Quantitative and Qualitative Mass Spectrometric
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QUANTITATIVE AND QUALITATIVE MASS SPECTROMETRIC ANALYSIS OF ANTICANCER AGENTS, DRUGS OF ABUSE AND ENZYME-INHIBITOR COMPLEXES KERRI M. SMITH Bachelor of Science in Chemistry Cleveland State University June 2006 submitted in partial fulfillment of requirements for the degree DOCTOR OF PHILOSOPHY IN CLINICAL AND BIOANALYTICAL CHEMISTRY at the CLEVELAND STATE UNIVERSITY NOVEMBER 2011 ©COPYRIGHT BY KERRI M. SMITH 2011 This dissertation has been approved for the Department of CHEMISTRY and the College of Graduate Studies by _____________________________________________ Dissertation Chairperson, Dr. Yan Xu ________________________ Department and Date __________________________________________________ Dr. Baochuan Guo ________________________ Department and Date __________________________________________________ Dr. Xue-Long Sun ________________________ Department and Date ____________________________________________________ Dr. John F. Turner ________________________ Department and Date ____________________________________________________ Dr. Yuping Wu ________________________ Department and Date MASS SPECTROMETRIC ANALYSIS OF ANTICANCER AGENTS AND DRUGS OF ABUSE IN BIOMATRICES, AS WELL AS CHARACTERIZATION OF INACTIVATION OF B-LACTAMASE ENZYMES KERRI M. SMITH ABSTRACT Mass spectrometry is a valuable tool in the analysis of many types of compounds. From small molecules to large proteins, mass spectrometry can help to interrogate samples and give insights to the origins of disease as well as calculate an exact concentration. Bioanalytical method development is an integral component in the measurement of such compounds. To facilitate proper analytical investigation, the process of method development, analysis, and interpretation must be understood. This work describes the basis and procedure for bioanalytical method development and its detailed application to preclinical studies of the antineoplastic agent hexamethylene bisacetamide and measurement of illicit drugs of abuse benzylpiperazine and trifluoromethylphenyl piperazine using liquid chromatography tandem mass spectrometry. Additionally, procedures for analyzing protein-drug interactions and their implication in antibiotic resistance are discussed. iv TABLE OF CONTENTS Page ABSTRACT ........................................................................................ iv LIST OF TABLES ................................................................................ ix LIST OF FIGURES ............................................................................... xi CHAPTER I INTRODUCTION OF LIQUID CHROMATOGRAPHY MASS SPECTROMETRY FOR BIOANALYTICAL ANALYSIS ..................................... 1 1.1. General introduction............................................................................... 1 1.2. Modern methods for sample analysis .................................................... 2 1.2.1. Liquid chromatographic separation ............................................... 3 1.2.2. Mass spectrometric detection ...................................................... 17 1.2.3. Quantitative bioanalytical HPLC-MS/MS method validation ........ 47 1.3. Conclusion ............................................................................................ 57 1.4. References ........................................................................................... 58 CHAPTER II DETERMINATION OF HEXAMTHYLENE BISACETAMIDE, AN ANTINEOPLASTIC COMPOUND, IN MOUSE AND HUMAN PLASMA BY LC– MS/MS ............................................................................................................... 63 2.1. Introduction ........................................................................................... 63 2.2. Materials and methods ........................................................................... 69 2.2.1. Chemicals and standard solutions ............................................... 69 v 2.2.2. Liquid chromatography tandem mass spectrometry .................... 71 2.2.3. Preparation of plasma calibrators and quality controls ................. 72 2.2.4. Animal study ................................................................................. 73 2.2.5. Sample preparation ...................................................................... 74 2.2.6. Stability studies ............................................................................ 75 2.3. Results ................................................................................................... 75 2.3.1. LC–MS/MS ................................................................................... 75 2.3.2. Matrix interference and specificity ................................................ 78 2.3.3. Method validation ......................................................................... 82 2.3.4. Application to animal study ........................................................... 93 2.4. Conclusion ............................................................................................. 98 2.5. References ............................................................................................. 99 CHAPTER III DEVELOPMENT OF A RAPID LIQUID CHROMATOGRAPHY TANDEM MASS SPECTROMETRY METHOD FOR THE ANALYTICAL DETERMINATION OF ILLICIT DRUGS BZP AND TFMPP IN HUMAN BLOOD, URINE, BILE, AND VITREOUS HUMOR ........................................................ 102 3.1. Introduction .......................................................................................... 102 3.1.1. History ........................................................................................ 106 3.1.2. Safety and legal status ............................................................... 108 3.1.3. Current LC-MS methods ............................................................ 110 3.2. Materials and methods ........................................................................ 110 3.2.1. Chemicals ................................................................................... 110 vi 3.2.2. HPLC–MS/MS ............................................................................ 111 3.2.3. Standard solution, calibrators, dilution, and quality control samples preparation ..................................................................................... 112 3.2.4. Sample preparation .................................................................... 113 3.2.5. Dilution and concentration studies ............................................. 114 3.2.6. Validation parameters ................................................................ 114 3.2.7. Method application ......................................................................116 3.3. Results and Discussion ........................................................................ 117 3.3.1. Optimization of MS/MS parameters ........................................... 117 3.3.2. Chromatographic separation and carryover ............................... 119 3.3.3. Method validation results ........................................................... 123 3.3.4. Application to ante- and post-mortem samples .......................... 134 3.4. Conclusion ........................................................................................... 137 3.5. References ........................................................................................... 138 CHAPTER IV INSIGHTS INTO THE INACTIVATION OF β-LACTAMASE ENZYMES FOR ANTIBIOTIC RESISTANCE BY MASS SPECTROMETRY ........................................................................................... 142 4.1 Introduction .......................................................................................... 142 4.1.1 Bacterial resistance .................................................................... 143 4.1.2 β-Lactamase classes .................................................................. 149 4.1.3 β-Lactam hydrolysis ................................................................... 151 4.2 Materials and methods ......................................................................... 156 vii 4.2.1 Preparation of enzymes for analysis .......................................... 156 4.2.2 Antibiotic susceptibility and kinetics ........................................... 156 4.2.3 Inactivation of β -lactamase enzymes ........................................ 157 4.2.4 Electrospray ionization mass spectrometry ................................ 157 4.3 Results and discussion ........................................................................ 158 4.3.1 Antibiotic susceptibility and kinetic analysis of sulbactam and clavulanate on SHV-2, KPC-2 and mutants ............................ 158 4.3.2 Mass Spectrometric studies of the inhibition of SHV-1 .......... 162 4.3.3 Mass Spectrometric studies of the inhibition of KPC-2 and KPC- 2 R220K ..................................................................................... 166 4.3.4 Proposed inactivation .............................................................. 171 4.4 Conclusion ........................................................................................... 174 4.5 References ........................................................................................... 175 viii LIST OF TABLES CHAPTER I Table 1.1 Common detectors for HPLC ......................................................... 6 Table 1.2 List of common mobile phase modifiers ....................................... 15 Table 1.3 List of common buffers ................................................................. 16 Table 1.4 Properties of