DEVELOPMENT OF BIOANALYTICAL METHODS FOR QUANTITATIVE MEASUREMENT OF ANTICANCER AGENTS SANDEEP REDDY KUNATI Bachelor of Pharmacy Kakatiya University December 2004 Master of Science in Chemistry Youngstown State University August 2008 submitted in partial fulfillment of requirements for the degree DOCTOR OF PHILOSOPHY IN CLINICAL AND BIOANALYTICAL CHEMISTRY at the CLEVELAND STATE UNIVERSITY MAY 2018 © COPYRIGHT BY SANDEEP REDDY KUNATI 2018 This dissertation has been approved for the Department of Chemistry and the College of Graduate Studies by ________________________________________________ Dissertation Committee Chairperson, Dr. Yan Xu ________________________________ Department & Date ________________________________________________ Dr. John F. Turner ________________________________ Department & Date ________________________________________________ Dr. Aimin Zhou ________________________________ Department & Date ________________________________________________ Dr. Bin Su ________________________________ Department & Date ________________________________________________ Dr. Siu-Tung Yau ________________________________ Department & Date ACKNOWLEDGMENTS I am grateful to God for his blessings throughout my life. I am indebted to my advisor and mentor, Dr. Yan Xu, for his support of my career goals, persistent help in every possible manner and immense guidance throughout my studies at CSU. This dissertation would not have been possible without him. I would like to thank my committee members Dr. John Turner, Dr. Aimin Zhou, Dr. Bin Su, and Dr. Siu-Tung Yau for their guidance and suggestions throughout my graduate research work. I would like to especially thank Dr. Shuming Yang, for his training, support and guidance throughout my research work. I would like to thank my fellow graduate students for their support throughout my graduate studies. I am grateful to my family for their love, support, and encouragement. DEVELOPMENT OF BIOANALYTICAL METHODS FOR QUANTITATIVE MEASUREMENT OF ANTICANCER AGENTS SANDEEP REDDY KUNATI ABSTRACT The ever-growing need to develop new anticancer agents due to increased incidence of cancer and its recurrence has led to a broad range of investigational efforts in anticancer research. Drug discovery and development processes involve evaluations of pharmacokinetic and pharmacodynamic parameters of potential drug candidates in order to assess their safety, toxicity, and efficacy profiles. By providing sensitivity, assay precision, reliability, and high-throughput, mass spectrometry has become an indispensable tool in quantitative and qualitative analysis, from early-stage drug discovery and development processes to routine laboratory analysis. Development of bioanalytical methods requires knowledge of separation science with instrumentation, data interpretation and sample preparation techniques from complex biological matrices. This work describes the theory, instrumentation, and method development processes behind these investigations. Further, detailed method development and validation procedures for investigational anticancer agents MLN0128, GS87, and curcumin using liquid chromatography-tandem mass spectrometry were presented. v TABLE OF CONTENTS Page ABSTRACT……………………………………………………………………………….v TABLE OF CONTENTS…………………………………………………………………vi LIST OF TABLES……………………………………………………………………….xii LIST OF FIGURES.……………………………………………………………………..xv CHAPTER I. INTRODUCTION TO BIOANALYTICAL METHOD DEVELOPMENT 1.1 General Introduction…………………………………………………………..1 1.2 High-Performance Liquid Chromatography (HPLC)…………………………3 1.2.1 Mobile phase………………………………………………………6 1.2.2 Column…………………………………………………………...10 1.3 Mass spectrometer…………………………………………………………...13 1.3.1 Ion source………………………………………………………...15 1.3.2 Mass analyzer.…………………………………………………....18 1.3.3 Ion detector.……………………………………………………...21 1.3.4 Tandem mass spectrometry (MS/MS)…………………………...21 1.4 Sample preparation…………………………………………………………..24 1.4.1 Protein precipitation (PPT)………………………………………24 1.4.2 Liquid-liquid extraction (LLE)…………………………………..27 vi 1.4.3 Solid-phase extraction (SPE)…………………………………….29 1.5 Method development………………………………………………………...31 1.6 Method validation……………………………………………………………31 1.7 Conclusion…………………………………………………………………...34 1.8 References……………………………………………………………………35 CHAPTER II. DETERMINATION OF MLN0128, AN INVESTIGATIONAL ANTINEOPLASTIC AGENT, IN HUMAN PLASMA BY LC-MS/MS 2.1 Introduction......................................................................................................41 2.2 Experimental....................................................................................................42 2.2.1 Chemicals.......................................................................................42 2.2.2 Instrumentation..............................................................................43 2.2.2.1 Liquid chromatography......................................................45 2.2.2.2 Tandem mass spectrometry................................................45 2.2.3 Preparation of stock and working solutions...................................46 2.2.4 Preparation of standard solutions...................................................46 2.2.5 Preparation of plasma calibrators and quality controls..................47 2.2.6 Plasma sample preparation............................................................47 2.2.7 Method validation..........................................................................48 2.2.7.1 Selectivity and lower limit of quantitation (LLOQ)..........48 2.2.7.2 Matrix factor (MF) and recovery.......................................49 2.2.7.3 Linear calibration curve.....................................................49 vii 2.2.7.4 Accuracy, precision, and dilution study.............................50 2.2.7.5 Stability..............................................................................50 2.3 Results and discussion.....................................................................................51 2.3.1 Method development.....................................................................51 2.3.1.1 Internal standard (IS).........................................................51 2.3.1.2 Chemical properties of the analyte and the IS...................52 2.3.1.3 Mass spectrometric detection............................................52 2.3.1.4 Liquid chromatographic separation...................................56 2.3.1.5 Plasma sample preparation................................................57 2.3.1.6 Reconstitution solution......................................................59 2.3.2 Method validation..........................................................................59 2.3.2.1 Selectivity and lower limit of quantitation.........................59 2.3.2.2 Matrix effect and recovery.................................................63 2.3.2.3 Calibration curve................................................................66 2.3.2.4 Assay accuracy, and precision, and dilution integrity.......69 2.3.2.5 Stability..............................................................................71 2.3.3 Method application........................................................................73 2.4 Conclusions......................................................................................................74 2.5 References........................................................................................................75 viii CHAPTER III. DEVELOPMENT AND VALIDATION OF AN LC-MS/MS METHOD FOR QUANTITATIVE DETERMINATION OF GS87, A NOVEL ANTINEOPLASTIC AGENT, IN MOUSE PLASMA 3.1 Introduction......................................................................................................79 3.2 Experimental....................................................................................................81 3.2.1 Chemicals and solutions................................................................81 3.2.2 Preparation of plasma calibrators and quality controls..................83 3.2.3 Plasma sample preparation............................................................84 3.2.4 Instrumentation..............................................................................84 3.2.5 Animal study..................................................................................86 3.3 Results and discussion.....................................................................................87 3.3.1 Method development.....................................................................87 3.3.1.1 Chemical properties of the analyte and the IS...................87 3.3.1.2 Mass spectrometric detection.............................................87 3.3.1.3 Liquid chromatography......................................................89 3.3.1.4 Plasma sample preparation and plasma volume................90 3.3.1.5 Influence of non-matrix solvent on plasma calibrators and QCs....................................................................................90 3.3.1.6 Matrix interferences...........................................................91 3.3.2 Method validation..........................................................................94 3.3.2.1 Selectivity and lower limit of quantitation.........................94 3.3.2.2 Matrix effect and Recovery...............................................97 3.3.2.3 Precision, accuracy, and dilution integrity studies...........100 ix 3.3.2.4 Calibration curve..............................................................102 3.3.2.5 Stability............................................................................105