Objectives of the Present Research work

52 26 LIST OF DRUGS SELECTED FOR PRESENT RESEARCH WORK S.NO Name of the Drug Substance Chemical Structure Classification 1. Rasagiline mesylate Parkinson's disease 2. Almotriptan malate Relief of migraine 3. Valacyclovir Hydrochloride Anti viral drug 4. Memantine Hydrochloride Alzheimer's disease Chapter 2 Aim & Objectives of the Present Research work

53 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 2 Aim & Objectives of the Present Research work

54 CHAPTER 3 Analytical method development and validation of Rasagiline by High performance Liquid chromatography with mass spectrometry

55 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 C 12 H 13 N with its molecular weight 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 (C max ) in approximately 1 hour. The absolute bioavailability of rasagiline is about 36%. Food does not affect the T max of rasagiline, although C max 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 Chapter 3 Rasagiline - Introduction

56 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 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 P 450 (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 minutes Literature survey reveals that only a few methods were reported to quantification of rasagiline in human plasma and pharmaceutical analysis 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 ng/mL and 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.al 38 with shorter runtimes at 3.5 minutes for each sample at a concentration range Chapter 3 Rasagiline - Introduction

57 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.al 37 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 - 13 C 3 mesylate ). This method can also be employed in Pharmacokinetic and Bio-equivalence study of Rasagiline. Chapter 3 Rasagiline - Introduction

58 Experimental Investigations Materials and reagents Rasagiline and Rasagiline- 13 C 3 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 Na 2 CO 3 were procured from Merck (Mumbai, India). Human plasma (K 2 EDTA) 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 Instrumentation and equipment An API 4000 LC-MS/MS system, 1200 Series HPLC system ( Agilent Technologies, Waldbronn, Germany), and Applied Biosystems Analyst Software version 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, mm (Tarson) polypropylene tubes, combitips ( Eppendorff) and variable size surgical gloves (Surgicare). Chapter 3 Rasagiline - Experimental

59 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. Mix 200 ml of Methanol with 800 ml 0.1% Formic Acid. Mobile phase Filter through 0.45 m filter Extraction Solvent Reconstitution solution (Autosampler wash) 80% Methanol Mix 750 L of Methyl tertiary butyl ether with 250 ml of Dichloro methane Mix 200 ml of Methanol with 800 ml 0.1% Formic Acid. Mix 800 ml of Methanol with 200 ml of water 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- 13 C 3 mesylate stock solution 50.0 µg/ml 50 ml Methanol Chapter 3 Rasagiline - Experimental

60 Preparation of standards and quality control (QC) samples Standard stock solutions of Rasagiline (50.0µg/ ml) and Rasagiline- 13 C 3 mesylate (5 0.0µg/ ml) were prepared in methanol. The IS spiking solutions (30.0 ng /ml) were prepared in 50% methanol from Rasagiline- 13 C 3 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, , , , , and pg/ml for analytical standards and 5.0, 15.0, and 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. Chapter 3 Rasagiline - Experimental

61 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 - 13 C 3 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- 13 C 3 mesylate molecules. A CAD product ion spectrum for Rasagiline and Rasagiline- 13 C 3 mesylate yielded high-abundance fragment ions of m/z(amu) and m/z(amu) respectively (Figure Figure 3.5) from its parent ion mass spectra. Chapter 3 Rasagiline Method Development

62 35 Figure 3.2 Parent ion mass spectra (Q 1 ) of Rasagiline Chapter 3 Rasagiline Method Development

63 36 Figure 3.3 Product ion mass spectra (Q 3 ) of Rasagiline Chapter 3 Rasagiline Method Development

64 37 Figure 3.4 Parent ion mass spectra (Q 1 ) of Rasagiline - 13 C 3 mesylate Chapter 3 Rasagiline Method Development

65 38 Figure 3.5 Product ion mass spectra (Q 3 ) of Rasagiline- 13 C 3 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 Chapter 3 Rasagiline Method Development

66 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- 13 C 3 mesylate.short length columns, such as Symmetry Shield RP 18 (50mm x 2.1 mm, 3.5 μm), Inertsil ODS-2V (50mm x 4.6 mm,5μm), Hypurity C 18 (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 C 18, (2.1 mm 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 C 18, 2.1 x 50 mm, 3.5 m, column. It gave satisfactory peak shapes for both Rasagiline and Rasagiline- 13 C 3 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 isotopelabeled or suitable analog drugs as an internal standard proves helpful when a significant matrix effect is possible. In our case, Rasagiline- 13 C 3 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 supp ression 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 Chapter 3 Rasagiline Method Development

67 40 with several organic solvents (ethyl acetate, chlorofo rm, 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- 13 C 3 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 C 18, 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- 13 C 3 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- 13 C 3 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 Chapter 3 Rasagiline Method Development

68 41 1M Na 2 CO 3 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 pg/ml in human plasma. Calibration curves were obtained by weighted linear regression (weighing factor: 1/ x 2 ). The ratio of Rasagiline peak area to Rasagiline- 13 C 3 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. Chapter 3 Rasagiline Method Development

69 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 Figure 3.6 MRM Chromatogram of Blank Human Plasma Sample Chapter 3 Rasagiline Method Validation

70 43 Figure 3.7 Chromatogram of Blank + IS Figure 3.8 Chromatogram of LOQ Sample (Rasagiline & IS) Chapter 3 Rasagiline Method Validation

71 44 Figure 3.9 Chromatogram of ULOQ Sample (Rasagiline & IS) Figure 3.10 Chromatogram of LLOQ Sample (Rasagiline & IS) Chapter 3 Rasagiline Method Validation

72 45 Figure 3.11 Chromatogram of LQC Sample (Rasagiline & IS) Figure 3.12 Chromatogram of MQC Sample (Rasagiline & IS) Chapter 3 Rasagiline Method Validation

73 46 Figure 3.13 Chromatogram of HQC Sample (Rasagiline & IS) Figure 3.14 Calibration Curve of Rasagiline Chapter 3 Rasagiline Method Validation

74 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 K 2 EDTA Plasma) for interference free Rasagiline blank plasma Matrix identification Blank plasma Area at Analyte (Rasagiline) RT Internal standard RT A A A A A A A A A A 0 0 Blank+IS with PL Blank(Blank Plasma Lot-1) LOQ with PL Blank (Blank Plasma Lot-1) 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 Chapter 3 Rasagiline Method Validation

75 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 K 2 EDTA Plasma) for Rasagiline Matrix Identification LLOQ Area Internal standard (IS) Area Interference with Analyte (% of LLOQ Response) Interference with IS (% of IS Response) A A A A A A 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. Chapter 3 Rasagiline Method Validation

76 49 Table 3.5 Limit of Quantitation for analyte (Rasagiline) Matrix identification Blank plasma area at Analyte RT LLOQ response LLOQ S/N RATIO AP/ A N Mean SD CV% LLOQ was spiked in A Acceptance criteria: 1. Mean S/N ratio of LLOQ should be 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 Chapter 3 Rasagiline Method Validation

77 50 Table 3.6 Intra batch (Within-Batch) Accuracy and Precision for determination of Rasagiline levels in human plasma Analytical Run ID P&A Batch 1 LLOQ 5.00 pg/ml Conc. Found % Nominal Low QC pg/ml Conc. Found % Nominal Mid QC pg/ml Conc. Found % Nominal High QC pg/ml Conc. Found % Nominal N Mean SD (±) CV (%) %Nominal Acceptance 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 ranged between 97.30% and %. The Inter batch percentage of coefficient of variation is 1.00% to 3.80% for Rasagiline. Results are presented in Table 3.7 Chapter 3 Rasagiline Method Validation

78 51 Table 3.7 Inter batch (Between-Bach) Accuracy and Precision for determination Analytical Run ID LLOQ 5.00 pg/ml Conc. Found of Rasagiline levels in human plasma % Nominal Low QC pg/ml Conc. % Found Nominal Mid QC pg/ml Conc. % Found Nominal High QC pg/ml Conc. % Found Nominal P&A Batch P&A Batch P&A Batch P&A Batch P&A Batch N Mean SD (±) CV (%) %Nominal Acceptance criteria: Same as presented intable 3.6 Chapter 3 Rasagiline Method Validation

79 52 Calibration Curve Calibration curves are found to be consistently accurate and precise for Rasagiline over 5.0 to pg/ml for calibration range. The correlation coefficient is greater than or equal to 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 human Plasma Analytical Run ID slope intercept r-squared P&A Batch P&A Batch P&A Batch P&A Batch P&A Batch N Mean SD(±) % CV Acceptance criteria: 1. Coefficient of regression (r) Chapter 3 Rasagiline Method Validation

80 53 Table 3.9 Back-calculated standard concentrations from each calibration curve for Rasagiline in human plasma. Analytical Run ID Nominal Concentration (ng/ml) CS1 CS2 CS3 CS4 CS pg/ml pg/ml pg/ml pg/ml pg/ml P&A Batch P&A Batch P&A Batch P&A Batch P&A Batch N Mean SD(±) % CV %Nominal Nominal Concentration(ng/mL) Analytical CS6 CS7 CS8 CS9 CS10 run ID pg/ml pg/ml pg/ml pg/ml pg/ml P&A Batch P&A Batch P&A Batch P&A Batch P&A Batch N Mean SD(±) % CV % Nominal 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%. Chapter 3 Rasagiline Method Validation

81 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- 13 C 3 mesylate for extracted is 3.40%. Results are presented in Table 3.10 Chapter 3 Rasagiline Method Validation

82 55 Table 3.10 Recovery of Analyte Rasagiline and Rasagiline- 13 C 3 mesylate from human plasma Standard Unextracted Rasagiline peak response Extracted Rasagiline peak response Unextracted Rasagiline 13 C 3 mesylate peak response Extracted Rasagiline 13 C 3 mesylate peak response Low QC: pg/ml N 6 6 Mean SD(±) % CV % Recovery Medium QC: pg/ml N 6 6 Mean SD(±) % CV % Recovery N 6 6 High QC: pg/ml Mean SD(±) % CV % Recovery 97.0 Mean recovery Mean % CV Chapter 3 Rasagiline Method Validation

83 56 Acceptance 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%. 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- 13 C 3 mesylate. Results are presented in Tables 3.11 and Chapter 3 Rasagiline Method Validation

84 57 Table 3.11 Assessment of Matrix Effect on determination of Rasagiline at LQC levels in human plasma Identification of matrix Drug response in Matrix at LQC Level Internal standard response Matrix factor A A A A A A A A A A A A A A A A A A N Grand Mean SD(±) % CV Acceptance criteria: 1. Mean % Nominal 100 ±15% of nominal value. 2. % CV 15%. Chapter 3 Rasagiline Method Validation

85 58 Table 3.12 Assessment of Matrix Effect on determination of Rasagiline at HQC levels in human plasma Identification of matrix Drug response in Matrix at HQC Level Internal standard response Matrix factor A A A A A A A A A A A A A A A A A A N Grand Mean SD(±) 0.26 % CV 3.80 Acceptance criteria: 1. Mean % Nominal 100 ±15% of nominal value. 2. % CV 15%. Chapter 3 Rasagiline Method Validation

86 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 % and 97.78% to % 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. Results are presented in Table 3.13 Table 3.13 Assessment of Dilution integrity for Rasagiline at DQC Concentration (pg/ml) DQC Dilution factor: ½ Nominal conc.: pg/ml DQC Dilution factor: ¼ Nominal conc.: pg/ml Conc. Found % Nominal Conc. Found %Nominal N 6 6 Mean SD(±) % CV Acceptance criteria 1. % CV 15%. 2. Mean % Nominal (100 ±15%). Chapter 3 Rasagiline Method Validation

87 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 % 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 during estimation of Rasagiline in human plasma Analytical Low QC 1.5 pg/ml High QC pg/ml Run ID comp Re-inj sample comp Re-inj sample sample sample N Mean SD(±) % CV %Accuracy Acceptance criteria: 1. % CV 15% Except LLOQ for which it is 20%. 2. Mean % Nominal (100±15% & for LLOQ 100±20%) % of the re-injected QCs at each level shall be within ± 20% of their previous concentration. Chapter 3 Rasagiline Method Validation

88 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 %. 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 Table 3.15 Ruggedness of the method for estimation of Rasagiline Plasma levels in human plasma with different Analyst LLOQ 5.00pg/mL Low QC pg/ml Mid QC High QC pg/ml pg/ml Analyst ID 1 Analyst ID 2 Analyst ID 1 Analyst ID 2 Analyst ID 1 Analyst ID 2 Analyst ID 1 Analyst ID N Mean Analytical Run ID P&A Batch 1 Analyst A 3 Analyst B SD(±) CV (%) %Accuracy Acceptance criteria: 1. % CV 15 % except LLOQ for which it is 20%. 2. Mean % Nominal (100±15% & for LLOQ 100 ±20%) Chapter 3 Rasagiline Method Validation

89 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 %. The Precision of Rasagiline QC samples within the range of 1.87% to 4.08%. These results indicated that the method is rugged and reproducible by different analyst. Results are presented in tables Table 3.16 Ruggedness of the method for estimation of Rasagiline in human plasma with different Analytical column Analytical Run ID LLOQ 5.00pg/mL Column ID LC/102 Column ID LC/115 Low QC pg/ml Column ID LC/102 Column ID LC/115 Mid QC pg/ml Column Column ID ID LC/102 LC/115 High QC pg/ml Column Column ID ID LC/102 LC/ P&A Batch N Mean SD (±) CV (%) %NOM Acceptance criteria: 1. % CV 15 % except LLOQ for which it is 20%. 2. Mean % Nominal (100 ±15% & for LLOQ 100 ±20%). Chapter 3 Rasagiline Method Validation

90 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 % 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 Table 3.17 Assessment of stability of Analyte (Rasagiline) in biological matrix at Room temperature Low QC pg/ml High QC pg/ml Comparison samples (0.00 hr) Stability samples (24.0 hrs) Comparison samples (0.00 hr) Stability samples (24.0 hrs) Conc. found % nominal Conc. found % nominal Conc. found % nominal Conc. found % nominal P&A Batch N Mean SD(±) CV (%) Accuracy Acceptance criteria: 1. % change should be ± 15 or % Ratio (stability/comparison) should be within %. 2. % CV 15%. 3. Mean % Nominal (100 ±15%). Chapter 3 Rasagiline Method Validation

91 64 FREEZE AND THAW STABILITY (after 3 rd 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 mean Accuracy for LQC and HQC samples of Rasagiline from comparison samples were % and % respectively. The plasma samples of Rasagiline e were found to be stable after 3 cycles at - 30±5 C Results are present in Table Table 3.18 Assessment of Freeze-Thaw stability of Analyte (Rasagiline) at -30 ± 5 C Low QC pg/ml High QC pg/ml Comparison samples Stability sample at 4 th cycle Comparison samples Stability sample at 4 th cycle Conc. found % nominal Conc. found % nominal Conc. found % nominal Conc. found % nominal N P&A Batch 5 Mean SD (±) % CV %NOM Acceptance criteria: % change should be ± 15 or % Ratio (stability/comparison) should be within %. 1. % CV 15%. 2. Mean % Nominal (100 ±15%). Chapter 3 Rasagiline Method Validation

92 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. The mean Accuracy for LQC and HQC samples of Rasagiline from comparison samples were 98.61% and 95.02% respectively. Rasagiline samples were stable for 55 hrs at 2-8 C in autosampler. Results are present in Table Table 3.19 Assessment of Auto sampler stability of Analyte (Rasagiline) at 2-8 C Low QC pg/ml High QC pg/ml Comparison samples (0.0 hr) Stability samples (55hrs) Comparison samples (0.0 hr) Stability samples (55 hrs) Conc. found % nominal Conc. found % nominal Conc. found % nominal Conc. found % nominal P&A Batch N Mean SD(±) % CV %NOM Acceptance criteria: 1. % change should be ± 15 or % Ratio (stability/comparison) should be within %. 2. % CV 15%. 3. Mean % Nominal (100 ±15%). Chapter 3 Rasagiline Method Validation

93 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 mean Accuracy for LQC and HQC samples of Rasagiline from comparison samples were % and 95.33% respectively. 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. Low QC pg/ml High QC pg/ml Comparison samples (0.0 hr) Stability samples (64days) Comparison samples (0.0 hr) Stability samples (64 days) Conc. found % nominal Conc. found % nominal Conc. found % nominal Conc. found % nominal N P&A Batch 5 Mean SD(±) CV (%) %NOM Acceptance criteria: 1. % change should be ± 15 or % Ratio (stability/comparison) should be within %. 2. % CV 15%. 3. Mean % Nominal (100 ±15%). Chapter 3 Rasagiline Method Validation

94 67 Short term stock solution stability of Rasagiline, Rasagiline - 13 C 3 mesylate and spiking solution of internal standard ( Rasagiline- 13 C 3 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 C 3 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 C 3 mesylate 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 Rasagiline stock solution from comparison samples was 0.4% and %Ratio (stability/comparison) was The %CV for Rasagiline 13 C 3 mesylate stock solution from comparison samples was 1.7% and %Ratio (stability/comparison) was The %CV for Rasagiline 13 C 3 mesylate working solution (Internal standard spiking solution) from comparison samples was 1.1% and %Ratio (stability/comparison) was Rasagiline, Rasagiline 13 C 3 mesylate stock solutions and Rasagiline 13 C 3 mesylate working solutions were found to be stable at room temperature for 9 hrs. Results are present in Tables 3.21 and Chapter 3 Rasagiline Method Validation

95 68 Table 3.21 Assessment of Short term stock solution stability of Analyte (Rasagiline) and internal standard (Rasagiline- 13 C 3 mesylate) at Room temperature Comparison standard stock solution response (0.0 hrs) Analyte Stability stock solution response (9hrs) Comparison stock solution response (0.0 hrs) Internal standard Stability standard stock Response (9hrs) Curve N Mean SD (±) CV (%) % Ratio Acceptance criteria: 1. % Ratio (stability/comparison) should be within %. Chapter 3 Rasagiline Method Validation

96 69 Table 3.22 Assessment of Short term solution stability of internal standard spiking solution (Rasagiline- 13 C 3 mesylate) at refrigerated conditions Comparison solution (Internal standard Spiking solution) Response (0.00 hrs) Stability solution (Internal standard Spiking solution) Response (10 days at refrigerated conditions) Curve N 6 6 Mean SD(±) % CV % Ratio Acceptance criteria: 1. % Ratio (stability/comparison) should be within %. 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. Chapter 3 Rasagiline Method Validation

97 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 K 2 EDTA. 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 Chapter 3 Rasagiline - Application

98 71 Table 3.23 Rasagiline Mean concentration (pg/ml) data for subject samples obtained from the LC-MS/MS Time in hours TEST Concentration (pg/ml) REFERENCE Chapter 3 Rasagiline - Application

99 72 Figure 3.15 Mean plasma concentration Vs time curve for Rasagiline Chapter 3 Rasagiline - Application

100 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 (t 1/2 ), 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 C max and T max values were determined. The AUC 0 t was obtained by the trapezoidal method. AUC 0 was calculated up to the last measureable concentration and extrapolations were obtained by the last measureable concentration and the terminal elimination rate constant (K el ). The K el was estimated from the slope of the terminal exponential phase of the plasma of the Rasagiline concentration-time curve using linear regression method. The t 1/2 was then calculated as 0.693/K el. The AUC 0 t, AUC 0 and C max 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 % for AUC 0 t, AUC 0 and C max 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 Chapter 3 Rasagiline - Pharmacokinetic Studies

101 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 Table 3.24 Rasagiline Pharmacokinetic data Rasagiline Pharmacokinetic data Pharmacokinetic Test Reference Parameter Mean±SD % CV Mean±SD % CV C max (pg/ml) ± ± AUC 0-t (pg/ml) ± ± AUC 0- (pg h/ml) ± ± T max (h) t 1/ Table 3.25 Rasagiline Pharmacokinetic data (Test /Reference) Pharmacokinetic Parameter C max AUC 0-t AUC 0- Test/Reference Chapter 3 Rasagiline - Pharmacokinetic Studies

102 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 AUC 0-t, AUC 0- C max, T max, and t 1/2. 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 3 Rasagiline - Pharmacokinetic Studies

103 CHAPTER 4 Analytical method development and validation of Almotriptan by High performance Liquid chromatography with mass spectrometry

104 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 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 D 6 malate As of now to our knowledge, several methods for the determination of Almotriptan in biological matrixes 42,45,46 pharmaceuticalcompounds by LC MS/MS 45,HPLC 47,48 HPTLC 49 and Fluorimetric and calorimetric 50 have been reported. However, Fleischhacker et.al 45 concentrated 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. Chapter 4 Almotriptan - Introduction

105 Experimental Investigations Materials and reagents Almotriptan malate was obtained from USP and Almotriptan malate- D 6 was obtained from clear synth Labs (P) Ltd, Mumbai, India. Human plasma (K 2 EDTA), 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 Instrumentation and equipment Refer Chapter Preparation of reagents and solvents Table 4.1 Preparation of Reagents and Solvents Reagents and Solvents preparation 50% Methanol Mix 500 ml of methanol with 500 ml of water. 10mM Ammonium formate P H : NSodium carbonate Mobile phase (Autosampler wash) 80% Acetonitrile Dissolve 1.26 g of ammonium formate into 2 L of water and adjust P H with formic acid Dissolve 26.5 g of anhydrous sodium carbonate into 1L of water. 10mM Ammonium formate p H :4.5: Acetonitrile in the ratio of 50:50 and Filter through 0.45 m filter Mix 800 ml of Acetonitrile with 200 ml of water. Reconstitution solution Extraction Solvent Mix 500 ml of 10mM ammonium formate with 500 ml of acetonitrile. MTBE (methyl-tertiary butyl ether) Chapter 4 Almotriptan - Experimental

106 Preparation of Stock solutions Table 4.2 Preparation of Stock solutions Name of the solution Concentration Volume (ml) Diluent Almotriptan stock solution µg/ml 25 ml Methanol Almotriptan-D 6 stock solution µg/ml 25 ml Methanol Preparation of standards and quality control (QC) Samples Standard stock solutions of Almotriptan (100.0µg/mL) and Almotriptan -D 6 (100.0µg/mL) were prepared in methanol. The spiking solution for Almotriptan-D 6 (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, and ng/ml for Analytical standards and 0.5, 1.5,75.0, and 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. Chapter 4 Almotriptan - Experimental

107 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 p H 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-D 6 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-D 6 yielded high-abundance fragment ions at m/z and (Figure ) 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 (p H 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 Chapter 4 Almotriptan - Method Development

108 80 method are most sensitive and accurate methods over the developed methods based on literature. Figure 4.2 Parent ion mass spectra (Q 1 ) of Almotriptan Chapter 4 Almotriptan - Method Development

109 81 Figure 4.3 Product ion mass spectra (Q 3 ) of Almotriptan Chapter 4 Almotriptan - Method Development

110 82 Figure 4.4 Parent ion mass spectra (Q 1 ) Almotriptan -D 6 malate Chapter 4 Almotriptan - Method Development

111 83 Figure 4.5 Product ion mass spectra (Q 3 ) of Almotriptan -D 6 Chapter 4 Almotriptan - Method Development

112 84 Chromatographic conditions A good separation and elution were achieved using Zorbax, SB C 18, 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-D 6 was found to be appropriate internal standard. Retention time of Almotriptan and Almotriptan-D 6 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-D 6 from human plasma. 100 µl of Almotriptan-D 6 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 (p H 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. Chapter 4 Almotriptan - Method Development

113 85 Calibration curve parameters and regression model The analytical curves were constructed using values ranging from ng/ml of Almotriptan in human plasma. Calibration curves were obtained by weighted 1/conc 2 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 r 2 = Coefficient of determination The ratio of Almotriptan peak area to Almotriptan -D 6 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%. Method Development Conclusion 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. Chapter 4 Almotriptan - Method Development

114 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 Representative chromatograms of Plasma blank, blank +IS, LOQ, ULOQ, LLOQC, LQC, MQC, HQC, Calibration curve are shown in Figure 4.6 to Figure Figure 4.6 MRM Chromatogram of Blank Human Plasma Sample Chapter 4 Almotriptan - Method Validation

115 87 Figure 4.7 Chromatogram of Blank + IS Chapter 4 Almotriptan - Method Validation

116 88 Figure 4.8 Chromatogram of LOQ Sample (Almotriptan & IS) Chapter 4 Almotriptan - Method Validation

117 89 Figure 4.9 Chromatogram of ULOQ Sample (Almotriptan & IS) Chapter 4 Almotriptan - Method Validation

118 90 Figure 4.10 Chromatogram of LLOQ Sample (Almotriptan & IS) Chapter 4 Almotriptan - Method Validation

119 91 Figure 4.11 Chromatogram of LQC Sample (Almotriptan & IS) Chapter 4 Almotriptan - Method Validation

120 92 Figure 4.12 Chromatogram of MQC Sample (Almotriptan & IS) Chapter 4 Almotriptan - Method Validation

121 93 Figure 4.13 Chromatogram of HQC Sample (Almotriptan & IS) Chapter 4 Almotriptan - Method Validation

122 94 Figure 4.14 Calibration Curve of Almotriptan Chapter 4 Almotriptan - Method Validation

123 95 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 4.3 Table 4.3 Screening of Different batches of blank matrix (Human K 2 EDTA Plasma) for interference free Almotriptan blank plasma Matrix identification Analyte (Almotriptan) RT Blank plasma Area Internal standard RT AP/3271/07/ AP/3272/07/ AP/3273/07/ AP/3274/07/ AP/3275/07/ AP/3276/07/ AP/3277/07/ AP/3278/07/ AP/3279/07/ AP/3280/07/ Blank+IS with AP/3271/07/ LOQ with AP/3271/07/ Blank Matrix Specificity and Limit of Quantification 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 Chapter 4 Almotriptan - Method Validation

124 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. The specificity experiment shall be considered for calculation of LOQ experiment. Results are presented in Table 4.5 Matrix Identification PL Blank- (K 2 EDTA- AP/3271/07/10) Table 4.4 Specificity of Different batches of blank matrix (Human K 2 EDTA Plasma) for Almotriptan LLOQ Area Internal standard (IS) area Interference with Analyte(% of LLOQ Response) Interference with IS(% of IS Response) AP/3271/07/ AP/3273/07/ AP/3274/07/ AP/3277/07/ AP/3280/07/ 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. Chapter 4 Almotriptan - Method Validation

125 97 Table 4.5 Limit of Quantification for analyte (Almotriptan) Matrix identification Blank plasma area at Analyte RT LLOQ response LLOQ S/N RATIO AP/3271/07/ N Mean LLOQ was spiked in AP/3271/07/10 Blank Plasma Lot Acceptance criteria: 1. Mean S/N ratio of LLOQ should be 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 Almotriptan was ranged between 98.67% and %. The Intra batch percentage of coefficient of variation is 0.79% to 3.05% for Almotriptan. Results are presented in Table 4.6 Chapter 4 Almotriptan - Method Validation

126 98 Table 4.6 Intra batch (Within-Batch) Accuracy and Precision for determination of Almotriptan levels in human plasma Analytical LLOQ 0.50 ng/ml Low QC 1.50 ng/ml Mid QC ng/ml High QC ng/ml Run ID Conc. found % nominal Conc. found % nominal Conc. found % nominal Conc. found % nominal P&A Batch N Mean SD (±) CV (%) %Accuracy Acceptance criteria: 1. % CV 15 % except LLOQ for which it is 20%. 2. Mean % Nominal (100 ±15% and 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 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 ranged between 98.62% and %. The Inter batch percentage of coefficient of variation is 1.23% to 2.65% for Almotriptan. Results are presented in Table 4.7 Chapter 4 Almotriptan - Method Validation

127 99 Table 4.7 Inter batch (Between-Batch) Accuracy and Precision for determination of Almotriptan levels in human plasma LLOQ 0.50 ng/ml Low QC 1.50 ng/ml Mid QC ng/ml High QC ng/ml Conc. found % nominal Conc. found % nominal Conc. found % nominal Conc. found % nominal N Analytical Run ID P&A Batch 1 P&A Batch 2 P&A Batch 3 P&A Batch 4 P&A Batch 5 Mean SD(±) CV (%) %Nominal Acceptance criteria: Same as Table 4.6 Chapter 4 Almotriptan - Method Validation

128 100 Calibration Curve Calibration curves are found to be consistently accurate and precise for Almotriptan over ng/mL for calibration range. The correlation coefficient is greater than for Almotriptan. Back calculations were made from the calibration curves to determine Almotriptan concentrations of each calibration standard. Results are presented in Tables 4.8 & 4.9. Table 4.8 Summary of calibration curve parameters for Almotriptan in human plasma Analytical Run Coefficient of Slope intercept ID regression (r 2 ) P&A Batch P&A Batch P&A Batch P&A Batch P&A Batch N Mean SD (±) CV (%) Regression Footnote (S): Resp.= Slope * Conc.+ Intercept Acceptance criteria: 1. Coefficient of regression (r) Chapter 4 Almotriptan - Method Validation

129 101 Table 4.9 Back-calculated standard concentrations from each calibration curve for Almotriptan in human plasma Nominal Concentration (ng/ml) Analytical Run CS1 CS2 CS3 CS4 CS5 ID 0.50 ng/ml 1.00 ng/ml 5.00 ng/ml ng/ml ng/ml P&A Batch P&A Batch P&A Batch P&A Batch P&A Batch N Mean SD(±) CV% %Nominal Nominal Concentration (ng/ml) Analytical run CS6 CS7 CS8 CS9 CS10 ID ng/ml ng/ml ng/ml ng/ml ng/ml P&A Batch P&A Batch P&A Batch P&A Batch P&A Batch N Mean SD(±) CV% %Nominal 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%. Chapter 4 Almotriptan - Method Validation

130 102 Recovery The percentage recovery of Almotriptan was determined by comparing the mean peak area of Almotriptan in extracted LQC, MQC, HQC samples with freshly prepared unextracted LQC, MQC, HQC samples respectively. The mean % recovery for LQC, MQC, HQC samples of Almotriptan were 97.73%, 90.97% and 93.35% respectively. 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%. 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 84.35%. The % CV recovery of IS Almotriptan D 6 for extracted is 4.3%. Results are presented in Table 4.10 Chapter 4 Almotriptan - Method Validation

131 103 Table 4.10 Recovery of Analyte (Almotriptan) and Almotriptan-D 6 from human plasma Standard Extracted peak response Unextracted peak response Drug IS Drug IS Low QC: ng/ml N % Recovery SD (±) 4.46 %CV 4.6 Medium QC: ng/ml N % Recovery SD(±) 3.04 %CV 3.3 High QC: ng/ml N % Recovery SD(±) 1.46 %CV 1.6 Drug IS Mean recovery Mean SD(±) Mean % CV Acceptance 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%. Chapter 4 Almotriptan - Method Validation

132 104 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 triplicate for each matrix were freshly prepared and analyzed in single run. No significant matrix effect found in different sources of human plasma tested for Almotriptan, Almotriptan -D 6. Results are presented in Table 4.11 and 4.12 Table 4.11 Assessment of Matrix Effect on determination of Almotriptan at LQC levels in human plasma Identification of Drug response Internal Matrix factor matrix in Matrix at LQC Level standard response AP/3271/07/ AP/3271/07/ AP/3271/07/ AP/3272/07/ AP/3272/07/ AP/3272/07/ AP/3273/07/ AP/3273/07/ AP/3273/07/ AP/3274/07/ AP/3274/07/ AP/3274/07/ AP/3275/07/ AP/3275/07/ AP/3275/07/ AP/3276/07/ AP/3276/07/ AP/3276/07/ N Grand Mean SD(±) CV (%) 3.15 Acceptance criteria: 1. Mean % Nominal 100 ±15% of nominal value. 2. % CV 15%. Chapter 4 Almotriptan - Method Validation

133 105 Table 4.12 Assessment of Matrix Effect on determination of Almotriptan at HQC levels in human plasma Identification of matrix Drug response in Matrix at HQC Level Internal standard response Matrix factor AP/3271/07/ AP/3271/07/ AP/3271/07/ AP/3272/07/ AP/3272/07/ AP/3272/07/ AP/3273/07/ AP/3273/07/ AP/3273/07/ AP/3274/07/ AP/3274/07/ AP/3274/07/ AP/3275/07/ AP/3275/07/ AP/3275/07/ AP/3276/07/ AP/3276/07/ AP/3276/07/ N Grand Mean SD(±) %CV 0.62 Acceptance criteria: 1. Mean % Nominal 100±15% of nominal value. 2. % CV 15%. Chapter 4 Almotriptan - Method Validation

134 106 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 Almotriptan nominal concentrations ranged between 94.35% and % for 1 in 4 dilutions and 1 in 2 dilutions respectively. The % CV is 1.54% to 2.01%. Results are presented in Table 4.13 Table 4.13 Assessment of Dilution integrity for Almotriptan at DQC Conc (ng/ml) DQC Dilution factor: ½ Nominal conc: ng/ml DQC Dilution factor: ¼ Nominal conc: ng/ml Conc. Found % Nominal Conc. Found %Nominal N 6 6 Mean %Nominal SD (±) CV (%) Acceptance criteria: 1. % CV 15%. 2. Mean % Nominal (100 ± 15%). Chapter 4 Almotriptan - Method Validation

135 107 Whole Batch Reinjection Reproducibility To evaluate the whole batch reinjection reproducibility experiment, samples of 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 calculated to determine precision and accuracy after reinjection. 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 between 1.16 % and 3.05%. Almotriptan was found to be stable at autosampler temperature post extraction (in reconstitution solution) for approx 26 hrs and reproducible after reinjection. Results are presented in Table 4.14 Table 4.14 Assessment of Whole Batch Re-injection Reproducibility during estimation of Almotriptan in human plasma Low QC 6.0 ng/ml High QC ng/ml Analytical Reinjection Reinjection Run ID Comp sample Comp sample sample sample N Mean SD(±) %CV %NOM Acceptance criteria: 1. % CV 15% Except LLOQ for which it is 20%. 2. Mean % Nominal (100 ±15% and for LLOQ 100±20%) % 0f the re-injected QCs at each level shall be within ±20% of their previous concentration. Chapter 4 Almotriptan - Method Validation

136 108 Ruggedness-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 Almotriptan QC samples within the range of 98.56% to %. The Precision of Almotriptan QC samples within the range of 0.79% to 3.05%. These results indicated that the method is rugged and reproducible by different analyst. Results are presented in Table 4.15 Table 4.15 Ruggedness of the method for estimation of Almotriptan Plasma levels in human plasma with different Analyst LLOQ Low QC Mid QC High QC 0.50 ng/ml 1.50 ng/ml ng/ml ng/ml Analytical Run ID P&A Batch 3 Analyst ID 1 Analyst ID 2 Analyst ID 1 Analyst ID 2 Analyst ID 1 Analyst ID 2 Analyst ID 1 Analyst ID N Mean SD (±) CV (%) %NOM Acceptance criteria: 1. % CV 15 % except LLOQ for which it is 20%. 2. Mean % Nominal (100±15% & for LLOQ 100 ± 20%). Chapter 4 Almotriptan - Method Validation

137 109 Ruggedness-Different Column To evaluate ruggedness experiment with different column, samples of P&A batch-5 were reinjected on different columns with same and specifications, Concentrations were calculated to determine precision and accuracy. The Accuracy of Almotriptan QC samples within the range of 97.56% to %. The Precision of Almotriptan QC samples within the range of 0.79% to 3.05%. These results indicated that the method is rugged and reproducible by different analyst. Results are presented in Table 4.16 Table 4.16 Ruggedness of the method for estimation of Almotriptan Plasma levels in human plasma with different Analytical column LLOQ Low QC Mid QC High QC 0.50 ng/ml 1.50 ng/ml ng/ml ng/ml Column Column Column Column Column Column Column Column ID ID ID ID ID ID ID ID LC/121 LC/145 LC/121 LC/145 LC/121 LC/145 LC/121 LC/145 Analytical Run ID N P&A Batch 5 Mean SD(±) CV (%) %NOM Acceptance criteria: 1. % CV 15 % except LLOQ for which it is 20%. 2. Mean % Nominal (100±15% & for LLOQ 100 ± 20%). Chapter 4 Almotriptan - Method Validation

138 110 Bench Top Stability (at room temp for 26 hrs) Spiked LQC and HQC samples were retrieved from deep freezer and were kept at room temperature for 26hrs 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 Almotriptan from comparison samples were 98.56% and % respectively. The plasma samples of Almotriptan were found to be stable for approximately 26 hrs min at room temperature. Results are present in Table 4.17 Table 4.17 Assessment of stability of Analyte (Almotriptan) in Biological matrix at Room temperature Comparison samples (0.00 hr) Conc. % Low QC 1.50 ng/ml Stability samples (26hrs) Comparison samples (0.00 hr) Conc. % High QC ng/ml Stability samples (26 hrs) Conc. % Conc. % found nominal found nominal found nominal found nominal N Mean SD(±) CV (%) %NOM Acceptance criteria: 1. % change should be ± 15 or % Ratio (stability/comparison) should be within %. 2. %CV 15%. 3. Mean % Nominal (100 ±15%). Chapter 4 Almotriptan - Method Validation

139 111 Freeze and Thaw Stability (after 3 rd 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 mean Accuracy for LQC & HQC samples of Almotriptan from comparison samples were 97.00% and % respectively. The plasma samples of Almotriptan were found to be stable after 3 cycles at -30 ±5 C. Results are present in Table 4.18 Table 4.18 Assessment of Freeze-Thaw stability of Analyte (Almotriptan) at -30±5 C Low QC 1.50 ng/ml High QC ng/ml Comparison samples Conc. % found nominal Stability sample at 4 th cycle Conc. % found nominal Comparison samples Conc. % found nominal Stability sample at 4 th cycle Conc. % found nominal N Mean SD(±) CV (%) %Accuracy Acceptance criteria 1. % change should be ± 15 or % Ratio (stability/comparison) should be within %. 2. % CV 15%. 3. Mean % Nominal (100 ±15%) Chapter 4 Almotriptan - Method Validation

140 112 Autosampler stability at 2-8 C in autosampler LQC 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 along with freshly prepared calibration standard samples. Concentrations were calculated to determine mean % change during stability period. The mean Accuracy change for LQC & HQC samples of Almotriptan from comparison samples were 98.11% and % 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 Low QC 1.50 ng/ml High QC ng/ml Comparison samples (0.0 hr) Stability samples (57 hr) Comparison samples (0.0 hr) Stability samples (57hr) Conc. found % nominal Conc. found % nominal Conc. found % nominal Conc. found Acceptance criteria: 1.%change should be ± 15 or % ratio (stability/comparison) should be within %. 2.%CV 15%. 3.Mean % Nominal (100 ±15%). Chapter 4 Almotriptan - Method Validation % nominal N Mean SD(±) CV (%) %NOM

141 113 Long term stability (at -30 C temp for 65 days) Spiked LQC and HQC samples were retrieved from deep freezer after 65 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 mean Accuracy for LQC and HQC samples of Almotriptan from comparison samples were 96.78% and % respectively. The plasma samples of Almotriptan were found to be stable for approximately 65 days at -30 C temp. Results are present in Table 4.20 Table 4.20 Assessment of Long term plasma stability of analyte (Almotriptan) at -30 C. Low QC 1.50 ng/ml High QC ng/ml Comparison samples (0.0 hr) Stability samples (65 days) Comparison samples (0.0 hr) Stability samples (65 days) Conc. found % nominal Conc. found % nominal Conc. found % nominal Conc. found % nominal N Mean SD(±) CV (%) %Accuracy Acceptance criteria: 1. % change should be ± 15 or % Ratio (stability/comparison) should be % 2. % CV 15%. 3. Mean % Nominal (100 ±15%). Chapter 4 Almotriptan - Method Validation

142 114 Short Term Stock Solution Stability of Almotriptan, and Almotriptan D6 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 Almotriptan and Almotriptan-D 6 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 The % CV for Almotriptan- D6 stock solution from comparison samples was 2.66% and % Ratio (stability/comparison) was The % CV for Almotriptan- D 6 working solution (Internal st andard spiking solution) from comparison samples was 1.53% and % Ratio (stability/ comparison) was Almotriptan, Almotriptan- D 6 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 Chapter 4 Almotriptan - Method Validation

143 115 Table 4.21 Assessment of Short term stock solution stability of Analyte (Almotriptan) and Internal standard (Almotriptan- D 6 ) at Room temperature Analyte Comparison Standard stock solution response (0.0 hr) Stability stock solution response (9.5 hr) Comparison stock solution response (0.0 hr) Internal standard Stability Standard stock Response (9.5 hr) N Mean SD (±) CV (%) % Ratio Acceptance criteria: 1. % change should be ± 5 % Table 4.22 Assessment of short term solution stability of internal standard spiking solution (Almotriptan- D 6 ) at refrigerated conditions Comparison solution (Internal standard Spiking solution) Response (0.0 hr) Stability solution (Internal standard spiking solution) Response (9.5 hr) N 6 6 Mean SD (±) CV (%) % Ratio Acceptance criteria: % change should be ± 5% Chapter 4 Almotriptan - Method Validation

144 116 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. Chapter 4 Almotriptan - Method Validation

145 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 K 2 EDTA. 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 IEC 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 Chapter 4 Almotriptan - Application

146 118 Table 4.23 Almotriptan Mean concentration (ng/ml) data for the subject samples obtained from the LC-MS/MS Time in hours Mean Plasma Concentration data Test Reference Figure 4.15 Mean plasma concentration Vs Time curve for Almotriptan Chapter 4 Almotriptan - Application

147 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 (t 1/2 ) and it was considered as area under the concentration time curve (AUC) ratio higher than 80% as per FDA guidelines Plasma Almotriptan concentration-time profiles were visually inspected C max and T max values were determined. The AUC 0 t was obtained by trapezoidal method. AUC 0- was calculated up to the last measureable concentration and extrapolations were obtained using the last measureable concentration and the terminal elimination rate constant (K el ). The terminal elimination rate constant (K el ), 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 halflife, t 1/2, was then calculated as 0.693/K el. Regarding AUC 0 t, AUC 0- and C max 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 AUC 0 t, AUC 0- and C max. Pharmacokinetic data is shown in Table 4.24 and Table 4.25 Chapter 4 Almotriptan - Pharmacokinetic Studies

148 120 Table 4.24 Almotriptan Pharmacokinetic data Almotriptan Pharmacokinetic data Pharmacokinetic Parameter Test Mean Reference Mean C max (ng/ ml) AUC 0-t (ng h/ml) AUC (ng h/ml) T max (h) t 1/ Table 4.25 Almotriptan Pharmacokinetic data (Test/Reference) Pharmacokinetic C max AUC 0-t AUC 0- Parameter Test/Reference Chapter 4 Almotriptan - Pharmacokinetic Studies

149 121 Pharmacokinetic Studies Conclusion The present study provides firm evidence to support that the in house 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 AUC 0-t, AUC 0-, C max, T max, t 1/2. The bioequivalence criteria are based on the 90% confidence intervals whose acceptance range is in between 80% -125%. 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 4 Almotriptan - Pharmacokinetic Studies

150 CHAPTER 5 Analytical method development and validation of Valacyclovir by High performance Liquid chromatography with mass spectrometry

151 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 C 13 H 20 N 6 O 4.HCl and a molecular weight of (Figure 5.1). Figure 5.1 Chemical structures of Valacyclovir (A), Valacyclovir-D 8 (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 P 450 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%. Chapter 5 Valacyclovir - Introduction

152 123 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 and pharmaceutical dosage forms 59-61,A number of methods were developed in animals such as rabbit 55 rat 56 and horse 57 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 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 ng/ml for Valacyclovir, ng/ ml for Acyclovir using the mobile phase ratio of 0.1% formic acid: methanol (30:70) on Gemini C 18 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. Chapter 5 Valacyclovir - Introduction

153 Experimental Investigations Materials and reagents Valacyclovir and Valacyclovir-D 8 TFA salt, Acyclovir were obtained from Synfine Research Canada. Formic acid, Acetic acid, Ammonium hydroxide, NH 4 OH, Ammonium formate were obtained from Merck Mumbai. Methanol, Acetonitrile (HPLC grade) were Obtained from J.T.Baker, Mumbai. Ammonium formate, Ammonia solution (NH 4 OH, 25%, Reagent grade), Formic acid, Glacial acetic acid (CH 3 COOH, 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 Instrumentation and equipment Refer Chapter Preparation of Reagents and Solvents Table 5.1 Preparation of Reagents and Solvents Reagents and Solvents preparation 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 acid Dilute 60 ml of glacial acetic acid to 1000 ml with water (prepare daily). 30% Formic acid Dilute 30 ml of formic acid to 100 ml with water. 2.5% NH 4 OH in methanol 10mM Ammonium formate, P H mM Ammonium formate, P H 3.1 Mix 10 ml of ammonia solution with 90 ml of methanol (prepare daily). Dissolve 1.26 g of ammonium formate into 2 L of water. Adjust P H to 5.0 ± 0.05 with formic acid. Dissolve 1.26 g of ammonium formate into 2 L of water. Adjust P H to 3.1 ± 0.05 with formic acid. 50% Methanol Mix 500 ml of methanol with 500 ml of water. Reconstitution solution 20% Methanol (Auto sampler wash) Acidified plasma Mobile phase Chapter 5 Mix 800 ml of 10mM ammonium formate, P H 3.1 with 200 ml of methanol. Mix 200 ml of methanol with 800 ml of water. Add approximately 5 ml of 30% formic acid to 100 ml of plasma. Mix 10mM Ammonium formate P H 5.0 : Methanol in the ratio of 80:20 and Filter through 0.45 m filter Valacyclovir - Experimental

154 Preparation of Stock solutions Table 5.2 Preparation of Stock solutions Name of the solution Concentration Volume (ml) Diluent Valacyclovir stock solution 100.o µg/ml 100 ml Methanol Valacyclovir- D 8 stock solution µg/ml 100 ml Methanol Acyclovir stock solution µg/ml 10 ml Methanol Preparation of standards and quality control (QC) Samples 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, and 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 (1.5 ng/ml) medium, (210.0 ng/ ml) and high (490.0 ng/ml) concentrations. All the samples were stored in -80 C freezer until analysis. Chapter 5 Valacyclovir - Experimental

155 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-D 8 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- D 8 molecules. A CAD product ion spectrum for Valacyclovir and Valacyclovir- D 8 yielded high-abundance fragment ions of m/z (amu) and m/z (amu) respectively Shown in Figure 5.2 and Figure 5.3. Chapter 5 Valacyclovir Method Development

156 127 Figure 5.2 Parent and Product ion mass spectra of Valacyclovir Chapter 5 Valacyclovir Method Development

157 128 Figure 5.3 Parent and Product ion mass spectra of Valacyclovir-D 8 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- D 8. Separation was tried using various combinations of mobile phase with variety of columns like YMC Pack pro C 18, RP-Amide, Ascentis Express RP-amide, X-Bridge, Discovery Chapter 5 Valacyclovir Method Development

158 129 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 (p H 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 - D 8 from the plasma samples. Chromatographic conditions Chromatographic separation was carried out on a reversed phase Zorbax, SB C 18, 4.6 x 75mm, 3.5 m column using a mixture of 10mM ammonium formate buffer (P H 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-D 8 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- D 8 (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 Chapter 5 Valacyclovir Method Development

159 130 conditioned with 2 ml of dichloromethane, 1.5 ml of 2.5% NH 4 OH 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% NH 4 OH 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 (P H 3.1): methanol 80:20) and vortexed briefly. Then the samples were transferred into auto sampler vials injected into the LC-MS/MS system. Calibration curve parameters and regression model The analytical curves of Valacyclovir were constructed in the concentrations ranging from ng/ml in human plasma. Calibration curves were obtained by weighted linear regression (weighing factor: 1/x 2 ). The ratio of Valacyclovir peak area to Valacyclovir-D 8 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. Method Development Conclusion 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. Chapter 5 Valacyclovir Method Development

160 Method 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 Representative chromatograms of Plasma blank, blank +IS, LOQ, ULOQ, LLOQC, LQC, MQC, HQC, Calibration curve are shown in Figure 5.6 to Figure 5.4 MRM Chromatogram of Blank Human Plasma Sample Chapter 5 Valacyclovir Method Validation

161 132 Figure 5.5 Chromatogram of Blank + IS Chapter 5 Valacyclovir Method Validation

162 133 Figure 5.6 Chromatogram of LOQ Sample (Valacyclovir & IS) Chapter 5 Valacyclovir Method Validation

163 134 Figure 5.7 Chromatogram of ULOQ Sample (Valacyclovir & IS) Chapter 5 Valacyclovir Method Validation

164 135 Figure 5.8 Chromatogram of LLOQ Sample (Valacyclovir & IS) Chapter 5 Valacyclovir Method Validation

165 136 Figure 5.9 Chromatogram of LQC Sample (Valacyclovir & IS) Chapter 5 Valacyclovir Method Validation

166 137 Figure 5.10 Chromatogram of MQC Sample (Valacyclovir & IS) Chapter 5 Valacyclovir Method Validation

167 138 Figure 5.11 Chromatogram of HQC Sample (Valacyclovir & IS) Figure 5.12 Calibration Curve of Valacyclovir Chapter 5 Valacyclovir Method Validation

168 139 Blank Matrix Screening Chapter 5 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 5.3. Table 5.3 Screening of Different batches of blank matrix (Human K 2 EDTA Plasma) for interference free Valacyclovir blank plasma Matrix identification Analyte (Valacyclovir) RT Blank plasma Area Internal standard RT AP/3451/09/ AP/3452/09/ AP/3453/09/ AP/3454/09/ AP/3455/09/ AP/3456/09/ AP/3457/09/ AP/3458/09/ AP/3459/09/ AP/3460/09/ Blank +IS with AP/3451/09/ LOQ with AP/3451/09/ Blank Matrix Specificity and Limit of Quantification 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 Valacyclovir Method Validation

169 140 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 5.5 The specificity experiment shall be considered for calculation of LOQ experiment. Results are presented in Table 5.4 Matrix Identification Table 5.4 Specificity of Different batches of blank matrix (Human K 2 EDTA Plasma) for Valacyclovir LLOQ Area Internal standard (IS) area Interference with Analyte(% of LLOQ Response) Interference with IS(% of IS Response) AP/3451/09/ AP/3452/09/ AP/3454/09/ AP/3456/09/ AP/3458/09/ AP/3459/09/ 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. Chapter 5 Valacyclovir Method Validation

170 141 Table 5.5 Limit of Quantitation for analyte (Valacyclovir) Matrix identification AP/3451/09/10 Blank plasma area at Analyte RT LLOQ response LLOQ S/N RATIO N Mean LLOQ was spiked in -AP/3451/09/10 Acceptance criteria: 1. Mean S/N ratio of LLOQ should be 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 Valacyclovir was ranged between 94.43% and 97.86%. The Intra batch percentage of coefficient of variation is 0.74% to 4.09% for Valacyclovir. Results are presented in Table 5.6 Chapter 5 Valacyclovir Method Validation

171 142 Table 5.6 Intra batch (Within-Batch) Accuracy and Precision for determination of Valacyclovir levels in human plasma Analytical LLOQ 0.50 ng/ml Low QC 1.50 ng/ml Mid QC ng/ml High QC ng/ml Run ID Conc. found % nominal Conc. found % nominal Conc. found % nominal Conc. found % nominal P&A Batch N Mean SD (±) CV (%) %Accuracy Acceptance criteria: 1. % CV 15 % except LLOQ for which it is 20%. 2. Mean % Nominal (100 ±15% and 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 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 ranged between 95.07% and %. The Inter batch percentage of coefficient of variation is 3.29% to 7.80% for Valacyclovir. Results are presented in Table 5.7 Chapter 5 Valacyclovir Method Validation

172 143 Table 5.7 Inter batch (Between-Batch) Accuracy and Precision for determination of Valacyclovir levels in human plasma Analytical LLOQ 0.50 ng/ml Low QC 1.50 ng/ml Mid QC ng/ml High QC ng/ml Run ID Conc. found % nominal Conc. found % nominal Conc. found % nominal Conc. found % nominal P&A Batch P&A Batch 2 P&A Batch 3 P&A Batch N P&A Batch 5 Mean SD(±) % CV %Nominal Acceptance criteria: Same as presented in Table 5.6 Chapter 5 Valacyclovir Method Validation

173 144 Calibration Curve Calibration curves are found to be consistently accurate and precise for Valacyclovir over ng/ml for calibration range. The correlation coefficient is greater than 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 C Coefficient of regression (r 2 ) P&A Batch P&A Batch P&A Batch P&A Batch P&A Batch N Mean SD (±) CV (%) Regression model y = ax 2 +bx+c where: y = peak area ratio (PAR) of valacyclovir to internal standard. x = concentration (ng/ml) of valacyclovir in plasma. Acceptance criteria: 1. Coefficient of regression (r) Chapter 5 Valacyclovir Method Validation

174 145 Table 5.9 Back-calculated standard concentrations from each calibration curve for Valacyclovir in human plasma. Nominal Concentration (ng/ml) Analytical Run CS1 CS2 CS3 CS4 CS5 ID 0.50 ng/ml 1.00 ng/ml 5.00 ng/ml ng/ml ng/ml P&A Batch P&A Batch P&A Batch P&A Batch P&A Batch N Mean SD(±) CV% %Nominal Nominal Concentration (ng/ml) Analytical run CS6 CS7 CS8 CS9 CS10 ID ng/ml ng/ml ng/ml ng/ml ng/ml P&A Batch P&A Batch P&A Batch P&A Batch P&A Batch N Mean SD(±) CV% %Nominal 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%. Chapter 5 Valacyclovir Method Validation

175 146 Recovery The percentage recovery of Valacyclovir was determined by comparing the mean peak area of Valacyclovir in extracted LQC, MQC, HQC samples with freshly prepared unextracted LQC, MQC, HQC samples respectively. The mean % recovery for LQC, MQC, HQC samples of Valacyclovir were 88.48%, % and % respectively. 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%. 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 %. The % CV recovery of IS Valacyclovir- D 8 for extracted is 7.9%. Results are presented in Table 5.10 Chapter 5 Valacyclovir Method Validation

176 147 Table 5.10 Recovery of Analyte Valacyclovir and Valacyclovir-D 8 from human plasma Standard Extracted peak response Unextracted peak response Drug IS Drug IS Low QC: 1.50 ng/ml N Mean SD (±) %CV 10.4 Medium QC: ng/ml N Mean SD(±) %CV 8.6 High QC: ng/ml N Mean SD(±) %CV 4.1 Drug IS Mean recovery of across QC levels Mean SD(±) of across QC levels The Mean % CV across QC levels Acceptance 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%. Chapter 5 Valacyclovir Method Validation

177 148 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. No significant matrix effect found in different sources of human plasma tested for Valacyclovir, Valacyclovir- D 8. Results are presented in Tables 5.11 and Table 5.11 Assessment of Matrix Effect on determination of Valacyclovir at LQC levels in human plasma Identification of matrix Drug response in Matrix at LQC Level Internal standard response Matrix factor AP/3451/09/ AP/3451/09/ AP/3451/09/ AP/3452/09/ AP/3452/09/ AP/3452/09/ AP/3453/09/ AP/3453/09/ AP/3453/09/ AP/3454/09/ AP/3454/09/ AP/3454/09/ AP/3455/09/ AP/3455/09/ AP/3455/09/ AP/3456/09/ AP/3456/09/ AP/3456/09/ N Grand Mean SD(±) % CV 9.36 Acceptance criteria: 1. Mean % Nominal 100±15% of nominal value. 2. % CV 15%. Chapter 5 Valacyclovir Method Validation

178 149 Table 5.12 Assessment of Matrix Effect on determination of Valacyclovir at HQC levels in human plasma Identification of matrix Drug response in Matrix at HQC Level Internal standard response Matrix factor AP/3451/09/ AP/3451/09/ AP/3451/09/ AP/3452/09/ AP/3452/09/ AP/3452/09/ AP/3453/09/ AP/3453/09/ AP/3453/09/ AP/3454/09/ AP/3454/09/ AP/3454/09/ AP/3455/09/ AP/3455/09/ AP/3455/09/ AP/3456/09/ AP/3456/09/ AP/3456/09/ N Grand Mean SD(±) % CV 1.80 Acceptance criteria: 1. Mean % Nominal 100 ±15% of nominal value. 2. % CV 15%. Chapter 5 Valacyclovir Method Validation

179 150 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 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%. Results are presented in Table Table 5.13 Assessment of Dilution integrity for Valacyclovir at DQC Conc (ng/ml) DQC Dilution factor: ½ Nominal conc: ng/ml DQC Dilution factor: ¼ Nominal conc: ng/ml Conc. Found % Nominal Conc. Found %Nominal N 6 6 Mean %Nominal SD (±) CV (%) Acceptance criteria: 1. % CV 15%. 2. Mean % Nominal (100 ±15%). Chapter 5 Valacyclovir Method Validation

180 151 Whole Batch Reinjection Reproducibility To evaluate the whole batch reinjection reproducibility experiment, samples of 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 calculated to determine precision and accuracy after reinjection. The Accuracy of Valacyclovir QC samples in reinjection was between % and %. 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 post extraction (in reconstitution solution) for approx. 26 hrs and reproducible after reinjection. Results are presented in Table 5.14 Table 5.14 Assessment of Whole Batch Re-injection Reproducibility during estimation of Valacyclovir in human plasma Low QC 1.50 ng/ml High QC ng/ml Analytical Reinjection Reinjection Run ID Comp sample Comp sample sample sample N Mean SD(±) %CV %NOM Acceptance criteria: 1. % CV 15% Except LLOQ for which it is 20%. 2. Mean % Nominal (100 ±15% and for LLOQ 100 ±20%) % 0f the re-injected QCs at each level shall be within ± 20% of their previous concentration. Chapter 5 Valacyclovir Method Validation

181 152 Ruggedness-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 Valacyclovir QC samples within the range of 98.43% to %. The Precision of Valacyclovir QC samples within the range of 0.74% to 4.81%. These results indicated that the method is rugged and reproducible by different analyst. Results are presented in Table 5.15 Table 5.15 Ruggedness of the method for estimation of Valacyclovir Plasma levels in human plasma with different Analyst. Analytical Run ID P&A Batch 3 LLOQ 0.50 ng/ml Low QC 1.50 ng/ml Mid QC ng/ml High QC ng/ml Analyst ID 1 Analyst ID 2 Analyst ID 1 Analyst ID 2 Analyst ID 1 Analyst ID 2 Analyst ID 1 Analyst ID N Mean SD (±) CV (%) %Accuracy Acceptance criteria: % CV 15 % except LLOQ for which it is 20%. 2. Mean % Nominal (100±15% & for LLOQ 100 ±20%). Chapter 5 Valacyclovir Method Validation

182 153 Ruggedness-Different Column To evaluate ruggedness experiment with different column, samples of P&A batch-5 were reinjected on different columns with same and specifications, Concentrations were calculated to determine precision and accuracy. The Accuracy of Valacyclovir QC samples within the range of 98.05% to %. The Precision of Valacyclovir QC samples within the range of 0.74% to 6.65%. These results indicated that the method is rugged and reproducible by different analyst. Results are presented in Table 5.16 Table 5.16 Ruggedness of the method for estimation of Valacyclovir Plasma levels in human plasma with different Analytical column LLOQ Low QC Mid QC High QC Analytical Run ID 0.50 ng/ml Column ID LC/312 Column ID LC/ ng/ml Column ID LC/312 Column ID LC/ ng/ml Column ID LC/312 Column ID LC/ ng/ml Column ID LC/312 Column ID LC/ N P&A Batch 5 Mean SD(±) CV (%) %Accuracy Acceptance criteria: 1. % CV 15 % except LLOQ for which it is 20%. 2. Mean % Nominal (100±15% & for LLOQ 100 ±20%). Chapter 5 Valacyclovir Method Validation

183 154 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 comparison samples were 96.55% and 97.48% respectively. The plasma samples of Valacyclovir were found to be stable for approximately 41.0 hrs min at room temperature. Results are present in Table 5.17 Table 5.17 Assessment of stability of Analyte (Valacyclovir) in Biological matrix at Room temperature Comparison samples (0.00 hr) Conc. % Low QC 1.50 ng/ml Stability samples (41.0 hrs) Comparison samples (0.00 hr) Conc. % High QC ng/ml Stability samples (41.0 hrs) Conc. % Conc. % found nominal found nominal found nominal found nominal N Mean SD(±) CV (%) %Accuracy Acceptance criteria: 1. % Ratio (stability/comparison) should be within %. 2. %CV 15%. 3. Mean % Nominal (100 ±15%). Chapter 5 Valacyclovir Method Validation

184 155 Freeze and Thaw Stability (after 3 rd 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 (sta bility 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 Valacyclovir from comparison samples were 95.22% and 98.37% respectively. The plasma samples of Valacyclovir were found to be stable after 3 cycles at - 30 ±5 C. Results are present in Table 5.18 Table 5.18 Assessment of Freeze-Thaw stability of Analyte (Valacyclovir) at -30±5 C Low QC 1.50 ng/ml High QC ng/ml Comparison samples Stability sample at 4 th cycle Comparison samples Stability sample at 4 th cycle Conc. found % nominal Conc. found % nominal Conc. found % nominal Conc. found % nominal N Mean SD(±) CV (%) %Accuracy Acceptance criteria Same as presented in Table 5.17 Chapter 5 Valacyclovir Method Validation

185 156 Autosampler stability at 2-8 C in autosampler LQC and HQC samples were prepared and processed. These processed samples were analyzed and kept in auto sampler for 79 hrs at 2-8 C and analyzed along with freshly prepared calibration standard samples. Concentrations were calculated to determine mean % change during stability period. The mean Accuracy change for LQC & HQC samples of Valacyclovir from comparison samples were 96.89% and 99.09% respectively. Valacyclovir samples were stable for 79 hrs at 2-8 C in autosampler. Results are present in Table 5.19 Table 5.19 Assessment of Autosampler stability of Analyte (Valacyclovir) at 2-8 C Low QC 1.50 ng/ml High QC ng/ml Comparison samples (0.0 hr) Stability samples (79 hr) Comparison samples (0.0 hr) Stability samples (79 hr) Conc. found % nominal Conc. found % nominal Conc. found % nominal Conc. found % nominal N Mean SD(±) CV (%) %Accuracy Acceptance criteria: Same as presented in Table 5.17 Chapter 5 Valacyclovir Method Validation

186 157 Long-term stability (at -30 C temp for 55 days) Spiked LQC and HQC samples were retrieved from deep freezer after 34 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 mean Accuracy for LQC and HQC samples of Valacyclovir from comparison samples were 93.56% and % respectively. The plasma samples of Valacyclovir were found to be stable for approximately 34 days at -30 C temp. Results are present in Table 5.20 Table 5.20 Assessment of Long term plasma stability of Analyte (Valacyclovir) at -30 C. Low QC 1.50 ng/ml High QC ng/ml Comparison samples (0.0 hr) Stability samples (34 days) Comparison samples (0.0 hr) Stability samples (34 days) Conc. found % nominal Conc. found % nominal Conc. found % nominal Conc. found % nominal N Mean SD(±) CV (%) %Accuracy Acceptance criteria: Same as presented in Table 5.17 Chapter 5 Valacyclovir Method Validation

187 158 Short Term Stock Solution Stability of Valacyclovir, and Valacyclovir- D 8 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 Valacyclovir and Valacyclovir-D 8 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- D 8 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 0.86% and % Ratio (stability/comparison) was The % CV for of Valacyclovir- D 8 stock solution from comparison samples was 1.06% and % Ratio (stability/comparison) was The % CV for Valacyclovir- D 8 working solution (Internal standard spiking solution) from comparison samples was 1.86% and % Ratio (stability/ comparison) was Valacyclovir, Valacyclovir- D 8 stock solutions and Valacyclovir- D 8 spiking solutions were found to be stable at room temperature for 9.0 hr. Results are present in Tables 5.21 and 5.22 Chapter 5 Valacyclovir Method Validation

188 159 Table 5.21 Assessment of Short term stock solution stability of Analyte (Valacyclovir) and Internal standard (Valacyclovir- D 8 ) at Room temperature Analyte Internal standard Comparison Standard stock solution response (0.0 hr) Stability stock solution response (9.0 hr) Comparison stock solution response (0.0 hr) Stability Standard stock Response (9.0 hr) N Mean SD (±) CV (%) % Ratio Acceptance criteria: 1. % change should be ± 15 % Table 5.22 Assessment of Short term solution stability of internal standard spiking solution (Valacyclovir- D 8 ) at refrigerated conditions Comparison solution (Internal standard Spiking solution) Response (0.0 hr) Stability solution (Internal standard spiking solution) Response (9.0 hr) N 6 6 Mean SD (±) CV (%) % Ratio Acceptance criteria: 1. % change should be ± 5% Chapter 5 Valacyclovir Method Validation

189 160 Method validation Conclusion As all the values obtained were within the Acceptance criteria. The method 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. Chapter 5 Valacyclovir Method Validation

190 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 Commi ttee) 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, twogroup 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 K 2 EDTA 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 Chapter 5 Valacyclovir - Application

191 162 Table Valacyclovir Mean concentration (ng/ml) data for the subject samples obtained from the LC-MS/MS Time in hours Mean Plasma Concentration data Test Reference Figure 5.13 Mean plasma Concentration Vs Time curve for Valacyclovir Chapter 5 Valacyclovir - Application

192 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 (t 1/2 ) and it was considered as area under the concentration time curve (AUC) ratio higher than 80% as per FDA guidelines Plasma Valacyclovir concentration-time profiles were visually inspected C max and T max values were determined. The AUC 0 t was obtained by trapezoidal method. AUC 0- was calculated up to the last measureable concentration and extrapolations were obtained using the last measureable concentration and the terminal elimination rate constant (K el ). The terminal elimination rate constant (K el ), 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, t 1/2, was then calculated as 0.693/K el. Regarding AUC 0 t, AUC 0- and C max 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 AUC 0 t, AUC 0- and C max. Pharmacokinetic data is shown in Table 5.24 and Table Chapter 5 Valacyclovir Pharmacokinetic Studies

193 164 Table 5.24 Valacyclovir Pharmacokinetic data Valacyclovir Pharmacokinetic data Pharmacokinetic Test Reference Parameter Mean±SD Mean±SD C max (ng/ml ) ± ± AUC 0-t (ng h/ml) ± ± AUC 0-inf (ng h/ml) ± ± T max (hr) t 1/ Table 5.25 Valacyclovir Pharmacokinetic data (Test/Reference) Pharmacokinetic Parameter Test/Reference C max AUC 0-t AUC Pharmacokinetic Studies Conclusion The present study provides firm evidence to support that the in house 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 and concentrations were found through Liquid Chromatography Tandem Mass Spectroscopy detection. The Pharmacokinetic parameters assessed were AUC 0-t, AUC 0-, C max, T max, t 1/2. The bioequivalence criteria are based on the 90% confidence intervals 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 5 Valacyclovir Pharmacokinetic Studies

194 CHAPTER 6 Analytical method development and validation of Memantine by High performance Liquid chromatography with mass spectrometry

195 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 receptors 62 Fig.6.1.Chemical structures of Memantine and Memantine-D 6 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 (C max ) ranging from 22 to 46 ng/ml following a single dose of 20 mg. The time to achieve maximum plasma concentration (T max ) following single doses of 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. Chapter 6 Memantine- Introduction

196 166 There were few methods established previously to determine Memantine in a variety of matrices. These methods include LC-MS HPLC GC MS 70 and Micellar electrokinetic chromatography 71 Among all methods LC-MS has gained more importance. M.Y. Liuet.al 62 developed the method with the Linear concentration range of 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.al 65 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.. Chapter 6 Memantine- Introduction

197 Experimental Investigations Materials and reagents Memantine hydrochloride was obtained from Varda biotech Pvt.Ltd. Andheri, Mumbai, India Memantine -D 6 hydrochloride obtained from Toronto Research Chemicals, Toronto, Canada. Human plasma (K 2 EDTA), 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 Instrumentation and equipment Refer Chapter Reagents and Solvents preparation Preparation of Reagents and Solvents Table 6.1 Preparation of Reagents and Solvents 0.1% Formic acid Dilute 1mL of formic acid to 1L with water. 10mM NaOH Extraction Solvent Dissolve 0.4 g of sodium hydroxide in 1000 ml of water. Mix 700 ml of diethyl ether with 300 ml of n-hexane. 50% Methanol Mix 500 ml of methanol with 500 ml of water. Reconstitution solution Autosampler wash Mobile phase Mix 350 ml of 0.1% formic acid with 650 ml of acetonitrile. Mix 800 ml of methanol with 200 ml of water. 0.1% Formic acid: Acetonitrile in the ratio of 35:65 and Filter through 0.45 m filter Chapter 6 Memantine - Experimental

198 Preparation of Stock solutions Table 6.2 Preparation of Stock solutions Name of the solutions Memantine stock solution Memantine -D 6 stock solution Concentration Volume (ml) Diluent g/ml 50 ml Methanol g/ml 50 ml Methanol Preparation of standards and quality control (QC) Samples Standard stock solutions of Memantine (100.00µg/mL) and Memantine -D 6 (100.00µg/mL) were prepared in methanol. The spiking solution for Memantine -D 6 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, , , , , , , , and pg/ml for Analytical standards and 50.0, 150.0, , 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. Chapter 6 Memantine - Experimental

199 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 -D 6 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-D 6. Chapter 6 Memantine Method Development

200 170 Figure 6.2 Parent ion mass spectra (Q 1 ) of Memantine Chapter 6 Memantine Method Development

201 171 Figure 6.3 Product ion mass spectra (Q 3 ) of Memantine Chapter 6 Memantine Method Development

202 172 Figure 6.4 Parent ion mass spectra (Q 1 ) Memantine-D 6 Chapter 6 Memantine Method Development

203 173 Figure 6.5 Product ion mass spectra (Q 3 ) of Memantine-D 6 Chapter 6 Memantine Method Development

204 174 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- D 6. 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 p H 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-C 18 (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 -D 6 was found to be appropriate internal standard. Retention time of Memantine and Memantine- D 6 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 Chapter 6 Memantine Method Development

205 175 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-D 6 from the plasma. Memantine-D 6 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-D 6 from human plasma. For this purpose,50 µl of Memantine-D 6 (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. Chapter 6 Memantine Method Development

206 176 Calibration curve parameters and regression model The analytical curves were constructed using values ranging from 50.0 to pg/ml of Memantine in human plasma. Calibration curves were obtained by weighted 1/Conc 2 quadratic regression analysis y = ax 2 +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- D 6 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%. Method Development Conclusion 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. Chapter 6 Memantine Method Development

207 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 Representative chromatograms of Plasma blank, blank +IS, LOQ, ULOQ, LLOQC, LQC, MQC, HQC, Calibration curve are shown in Figure 6.6 to Figure 6.6 MRM Chromatogram of Blank Human Plasma Sample Chapter 6 Memantine Method Validation

208 178 Figure 6.7 Chromatogram of Blank + IS Chapter 6 Memantine Method Validation

209 179 Figure 6.8 Chromatogram of LOQ Sample (Memantine & IS) Chapter 6 Memantine Method Validation

210 180 Figure 6.9 Chromatogram of ULOQ Sample (Memantine & IS) Chapter 6 Memantine Method Validation

211 181 Figure 6.10 Chromatogram of LLOQ Sample (Memantine & IS) Chapter 6 Memantine Method Validation

212 182 Figure 6.11 Chromatogram of LQC Sample (Memantine & IS) Chapter 6 Memantine Method Validation

213 183 Figure 6.12 Chromatogram of MQC Sample (Memantine & IS) Chapter 6 Memantine Method Validation

214 184 Figure 6.13 Chromatogram of HQC Sample (Memantine & IS) Chapter 6 Memantine Method Validation

215 185 Figure 6.14 Calibration Curve of Memantine Chapter 6 Memantine Method Validation

216 186 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 6.3. Table 6.3 Screening of Different batches of blank matrix (Human K 2 EDTA Plasma) for interference free Memantine blank plasma Matrix identification Blank plasma Area Analyte (Memantine) RT AP/ A 469 AP/ A 0 AP/ A 0 AP/ A 0 AP/ A 0 AP/ A 121 AP/ A 22 AP/ A 46 AP/ A 0 AP/ A 57 Blank+IS with AP/ A 0 Internal standard RT LOQ with AP/ A Chapter 6 Memantine Method Validation

217 187 Blank Matrix Specificity and Limit of Quantification 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 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 6.4. The specificity experiment shall be considered for calculation of LOQ experiment. Results are presented in Table 6.5 Chapter 6 Memantine Method Validation

218 188 Table 6.4 Specificity of Different batches of blank matrix (Human K 2 EDTA Plasma) for Memantine Matrix Identification Blank Area LLOQ Response) AP/ A AP/ A AP/ A AP/ A AP/ A AP/ A AP/ A AP/ A AP/ A AP/ A Internal standard (IS) area Interference with Analyte (% of LLOQ Response) Interference with IS(% of IS Response) 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. Chapter 6 Memantine Method Validation

219 189 Table 6.5 Limit of Quantitation for analyte (Memantine) Matrix identification Blank plasma area at LLOQ LLOQ S/N Analyte RT response RATIO AP/ A N Mean LLOQ was spiked in AP/ A Acceptance criteria: 1. Mean S/N ratio of LLOQ should be 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 Memantine was ranged between 87.8% and 99.8%. The Intra batch percentage of coefficient of variation is 2.1% to 3.7% for Memantine. Results are presented in Table 6.6. Chapter 6 Memantine Method Validation

220 190 Table 6.6 Intra batch (Within-Batch) Accuracy and Precision for determination Memantine levels in human plasma Analytical LLOQ pg/ml Low QC pg/ml Mid QC pg/ml High QC pg/ml Run ID Conc. found % nominal Conc. found % nominal Conc. found % nominal Conc. found % nominal P&A Batch N Mean SD (±) % CV %Accuracy Acceptance criteria: 1. % CV 15 % except LLOQ for which it is 20%. 2. Mean % Nominal (100 ±15% and 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 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 ranged between 95.70% and 99.10%. The Inter batch percentage of coefficient of variation is 1.40% to 7.80% for Memantine. Results are presented in Table 6.7. Chapter 6 Memantine Method Validation

221 191 Table 6.7 Inter batch (Between-Batch) Accuracy and Precision for determination Memantine levels in human plasma Analytical LLOQ pg/ml Low QC pg/ml Mid QC pg/ml High QC pg/ml Run ID Conc. found % nominal Conc. found % nominal Conc. found % nominal Conc. found % nominal P&A Batch P&A Batch 2 P&A Batch 3 P&A Batch N P&A Batch 5 Mean SD(±) % CV %Nominal Acceptance criteria: Same as presented in Table 6.6 Chapter 6 Memantine Method Validation

222 192 Calibration Curve Calibration curves are found to be consistently accurate and precise for Memantine over pg/ml for calibration range. The correlation coefficient is greater than for Memantine. Back calculations were made from the calibration curves to determine Memantine concentrations of each calibration standard. Results are presented in Tables 6.8 & 6.9. Table 6.8 Summary of calibration curve parameters for Memantine in human plasma Coefficient Analytical of A B C Run ID regression (r 2 ) P&A Batch P&A Batch P&A Batch P&A Batch P&A Batch N Mean SD (±) CV (%) Acceptance criteria: Regression Footnote(s): Resp. = A * (Conc. 2 ) + B * Conc. + C 1. Coefficient of regression (r) Chapter 6 Memantine Method Validation

223 193 Table 6.9 Back-calculated standard concentrations from each calibration curve Analytical Run ID for Memantine in human plasma. Nominal Concentration (ng/ml) CS1 CS2 CS3 CS4 CS pg/ml pg/ml pg/ml pg/ml pg/ml P&A Batch P&A Batch P&A Batch P&A Batch P&A Batch N Mean SD(±) % CV %Nominal Nominal Concentration (ng/ml) Analytical run CS6 CS7 CS8 CS9 CS10 ID pg/ml pg/ml pg/ml pg/ml pg/ml P&A Batch P&A Batch P&A Batch P&A Batch P&A Batch N Mean SD(±) CV% %Nominal 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%. Chapter 6 Memantine Method Validation

224 194 Recovery The percentage recovery of Memantine was determined by comparing the mean peak area of Memantine in extracted LQC, MQC, HQC samples with freshly prepared unextracted LQC, MQC, HQC samples respectively. The mean % recovery for LQC, MQC, HQC samples of Memantine were 79.45%, 91.25% and 87.52% respectively. 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%. 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 80.31%. The %CV recovery of IS Memantine -D 6 for extracted is 19.60%. Results are presented in Table Chapter 6 Memantine Method Validation

225 195 Table 6.10 Recovery of Analyte (Memantine) and Memantine D 6 from human plasma Standard Extracted peak response Unextracted peak response Drug IS Drug IS Low QC: pg/ml N % Recovery SD (±) % CV Medium QC: pg/ml N % Recovery SD(±) % CV High QC: pg/ml N % Recovery SD(±) % CV 14.4 Drug IS Mean recovery of across QC levels Mean SD(±) of across QC levels The Mean % CV across QC levels Acceptance 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%. Chapter 6 Memantine Method Validation

226 196 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. No significant matrix effect found in different sources of human plasma tested for Memantine, Memantine-D 6. Results are presented in Tables 6.11 and Table 6.11 Assessment of Matrix Effect on determination of Memantine at LQC levels in human plasma Identification of matrix Drug response in Matrix at LQC Level Internal standard response Matrix factor AP/ A AP/ A AP/ A AP/ A AP/ A AP/ A AP/ A AP/ A AP/ A AP/ A AP/ A AP/ A AP/ A AP/ A AP/ A AP/ A AP/ A AP/ A N Grand Mean SD(±) CV (%) 3.16 Acceptance criteria: 1. Mean % Nominal 100 ± 15% of nominal value. 2. %CV 15%. Chapter 6 Memantine Method Validation

227 197 Table 6.12 Assessment of Matrix Effect on determination of Memantine at HQC levels in human plasma Identification of matrix Drug response in Matrix at HQC Level Internal standard response Matrix factor AP/ A AP/ A AP/ A AP/ A AP/ A AP/ A AP/ A AP/ A AP/ A AP/ A AP/ A AP/ A AP/ A AP/ A AP/ A AP/ A AP/ A AP/ A N Grand Mean SD(±) % 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 matrix Peak response ratio (analyte/is) in the absence of extracted matrix Chapter 6 Memantine Method Validation

228 198 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 Memantine nominal concentrations ranged between % and % for 1 in 2 dilutions and 1 in 4 dilutions respectively. The % CV is 0.66% to 0.63%. Results are presented in Table Table 6.13 Assessment of Dilution integrity for Memantine at DQC Conc (Pg/mL) DQC Dilution factor: ½ Nominal conc: pg/ml DQC Dilution factor: ¼ Nominal conc: Pg/mL Conc. Found % Nominal Conc. Found %Nominal N 6 6 Mean %Nominal SD (±) CV (%) Acceptance criteria: 1. % CV 15%. 2. Mean % Nominal (100 ±15%). Chapter 6 Memantine Method Validation

229 199 Whole Batch Reinjection Reproducibility To evaluate the whole batch reinjection reproducibility experiment, samples of 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 calculated to determine precision and accuracy after reinjection. 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. Results are presented in Table Table 6.14 Assessment of Whole Batch Re-injection Reproducibility during estimation of Memantine in human plasma Low QC pg/ml High QC pg/ml Analytical Reinjection Reinjection Run ID Comp sample Comp sample sample sample N Mean SD(±) %CV %NOM Acceptance criteria: 1. % CV 15% Except LLOQ for which it is 20%. 2. Mean % Nominal (100±15% and for LLOQ 100 ± 20%) % 0f the re-injected QCs at each level shall be within ±20% of their previous concentration. Chapter 6 Memantine Method Validation

230 200 Ruggedness-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 Memantine QC samples within the range of 95.60% to %. The Precision of Memantine QC samples within the range of 0.61% to 3.58%. These results indicated that the method is rugged and reproducible by different analyst. Results are presented in Table 6.15 Table 6.15 Ruggedness of the method for estimation of Memantine Plasma levels in human plasma with different analyst Analytical LLOQ pg/ml Low QC pg/ml Mid QC pg/ml High QC pg/ml Run ID Analyst ID 1 Analyst ID 2 Analyst ID 1 Analyst ID 2 Analyst ID 1 Analyst ID 2 Analyst ID 1 Analyst ID P&A Batch N Mean SD (±) CV (%) %NOM Acceptance criteria: 1. % CV 15 % except LLOQ for which it is 20%. 2. Mean % Nominal (100±15% & for LLOQ 100 ± 20%). Chapter 6 Memantine Method Validation

231 201 Ruggedness-Different Column To evaluate ruggedness experiment with different column, samples of P&A batch-5 were reinjected on different columns with same and specifications, Concentrations were calculated to determine precision and accuracy. 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%. These results indicated that the method is rugged and reproducible by different analyst. Results are presented in Table 6.16 Table 6.16 Ruggedness of the method for estimation of Memantine Plasma levels in human plasma with different Analytical column LLOQ Low QC Mid QC High QC Analytical Run ID 5.00 ng/ml Column ID LC/221 Column ID LC/ ng/ml Column ID LC/221 Column ID LC/ ng/ml Column ID LC/221 Column ID LC/ ng/ml Column ID LC/221 Column ID LC/ N P&A Batch 5 Mean SD(±) CV (%) %NOM Acceptance criteria: 1. % CV 15 % except LLOQ for which it is 20%. 2. Mean % Nominal (100 ± 15% & for LLOQ 100 ± 20%). Chapter 6 Memantine Method Validation

232 202 Bench Top Stability (at room temp for 26.0 hrs) Spiked LQC and HQC samples were retrieved from deep freezer and were kept at room temperature for 26 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 Memantine from comparison samples were 92.67% and 98.00% respectively. The plasma samples of Memantine were found to be stable for approximately Chapter hrs min at room temperature. Results are present in Table Table 6.17 Assessment of stability of Analyte (Memantine) in Biological matrix at Room temperature Low QC ng/ml High QC ng/ml Comparison Comparison Stability samples Stability samples samples samples (26.0 hrs) (26.0 hrs) (0.00 hr) (0.00 hr) Conc. found % nominal Conc. found % nominal Conc. found % nominal Conc. found % nominal N Mean SD(±) CV (%) %NOM Acceptance criteria: 1. % change should be ± 15 or % Ratio (stability/comparison) should be within %. 2. % CV 15%. 3. Mean % Nominal (100 ± 15%). Memantine Method Validation

233 203 Freeze and Thaw Stability (after 3 rd 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 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 Table 6.18 Assessment of Freeze-Thaw stability of Analyte (Memantine) at -30±5 C Low QC pg/ml High QC pg/ml Comparison samples Stability sample at 4 th cycle Comparison samples Stability sample at 4 th cycle Conc. found % nominal Conc. found % nominal Conc. found % nominal Conc. found % nominal N Mean SD(±) CV (%) %Accuracy Acceptance criteria 1. % change should be ± 15 or % Ratio (stability/comparison) should be within %. 2. %CV 15%. 3. Mean % Nominal (100±15%) Chapter 6 Memantine Method Validation

234 204 Autosampler stability at 2-8 C in autosampler LQC and HQC samples were prepared and processed. These processed samples were analyzed and kept in auto sampler for 79 hrs at 2-8 C and analyzed along with freshly prepared calibration standard samples. Concentrations were calculated to determine mean % change during stability period. The mean Accuracy change for LQC & HQC samples of Memantine from comparison samples were 92.67% and 98.72% respectively. Memantine samples were stable for 79 hrs at 2-8 C in autosampler. Results are present in Table 6.19 Table 6.19 Assessment of Autosampler stability of Analyte (Memantine) at 2-8 C Low QC pg/ml High QC pg/ml Comparison samples (0.0 hr) Stability samples (79 hr) Comparison samples (0.0 hr) Stability samples (79 hr) Conc. found % nominal Conc. found % nominal Conc. found % nominal Conc. found % nominal N Mean SD(±) CV (%) %NOM Acceptance criteria: 1. % change should be ± 15 or % ratio (stability/comparison) should be within %. 2. %CV 15%. 3. Mean % Nominal (100 ±15%). Chapter 6 Memantine Method Validation

235 205 Longterm stability (at -30 C temp for 76 days) Spiked LQC and HQC samples were retrieved from deep freezer after 76days 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 Memantine from comparison samples were 92.67% and 97.67% respectively. The plasma samples of Memantine were found to be stable for approximately 76 days at -30 C temp. Results are present in Table 6.20 Table 6.20 Assessment of Long term plasma stability of Analyte (Memantine) at -30 C. Low QC pg/ml High QC pg/ml Comparison samples (0.0 hr) Stability samples (76 days) Comparison samples (0.0 hr) Stability samples (76 days) Conc. found % nominal Conc. found % nominal Conc. found % nominal Conc. found % nominal N Mean SD(±) CV (%) %Accuracy Acceptance criteria: 1. % change should be ± 15 or % Ratio (stability/comparison) should be within %. 2. %CV 15%. 3. Mean % Nominal (100±15%). Chapter 6 Memantine Method Validation

236 206 Short Term Stock Solution Stability of Memantine and Memantine D6 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 Memantine and Memantine-D 6 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 D 6 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 2.13% and % Ratio (stability/comparison) was The % CV for of Memantine- D 6 stock solution from comparison samples was 1.00% and % Ratio (stability/comparison) was The % CV for Memantine- D 6 working solution (Internal standard spiki ng solution) from comparison samples was 1.30% and % Ratio (stability/ comparison) was Memantine, Memantine- D 6 stock solutions and Memantine D 6 working solutions were found to be stable at room temperature for 9.5 hr. Results are present in Table 6.21 and Chapter 6 Memantine Method Validation

237 207 Table 6.21 Assessment of Short term stock solution stability of Analyte (Memantine) and Internal standard (Memantine -D 6 ) at Room temperature Analyte Internal standard Comparison Standard stock solution response (0.0 hr) Stability stock solution response (9.0 hr) Comparison stock solution response (0.0 hr) Stability Standard stock Response (9.0 hr) N Mean SD (±) CV (%) % Ratio Acceptance criteria: 1. % change should be ± 5 % Table 6.22 Assessment of short term solution stability of internal standard Spiking solution (Memantine -D 6 ) at refrigerated conditons Comparison solution (Internal standard Spiking solution) Response (0.0 hr) Stability solution (Internal standard spiking solution) Response (9.0 hr) N 6 6 Mean SD (±) CV (%) % Ratio Acceptance criteria: 1. % change should be ± 5% Chapter 6 Memantine Method Validation

238 208 Method validation Conclusion As all the values obtained were within the Acceptance criteria. The method 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. Chapter 6 Memantine Method Validation

239 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 K 2 EDTA. 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 Chapter 6 Memantine - Application

240 210 Table 6.23 Memantine Mean concentration (pg/ml) data for the subject samples obtained from the LC-MS/MS Time in hours Mean Concentration data Period 1 Period Chapter 6 Memantine - Application

241 211 Figure 6.15 Mean plasma concentration Vs time curve for Memantine Chapter 6 Memantine - Application

242 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 (t 1/2 ) and it was considered as area under the concentration time curve (AUC) ratio higher than 80% as per FDA guideline Plasma Memantine concentration-time profiles were visually inspected C max and T max values were determined. The AUC 0 t was obtained by trapezoidal method. AUC 0- was calculated up to the last measureable concentration and extrapolations were obtained using the last measureable concentration and the terminal elimination rate constant (K el ). The terminal elimination rate constant (K el ), 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 halflife, t 1/2, was then calculated as 0.693/K el. Regarding AUC 0 t, AUC 0- and C max 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 AUC 0 t, AUC 0- and C max. Pharmacokinetic data is shown in Table 6.24 and Table Chapter 6 Memantine Pharmacokinetic Studies

243 213 Pharmacokinetic Parameter C max (Pg/ml) Table 6.24 Memantine Pharmacokinetic data Memantine Pharmacokinetic data Test Reference Mean±SD Mean±SD ± ± AUC 0-t ± ± (Pg hr/ml) AUC ± ± (Pg hr/ml) T max (h) t 1/ Table 6.25 Memantine Pharmacokinetic data (Test/Reference) Pharmacokinetic Parameter C max AUC 0-t AUC 0- Test/Reference Pharmacokinetic Conclusion The present study provides firm evidence to support that the in house 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 AUC 0-t, AUC 0-, C max, T max, t 1/2. The bioequivalence criteria are based on the 90% confidence intervals whose acceptance range is in between 80% -125%. 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 6 Memantine Pharmacokinetic Studies

244 CHAPTER 7 Summary & Conclusion

245 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 Chapter 7 Summary and Conclusion

246 215 Assay Precision and Accuracy, Recovery, Stability parameters like Short time stability, Long time stability, Auto sampler stability, Bench Top Stability and Freezethaw 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 pg/ml using a simple liquid-liquid extraction technique for drug and internal standard within 3minutes of analysis time in biological fluids. Rasagiline- 13 C 3 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 ng/ml using a Liquid-Liquid Extraction technique. Deuterated compound Almotriptan- D 6 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-D 8, in plasma samples over a Chapter 7 Summary and Conclusion

247 216 concentration range of 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 pg/ml for by a simple liquid-liquid extraction technique for drug and internal standard. Deuterated compound Memantine- D 6 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 C max, T max, AUC 0-t and AUC 0- were within the acceptance criteria for selected drugs. The overall pharmacokinetic parameters were within the range of % 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. Chapter 7 Summary and Conclusion

248 217 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 7 Summary and Conclusion

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261 Appendix

262 LIST OF PUBLICATIONS - RELAVENT TO RESEARCH WORK Publication type Full Length Article Full Length Article Full Length Article Full Length Article Title 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 Konda, Babu Rao Chandu, B.R.Challa, Chandrasekhar.K.B, Method Development and Validation of Almotriptan in Human Plasma by HPLC Tandem Mass spectrometry: Application to Pharmacokinetic Study Ravi kumar Konda, Babu.Rao Chandu, B.R.Challa, Chandrasekhar.K.B, Development and Validation of a sensitive LC- MS/MS method for determination of Valacyclovir in Human Plasma: Application to a Bioequivalence Study. Ravi kumar Konda, Babu Rao Chandu, B.R.Challa, Chandrasekhar.K.B, Bioanalytical Method Development and Validation of Memantine in Human Plasma by High Performance Liquid chromatography with Tandem Mass spectrometry: Application to Bioequivalence Study Ravi kumar Konda, B.R.Challa, Babu Rao Chandu, Chandrasekhar.K.B, Status Journal of Pharmaceutical Analysis Elsevier publication. - Published /j.jpha Scientia Pharmaceutica Peer reviewed- Austria Journal. - Published /sciphar m Acta Chromato Graphica International Peer reviewed journal- - Accepted Journal of Analytical Methods in Chemistry Hindawi publishing corporation. -Published doi: / 2012/ Appendix Research Publications

263 Journal of Pharmaceutical Analysis ]]]];](]):]]] ]]] Contents lists available at ScienceDirect Journal of Pharmaceutical Analysis 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 Konda a,d, Babu Rao Chandu b, B.R. Challa c,n, Chandrasekhar B. Kothapalli d a Hindu College of Pharmacy, Amaravathi Road, Guntur, Andhra Pradesh , India b Don Bosco College of Pharmacy, Pulladigunta, Guntur, Andhra Pradesh , India c Nirmala College of Pharmacy, Madras Road, Kadapa, Andhra Pradesh , India d Jawaharlal Nehru Technological University, Anantapur, Andhra Pradesh , 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 been developed and validated for quantification of Rasagiline in human plasma. Rasagiline- 13 C 3 mesylate was used as an internal standard for Rasagiline. Zorbax Eclipse Plus C18 (2.1 mm 50 mm, 3.5 mm) column provided chromatographic separation of analyte followed by detection with mass spectrometry. The method involved simple isocratic chromatographic condition and mass spectrometric detection in the positive ionization mode using an API-4000 system. The total run time was 3.0 min. The proposed method has been validated with the linear range of pg/ml for 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- 13 C 3 mesylate analog was 96.9% and 96.7% respectively. This validated method was successfully applied to the bioequivalence and pharmacokinetic study of human volunteers under fasting condition. & 2012 Xi an Jiaotong University. Production and hosting by Elsevier B.V. All rights reserved. n Corresponding author. Tel.: þ address: baluchalla_99@yahoo.com (B.R. Challa) & 2012 Xi an Jiaotong University. Production and hosting by Elsevier B.V. All rights reserved. Peer review under responsibility of Xi an Jiaotong University 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 C 12 H 13 N with its molecular weight (Fig. 1). Rasagiline is rapidly absorbed, reaching peak plasma concentration (C max ) in approximately 1 h. The absolute bioavailability of Rasagiline is about 36%. Food does not affect the t max of Rasagiline, although C max and exposure (AUC) decreased by 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),

264 2 R.K. Konda et al triple quadrupole instrument (ABI-SCIEX, Toronto, Canada) was used. Data processing was performed with Analyst software package (SCIEX) Detection Figure 1 Chemical structures of Rasagiline (A) and Rasagiline- 13 C 3 mesylate (B). 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 ng/ml. Rasagiline is almost metabolized in the liver and undergoes urinary excretion. Half-life of Rasagiline is about 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 ng/ml in human plasma and 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 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 smallamount of plasma sample(100 ml plasma) utilization during sample processing, simple extraction and analyte comparison with isotope labeled internal standard (Rasagiline- 13 C 3 mesylate). 2. Experimental Rasagiline and Rasagiline- 13 C 3 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 Na 2 CO 3 were procured from Merck (Mumbai, India). Human plasma (K 2 EDTA) was obtained from Doctors Pathological Lab, Hyderabad. The AZILECT s tablets, containing 1 mg Rasagiline per tablet, were obtained from Teva Pharma (USA) Instrumentation An HPLC system (1200 series model, Agilent Technologies, Waldbronn, Germany) connected with mass spectrometer API 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 parameters 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- 13 C 3 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- 13 C 3 mesylate. The mass transitions were selected as m/z for Rasagiline and m/z for Rasagiline- 13 C 3 mesylate. The data acquisition was ascertained by Analyst software 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- 13 C 3 mesylate were eluted at min, with a total run time of 3.0 min for each sample 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- 13 C 3 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- 13 C 3 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, , , , , and pg/ml and quality control samples were made at concentrations of 5.0, 15.0, and pg/ml for Rasagiline Sample preparation For sample preparation, 100 ml of plasma sample or Rasagiline spiked standard or quality control plasma sample was added to 5 ml ria vial tubes. 50 ml of internal standard and 200 ml of 1M Na 2 CO 3 solution were added and vortexed 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),

265 Bio-analytical method development and validation of Rasagiline 3 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 approximately 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 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 Matrix effect Matrix effect for Rasagiline and internal standard was evaluated by comparing the peak area ratio in the postextracted 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% 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% Recovery The extraction efficiencies of Rasagiline and Rasagiline- 13 C 3 mesylate were determined by analysis of six replicates at each quality control concentration level for Rasagiline and at one concentration for Rasagiline- 13 C 3 mesylate. The percentage recovery was evaluated by comparing the peak areas of extracted standards to the peak areas of nonextracted standards (spiked into mobile phase) 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 concentrations obtained after 78 days were compared with initial concentrations 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 AZILECT s 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 K 2 EDTA 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 pharmacokinetic parameters were computed using Win-Nonlin s software version 5.2 and 90% confidence interval was computed using SAS s software version Results and discussion 3.1. Method development During method development, different options were evaluated to optimize mass spectrometry detection parameters, chromatography and sample extraction 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 optimized 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 predominant peaks in the primary ESI spectra of Rasagiline and Rasagiline- 13 C 3 mesylate correspond to the MHþ ions at 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),

266 4 R.K. Konda et al. m/z and respectively (Fig. 2A and C). Product ions of Rasagiline and Rasagiline- 13 C 3 mesylate scanned in quadrupole 3 after a collision with nitrogen in quadrupole 2 had an m/z of and respectively (Fig. 2B and D) 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- 13 C 3 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- 13 C 3 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, 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- 13 C 3 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 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 extraction(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- 13 C 3 mesylate Parent ion, and (D) Rasagiline- 13 C 3 mesylate Product ion. 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),

267 Bio-analytical method development and validation of Rasagiline 5 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 compounds were investigated to find a suitable internal standard, and finally Rasagiline- 13 C 3 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 procedure resulted in reduced analysis time with accurate and precise detection of Rasagiline in human plasma 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 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- 13 C 3 mesylate. The mean % interference observed at the retention time of analytes between six different lots of human plasma, including hemolyzed and lipedemic plasma containing K 2 EDTA as an anti-coagulant was 0.00% and 0.00% for Rasagiline and Rasagiline- 13 C 3 mesylate respectively, 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 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 Linearity The peak area ratios of calibration standards were proportional to the concentration of Rasagiline in each assay over the nominal concentration range of pg/ml. The calibration curves appeared linear and were well described by leastsquares linear regression lines. As compared to the 1/x Figure 3 Blank plasma chromatograms of Rasagiline and Rasagiline- 13 C 3 mesylate in human plasma. 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),

268 6 R.K. Konda et al. Figure 4 LLOQ chromatograms of Rasagiline and Rasagiline- 13 C 3 mesylate in human plasma. Table 1 Concentration (pg/ml) Calibration curve details. Mean (pg/ml) weighing factor, a weighing factor of 1/x2 achieved the best result and was chosen to achieve homogeneity of variance. The correlation coefficient was Z 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 concentrations were less than 15%. This validated linearity range justifies the concentration observed during real sample analysis 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. SD CV (%) Accuracy SD: Standard deviation Recovery Six aqueous replicates (samples spiked in reconstitution solution) at low, medium and high quality control concentration levels for Rasagiline were prepared for recovery determination, 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- 13 C 3 mesylate was 96.7% with a precision of 2.1%. This indicates that the extraction efficiency for Rasagiline as well as Rasagiline- 13 C 3 mesylate was consistent and reproducible 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 Stabilities Stock solution stability was performed to check stability of Rasagiline and Rasagiline- 13 C 3 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- 13 C 3 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 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),

269 Bio-analytical method development and validation of Rasagiline 7 Table 2 Within-run and between-run precision and accuracy. Nominal added concentration (pg/ml) Within-run (n¼6) Mean (pg/ml) SD Precision (CV, %) Accuracy Between-run (n¼36) Mean (pg/ml) SD Precision (CV, %) Accuracy 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) Autosampler stability (55 h) Long-term stability (78 days) Freeze thaw stability (cycle 3, 48 h) 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 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 containing 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 (independent ethics committee) as per DCGI (Drug Control General of India) guidelines. The pharmacokinetic parameters evaluated were C max (maximum observed drug concentration during the study), AUC (area under the plasma concentration time curve measured 6.5 h, using the trapezoidal rule), t max (time to observe maximum drug concentration), K el (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 t 1/2 (terminal half-life as determined by the quotient 0.693/K el, Table 4). The Test/Reference ratios for C max, AUC 0 6.5, and AUC 0 N were 80.22, and 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 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 human plasma from 22 subjects that are receiving 1 1mg oral dose of Rasagiline tablet as test and reference is shown in Figure Conclusion Rasagiline Test Reference AUC 0 t (pg h/ml) C max (pg/ml) AUC 0 N (pg h/ml) K el t 1/ t max (h) AUC 0 N : area under the curve extrapolated to infinity; AUC 0 t : area under the curve up to the last sampling time; C max :the maximum plasma concentration; t max : the time to reach peak concentration; K el : the apparent elimination rate constant. t 1/2 : 0.693/K el 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. 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),

270 8 R.K. Konda et al. Figure 5 Mean plasma concentrations of test versus reference after 1 mg dose (1 1 mg tablet) in 22 healthy volunteers. 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 antiparkinsonian monoamine oxidase-b inhibitor with neuroprotective activity, Prog. Neurobiol. 92 (3) (2010) [2] J. Leegwater-Kim, E. Bortan, The role of rasagiline in the treatment of Parkinson s disease, Clin. Interv. Aging 5 (2010) [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) [4] S. Won Hyuk, K.S. Suslick, S. Yoo-Hum, Therapeutic agents for Alzheimer s disease, Curr. Med. Chem. Cent. Nerv. Syst. Agents 5 (2005) [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) [6] D. Haberle, K. Magyar, E. Szoko, Determination of the norepinephrine 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) [7] C. Binda, F. Hubalek, M. Li, et al., Binding of rasagiline-related inhibitors to human monoamine oxidases: a kinetic and crystallographic analysis, J. Med. Chem. 48 (26) (2005) [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) [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) [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 [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 [12] Guidance for Industry: Bioavailability and Fed Bioequivalence Studies for Orally Administered Drug Products General Considerations, U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER), March 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),

271 Sci Pharm 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, , India. 2 Jawaharlal Nehru Technological University, Anantapur, , India. 3 Donbosco college of Pharmacy, Pulladigunta, Guntur, , India. 4 Nirmala college of Pharmacy, Madras road, Kadapa, Andhrapradesh, , India. * Corresponding author. baluchalla_99@yahoo.com (B. R. Challa) Sci Pharm. 2012; 80: doi: /scipharm Published: February 27 th 2012 Received: December 1 st 2011 Accepted: February 27 th 2012 This article is available from: 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 ( 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-d 6 (ALD6) as an internal standard. Almotriptan and Almotriptan-d 6 were detected with proton adducts at m/z and in multiple reaction monitoring (MRM) positive mode, respectively. The method was linear over a concentration range of 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 betweenrun accuracy of the method throughout its linear range was to % and to %, respectively. The mean recovery of drug and internal standard from human plasma was ± 4.32% and ± 6.32%. It can be applied for clinical and pharmacokinetic studies. Keywords HPLC MS/MS Almotriptan Human plasma Pharmacokinetic study LLE

272 368 K. Ravikumar et al.: 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 halflife 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]. O S N O N H N HO O HO O OH O S N O D 3 C N N H CD 3 HO O HO O OH Almotriptan malate Almotriptan-d 6 malate Fig. 1. Chemical structure of Almotriptan malate and Almotriptan-d 6 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-d 6 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 software package (SCIEX). Sci Pharm. 2012; 80:

273 Method Development and Validation of Almotriptan in Human Plasma by HPLC Tandam Mass 369 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: 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: 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-d 6 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, and 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-d 6 (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 ng/ml, (0.5, 1.0, 5.0, 15.0, 30.0, 45.0, 60.0, 90.0, and ng/ml), four non-zero quality control standards (0.5, 1.5, Sci Pharm. 2012; 80:

274 370 K. Ravikumar et al.: 75.0 and 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/x 2 linear regression model (y = mx + c). The ratio of Almotriptan peak area to Almotriptan-d 6 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 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 betweenrun coefficients of variations of the measured concentrations at each level (CV%). Recovery The extraction recovery of Almotriptan and Almotriptan-d 6 from human plasma was determined by analyzing quality control samples. Recovery at three concentrations (1.5, 75.0 and 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, twoperiod, 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 K 2 EDTA as an anticoagulant. The plasma from these samples was separated by centrifugation at Sci Pharm. 2012; 80:

275 Method Development and Validation of Almotriptan in Human Plasma by HPLC Tandam Mass 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-d 6 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-d 6 yielded highabundance fragment ions at m/z and 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:

276 372 K. Ravikumar et al.: Fig. 3. Mass-spectra of Almotriptan-d 6 (Q1), Almotriptan-d 6 (Q3) Method Validation Linearity and Range The method produced highly linear responses within the wide concentration range of 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 (%) Sci Pharm. 2012; 80:

277 Method Development and Validation of Almotriptan in Human Plasma by HPLC Tandam Mass 373 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-d 6 (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. Sci Pharm. 2012; 80:

278 374 K. Ravikumar et al.: 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 ± ± ± ± Spiked plasma Between-run concentration Concentration measured (ng/ml) (n=30) (ng/ml) (mean ± SD) % CV % Accuracy ± ± ± ± 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-d 6 were found to be ± 4.32 % and ± 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 Concentration measured (n=6) (ng/ml) (mean ±SD) Processed sample stability 26.0 h 57 h % CV (n=6) Concentration measured (n=6) (ng/ml) (mean ± SD) % CV (n=6) ± ± ± ± Spiked plasma Long term stability Freeze and thaw stability concentration (ng/ml) 65 days Cycle 3 (48 h) Concentration measured (n=6) (ng/ml) (mean ± SD) % CV (n=6) Concentration measured (n=6) (ng/ml) (mean ± SD) % CV (n=6) ± ± ± ± Sci Pharm. 2012; 80:

279 Method Development and Validation of Almotriptan in Human Plasma by HPLC Tandam Mass 375 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), AUC 0 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 AUC 0 t (ng h/ml) Cmax (ng/ml) AUC 0 (ng h/ml) Kel Tmax (h) AUC 0 : area under the curve extrapolated to infinity; AUC 0 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 %, (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. Sci Pharm. 2012; 80:

280 376 K. Ravikumar et al.: 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 AUC 0-t AUC 0 Test/Ref 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 ng/ml. Almotriptan-d 6 (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. Sci Pharm. 2012; 80:

281 Method Development and Validation of Almotriptan in Human Plasma by HPLC Tandam Mass 377 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. References [1] Jansat JM, Costa J, Salvà P, Fernandez FJ, Martinez-Tobed A. Absolute Bioavailability, Pharmacokinetics and Urinary Excretion of the Novel Antimigraine Agent Almotriptan in Healthy Male Volunteers. J Clin Pharmacol. 2002; 42: [2] Villalón CM, Centurión D, Valdivia LF, de Vries P, Saxena PR. Migraine: Pathophysiology, Pharmacology, Treatment and Future Trends. Curr Vasc Pharmacol. 2003; 1: [3] Jhee SS, Shiovitz T, Crawford AW, Cutler NR.. Pharmacokinetics and Pharmacodynamics of the Triptan Antimigraine Agents: A Comparative Review. Clin Pharmacokinet. 2001; 40: [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: [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: [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: [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: [8] Suneetha A, Syamasundar B. Fluorimetric and colorimetric methods for the determination of some antimigraine drugs. Indian J Pharm Sci. 2010; 72: [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: [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 [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 Sci Pharm. 2012; 80:

282 378 K. Ravikumar et al.: [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). Sci Pharm. 2012; 80:

283 On Sun, 4/15/12, wrote: From: Subject: Final decision To: Date: Sunday, April 15, 2012, 1:51 PM Dear R.K.Konda, Your revised re-submission was repeatedly evaluated for its quality and now I am very pleased to let you know that it finally conforms with scientific and formal demands of our journal. I remain in full agreement with the Reviewers recommendations and therefore my decision is to accept your manuscript for publication in Acta Chromatographica. Your accepted manuscript is scheduled for the Acta Chromatographica issue no. 4 / However, beginning from January, 2012, the electronic ONLINE PREVIEW of the manuscripts accepted to our journal in the DOI system starts ( so that your accepted paper will appear in the Internet much earlier and owing to the DOI ID, it will immediately obtain the status of a published paper. Kind regards, Danica Agbaba Editor, Acta Chromatographica

284 DEVELOPMENT AND VALIDATION OF A SENSITIVE LC- MS/MS METHOD FOR DETERMINATION OF VALACYCLOVIR IN HUMAN PLASMA: APPLICATION TO A BIOEQUIVALENCE STUDY Ravi Kumar.Konda 1,4*, Babu.Rao.Chandu 2, B.R.Challa 3,Chandrasekhar.K.B 4, 1 Department of pharmaceutical chemistry,hindu college of Pharmacy, Amaravathi Road, Guntur,Andhrapradesh, India Department of pharmaceutical Sciences, Donbosco college of Pharmacy, pulladigunta, Guntur, India Department of pharmaceutical Analysis, Nirmala college of Pharmacy, Madras road, Kadapa, Andhrapradesh, India Department of chemistry, Jawaharlal Nehru Technological University, Anantapur, India *Correspondence to: Ravi Kumar.Konda, Department of pharmaceutical chemistry Hindu college of Pharmacy, Amaravathi Road, Guntur,Andhrapradesh, India baluchalla_99@yahoo.com & victory2ravi@yahoo.co.in #Tel#

285 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 and in multiple reaction monitoring (MRM) positive mode respectively. The method was linear over the concentration range of 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 withinrun 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 ± % and ± 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

286 Hindawi Publishing Corporation Journal of Analytical Methods in Chemistry Volume 2012, Article ID , 8 pages doi: /2012/ Research Article Bioanalytical Method Development and Validation of Memantine in Human Plasma by High Performance Liquid Chromatography with Tandem Mass Spectrometry: Application to Bioequivalence Study Ravi Kumar Konda, 1, 2 B. R. Challa, 3 Babu Rao Chandu, 4 and Kothapalli B. Chandrasekhar 2 1 Department of Pharmaceutical Chemistry, Hindu College of Pharmacy, Amaravathi Road, Guntur, Andhrapradesh , India 2 Department of Chemistry, Jawaharlal Nehru Technological University, Anantapur , India 3 Department of Pharmaceutical Analysis, Nirmala College of Pharmacy, Madras Road, Kadapa, Andhrapradesh , India 4 Department of Pharmaceutical Sciences, Donbosco College of Pharmacy, Pulladigunta, Guntur , India Correspondence should be addressed to Ravi Kumar Konda, drchalla2121@yahoo.com 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 Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. A simple, sensitive, and rapid HPLC-MS/MS method was developed and validated for quantitative estimation of memantine in human plasma. Chromatography was performed on Zorbax SB-C 18 ( mm, 3.5 µm) column. Memantine (ME) and internal standard Memantine-d6(MED6) were extracted by using liquid-liquid extraction and analyzed by LC-ESI-MS/MS using multiplereaction monitoring (MRM) mode. The assay exhibited a linear dynamic range of pg/ml for ME in human plasma. This method demonstrated an intra- and interday precision within the range of and %, respectively. Further intraand interday accuracy was within the range of and % correspondingly. The mean recovery of ME and MED6 was ± 6.87 and ± 5.70%, respectively. The described method was successfully employed in bioequivalence study of ME in Indian male healthy human volunteers under fasting conditions. 1. Introduction Memantine (1-amino-3,5-dimethyladamantane hydrochloride) (Figure 1) acting on the glutamatergic system by blocking N-methyl-D-aspartate (NMDA) glutamate receptors [1]. Memantine (ME) is used in Parkinson s disease and 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. ME is well absorbed, with peak plasma concentrations (C max ) ranging from 22 to 46 ng/ml following a single dose of 20 mg. The time to achieve maximum plasma concentration (T max ) following single doses of 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 [1]. There were few methods established previously to determine 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].

287 2 Journal of Analytical Methods in Chemistry NH 2 HCl D D NH 2 HCl H 3 C D CH 3 D D D (a) (b) Figure 1: Chemical structures of (a) Memantine hydrochloride and (b) Memantine-D 6 HCL. Among all methods LC-MS [1 4] has gained more importance. Liu et al. [1] developed the method with the linear concentration range of ng/ml, with 0.2 ng/ml sensitivity. This sensitivity was improved by Almeida et al. [2]. They developed the method with the linear concentration range of 0.1 to 50 ng/ml, with 0.1 ng/ml sensitivity. Pan et al. [3] 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. Koeberle et al. [4] developed the method indifferent melanins. The reported methods do 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. The purpose of this investigation was to develop a rapid, simple, sensitive, and selective LC-MS/MS method for the quantitative estimation of ME 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, and/or bioequivalence studies of ME. 2. Materials and Methods 2.1. Chemicals. ME (99.9%) was obtained from Varda biotech Pvt. Ltd. Andheri, Mumbai, India. MED6 (99.0%) was obtained from the Toronto Research Chemicals, Toronto, Canada. Blank plasma lots were purchased from Navjeevan blood bank, Hyderabad. HPLC-grade methanol and acetonitrile were purchased from Jt. Baker, USA. Diethyl ether and n-hexane were purchased from Lab Scan, Asia Co. Ltd, Bangkok, Thailand. Formic acid and sodium hydroxide were purchased from Merck Mumbai, India. HPLC-grade water from Milli-Q System was used. All other chemicals used were analytical grade Instrumentation and Chromatographic Conditions. HPLC system (1200 series, Agilent Technologies, Germany) is connected with API 4000 triple quadrupole mass spectrometer (ABI-SCIEX, Toronto, Canada) using multiple reaction monitoring (MRM). A turbo electrospray interface in positive ionization mode was used. Data processing was performed on Analyst software package (SCIEX). The chromatography was performed on a Zorbax SB-C 18 ( mm, 3.5 µm) (Agilent technologies,germany) at 40 C temperature. The mobile phase composition was a mixture of 0.1% formic acid : acetonitrile (35 : 65 v/v) which was pumped at a flow-rate of 0.6 ml/min without split Preparation of Calibration Standards and Quality Control Samples. Standard stock solutions of ME ( µg/ml) and MED6 ( µg/ml) were separately prepared in methanol. MED6 dilution (25.00 ng/ml) was made from MED6 standard stock solution with diluent (methanol: water 50 : 50 v/v). Standard stock solution of ME was added to drug-free human plasma to obtain ME calibration standards of 50.00, , , , , , , , , and pg/ml. Quality control (QC) samples were also prepared as a bulk on an independent weighing of standard drug at concentrations of (LLOQ), (LQC), (MQC), and pg/ml (HQC) from standard stock solutions of ME. The calibration standards and quality control samples were divided into aliquots in 5 ml Ria vials and stored in the freezer at below 30 C until analysis Sample Preparation. 50 µl of MED6 (25 ng/ml), 100 µl of plasma sample, and 100 µl of10mmnaohwereadded into 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 conditions. Then, the supernatant from each sample was transferred into labelled vials by using the dry-ice acetone flash freeze technique and evaporated to dryness under nitrogenstreamat40 C.Thedriedresiduewasreconstituted with 400 µl of 0.1% of formic acid: acetonitrile (35 : 65 v/v) mixture and vortexed until dissolved. Finally, a 20 µlofeach sample was transferred into auto sampler vials and injected into HPLC connected with mass spectrometer Recovery. Recovery of ME was evaluated by comparing the mean peak area of six extracted low, medium, and high (150.00, , and pg/ml) quality control samples to the mean peak area of six aqueous standards

288 Journal of Analytical Methods in Chemistry 3 with the same concentrations of low, medium, and high ME quality control samples. Similarly the recovery of MED6 was evaluated by comparing the mean peak area of extracted quality control samples to the mean peak area of MED6 in aqueous standards samples with the same concentrations of MED Selectivity. The selectivity of the method was determined by blank human plasma samples from six different healthy human volunteers to test the potential interferences of endogenous compounds coeluted with ME and MED6. The Chromatographic peaks of ME and MED6 were identified on the basis of their retention times and MRM responses. The mean peak area of LOQ for ME and MED6 at corresponding retention time in blank samples should not be more than 20 and 5%, respectively Limit of Quantification (LOQ). The LOQ was estimated in accordance with the baseline noise method at a signal-tonoise ratio (S/N) of 5. It was experimentally determined by injecting six samples with ME at the LLOQ concentration. The acceptance criterion for S/N was 5 and calculated by selecting the noise region as close as possible to the signal peak, which was at least 8 times of the signal peak width at half height Analytical Curves. The analytical curves of ME were constructed in the concentrations ranging from to pg/ml in human plasma. The calibration curve was constructed by using instrument response (ratio of ME peak area to MED6 peak area) against the ME concentration (pg/ml) for four consecutive days by weighted 1/x 2 quadratic regression model. The fitness of calibration curve was confirmed by back-calculating the concentrations of calibration standards Calibration Curve Standards, Regression Model, Precision, and Accuracy Batches. Calibration curve standard samples and QC samples were prepared in replicates (n = 6) for analysis. Correlation coefficients (r 2 ) were obtained by using quadratic regression model in whole range of tested concentrations. The accuracy and precision for the back calculated concentrations of the calibration points should be within ±15% whereas those of LLOQ should be within ±20% of their nominal values Stability. Low and high QC samples (n = 6) were retrieved from the deep freezer; samples were processed for three freeze/thaw cycles according to the clinical protocols. The samples were stored at 10 Cto 30 C in three cycles of 24, 36, and 48 hr. In addition, the long-term stability of ME in QC samples was also evaluated after 76 days of storage at 10 to 30 C. The stability at refrigerated temperature was studied following 79 hr storage period in the autosampler tray. Bench top stability was studied for 26-hour period. Stability samples were processed and extracted along with the freshly spiked calibration curve standards. Stability of the stock solutions was proved for 24 days. The precision and accuracy for the stability samples were maintained within 15 and ±15%, respectively, of their nominal concentrations Matrix Effect. The matrix effect due to plasma matrix was used to evaluate ion suppression/enhancement in a signal by comparing the absolute response of QC samples after pretreatment (liquid-liquid extraction) with that of reconstituted samples extracted blank plasma sample spiked with analyte. Experiments were performed at low and high concentration levels in triplicate. The acceptable precision (%CV) should be 15% Analysis of Human Plasma Samples. The bioanalytical method described previously was applied to determine ME concentrations in plasma following oral administration to healthy adult human male volunteers below 25 years of age. The volunteers were contracted by Micro Therapeutics Research Labs Pvt Ltd., Chennai, India. They were screened before participation in the study and an informed consent was taken from them. These volunteers, were not undergone any other medication before conducting this study. To each of the 20 volunteers a tablet containing 10 mg of ME was orally administered along with a 240 ml of drinking water. Proper diet was provided to each volunteer as per the protocol. The reference product (Namenda tablets 10 mg, Forest laboratories, Ireland) and test product (Memantine tablets 10 mg) were used in the study. The study protocol was approved by IEC (Institutional Ethical Committee) and by ICMR (Indian Council of Medical Research). Blood samples were collected as predose (0 hr) 5 minutes 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 hr. After dosing, a 5 ml blood sample was collected each preestablished time in vacutainers containing K 2 EDTA. A total of 34 samples (17 time points each for reference and test) were collected and centrifuged at 3200 rpm and10 C for 10 min. Then they were stored at 30 C until further analysis. Test and reference were administered to the same human volunteers under fasting conditions separately after a washing period of 18 days as per protocol approved by IEC Pharmacokinetics and Statistical Analysis. Pharmacokinetics parameters were calculated from plasma levels applying a noncompartmental statistics model using WinNon-Lin 5.0 software (Pharsight, USA). Following Food and Drug Administration (F.D.A) guideline [11, 12], blood samples were drawn up to a period of three to five times the terminal elimination half-life (t 1/2 ) and it was considered as the area under the concentration time curve (AUC) ratio higher than 80%. The C max and T max values were determined by visual inspection of the plasma ME concentration-time profiles. The area under the concentration-time curve (AUC 0 t )was obtained by the trapezoidal method. The total area under the curve (AUC 0 ) was calculated up to the last measureable concentration, and extrapolations were obtained by the last measureable concentration and the terminal elimination rate constant (K e ). The K e was estimated from the slope of the terminal exponential phase of the plasma of ME

289 4 Journal of Analytical Methods in Chemistry Intensity (cps) 2.4e6 2.3e6 2.2e6 2.1e6 2e6 1.9e6 1.8e6 1.7e6 1.6e6 1.5e6 1.4e6 1.3e6 1.2e6 1.1e6 1e6 9e5 8e5 7e5 6e5 5e5 4e5 3e5 2e m/z (amu) Intensity (cps) 5.7e4 5.6e4 5.5e4 5.4e4 5.3e4 5.2e4 5.1e4 5e4 4.9e4 4.8e4 4.7e4 4.6e4 4.5e4 4.4e4 4.3e4 4.2e4 4.1e4 4e4 3.9e4 3.8e4 3.7e4 3.6e4 3.5e4 3.4e4 3.3e4 3.2e4 3.1e m/z (amu) (a) (b) Intensity (cps) 1.25e6 1.2e6 1.15e6 1.1e6 1.05e6 1e6 9.5e5 9e5 8.5e5 8e5 7.5e5 7e5 6.5e5 6e5 5.5e5 5e5 4.5e5 4e5 3.5e5 3e5 2.5e5 2e5 1.5e m/z (amu) Intensity (cps) 2.6e5 2.5e5 2.4e5 2.3e5 2.2e5 2.1e5 2e5 1.9e5 1.8e5 1.7e5 1.6e5 1.5e5 1.4e5 1.3e5 1.2e5 1.1e5 1e5 9e4 8e4 7e4 6e4 5e4 4e4 3e4 2e4 1e m/z (amu) (c) (d) Figure 2: (a)massspectra of Memantine parent ion (Q1). (b)massspectra of Memantine product ion (Q3). (c)massspectra ofmemantine- D 6 parent ion. (d) Mass spectra of Memantine-D 6 product ion (Q3). concentration-time curve using linear regression method. The t 1/2 was then calculated as 0.693/K e. The AUC 0 t, AUC 0,andC max bioequivalence were assessed by analysis of variance (ANOVA), and the standard 90% confidence intervals (90% CIs) of the ratios test/reference were calculated after transforming the data logarithmically. The bioequivalence was considered when the ratio of averages of log transformed data was within % for AUC 0 t, AUC 0,andC max [11, 12]. 3. Results and Discussion 3.1. Method Development and Validation. 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 ME and MED6 which were shown in Figures 2(a), 2(b), 2(c), and 2(d). ESI-LC-MS/MS is a very powerful technique for pharmacokinetic studies since it provides sensitivity

290 Journal of Analytical Methods in Chemistry 5 and selectivity requirements for analytical methods. 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 was 400 milliseconds for both ME and MED6. The product ion (Q3) mass spectra of ME and the MED6 are shown in Figures 2(b) and 2(d). [M+H] + was the predominant ion in the Q1 spectrum. The Q1 for ME and MED6 was and 186.1, respectively, and were used as the precursor ion to obtain product ion spectra. The collisionally associated dissociation (CAD) mass spectrum of ME shows formation of characteristic product ions at m/z 161.8, 163.2, and The major product ion at m/z for ME could be explained by the splitting of 1-amino-3-,5-dimethyladamantane hydrochloride from the protonated precursor molecule. The CAD mass spectrum of MED6 shows formation of characteristic product ions at m/z The major product ion at m/z arose from 3,5- Dimethyl-d6-tricyclo-[3,3,1,13,7]decan-1-amine,3,5-Dimethyl-d6-1-adamantanamine from the protonated precursor molecule. The most sensitive mass transitions were from m/z to for ME and m/z to m/z for the MED6. The proposed fragmentation pattern is Figure 2(a) Figure2(b), Figure2(c) Figure 2(d). The inherent selectivity of MS-MS detection was also expected to be beneficial in developing a selective and sensitive method. The chromatographic conditions particularly the composition of mobile phase, flow-rate of mobile phase, choosing of suitable column, injection volume, column oven temperature, autosampler 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 ME and MED6. 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 ME 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. The high proportion of organic solvent eluted both the ME and the MED6 at retention time 1.45 ± 0.2min at a flow rate of 0.6 ml/min, produced good peak shapes, and permitted a run time of 3.5 min. Liquid-liquid extraction (LLE) was used for the sample preparation in this work. LLE can be helpful to clean the samples. Clean samples are essential for minimizing ion suppression and matrix effectin LC-MS/MS analyses. Several organic solvents and their mixtures in different combinations and ratios were evaluated. Finally, diethyl ether/n-hexane (70 : 30) was found to be optimal, which produced a clean chromatogram for a blank plasma sample and yielded the Spiked plasma concentration (pg/ml) Table 1: Concentration data form validation. Concentration measured (pg/ml) Mean ± Sd n = 5 Precision (% CV) Accuracy % ± ± ± ± ± ± ± ± ± ± highest recovery for the ME and MED6 from the plasma. Memantine-D 6 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 Selectivity. The selectivity of the method was examined by analyzing blank human plasma extracts (n = 6). The result of one blank (Figure 3(a)) plasma is shown and the lack of interference is similar to other samples which were studied which shows no significant direct interference in the blank plasma traces as observed from endogenous substances in drug-free human plasma at the retention time of the analyte Limit of Quantification (LOQ). The LOQ signal-to-noise (S/N) value found for 6 injections of ME at LOQ concentration was Figure 3(b) shows a representative ionchromatogram for the LOQ (50 pg/ml) with 20 µl injection volume Linearity, Precision, and Accuracy. The ten-point calibration curve was linear over the concentration range pg/ml. The calibration model was selected based on the analysis of the data by quadratic regression with intercepts and weighting factor 1/x 2. The best quadratic regression for the calibration curve was achieved with a 1/x 2 weighing factor, giving a mean quadratic regression equation for the calibration curve of y = x x (y = ax 2 + bx + c) where y is the peak-area ratio of the ME to the MED6 and x is the concentration of the ME in plasma (Table 1). For the between-batch experiments, the precision and accuracy ranged from 1.4 to 2.7% and 95.7 to 99.1%, respectively (Table 2). Further, in within-batch experiments the precision and accuracy ranged from 2.1 to 2.3% and 95.6 to 99.8% correspondingly.

291 6 Journal of Analytical Methods in Chemistry Intensity (cps) Time (min) (a) Intensity (cps) e5 1.3e5 1.2e5 1.1e5 1e5 9e4 8e4 7e4 6e4 5e4 4e4 3e4 2e4 1e Time (min) Time (min) Memantine Memantine D 6 Intensity (cps) 3 (b) 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 plasma concentration (pg/ml) Concentration measured (n = 6) (pg/ml) (mean ± Sd.) Within-run (n = 6) Between-run (n = 30) Precision (%CV) Accuracy % Concentration measured (n = 30) (pg/ml) (mean ± Sd.) Precision (%CV) Accuracy % ± ± ± ± ± ±