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Development of Liquid Chromatography-Mass Spectrometric DEVELOPMENT OF LIQUID CHROMATOGRAPHY-MASS SPECTROMETRIC ASSAYS AND SAMPLE PREPARATION METHODS FOR THE BIOLOGICAL SAMPLE ANALYSIS SUJATHA CHILAKALA Master of Science in Pharmaceutical Sciences University of Greenwich, UK July 2007 submitted in partial fulfillment of requirements for the degree DOCTOR OF PHILOSOPHY IN CLINICAL-BIOANALYTICAL CHEMISTRY at CLEVELAND STATE UNIVERSITY November 2017 We hereby approve this dissertation for Sujatha Chilakala Candidate for the Doctor of Philosophy in Clinical-Bioanalytical Chemistry degree for the Department of Chemistry and the CLEVELAND STATE UNIVERSITY College of Graduate Studies _________________________________________________________________ Dissertation Chairperson, Dr. Yan Xu _____________________________________________ Department & Date ____________________________________________________________________ Dissertation Committee Member, Dr. Bin Su _____________________________________________ Department & Date ____________________________________________________________________ Dissertation Committee Member, Dr. Aimin Zhou _____________________________________________ Department & Date ____________________________________________________________________ Dissertation Committee Member, Dr. David Anderson _____________________________________________ Department & Date ____________________________________________________________________ Dissertation Committee Member, Dr. Petru Fodor _____________________________________________ Department & Date Student’s Date of Defense: November 27th, 2017 Dedicated to my loving husband Amar and to my dearest son Nushyanth Acknowledgement This thesis would not have been possible without the support of many people. First and foremost, I would like to thank my adviser, Dr. Yan Xu, for his intellectual supervision, continuous support, endless patience, motivation and encouragement throughout my graduate studies. I am very grateful for the time he spent on helping me with every aspect of this research, for his invaluable suggestions, careful guidance and kindness. I am fortunate to have Dr. Xu, a knowledgeable and excellent professor as my adviser. I am pleased to thank my committee members, Dr. David Anderson, Dr. Aimin Zhou, Dr. Bin Su and Dr. Petru S Fodor, for their support, sound advice and fruitful comments on my research. I also would like to thank our collaborator from Cleveland clinic Dr. Yogen Saunthararajah and his group for providing us samples, support and suggestions. I thank Dr. Xiang Zhou for his continuous support with instrument training and handling problems. A special thanks to Dr. Mundell, for all his help and support during my duties as a TA. I would also thank Richelle Emery, Michelle Jones and Janet in the Chemistry office for all their administrative support over the years. I would also like to express my sincere thanks to all of my colleagues and friends for their encouragement and invaluable scientific discussions. A big thanks to all fellow graduate students and other staff at Cleveland State University. Finally, my special thanks go to my family. Words fail me when I try to express my gratitude towards my family. I am deeply and forever indebted to my husband for his everlasting love, support and encouragement, for giving of himself beyond the call of duty. I would also like to thank my parents, mother-in-law, my siblings for their unconditional love and support. I owe all of my achievements to my family, especially to my husband and my dearest son. DEVELOPMENT OF LIQUID CHROMATOGRAPHY-MASS SPECTROMETRIC ASSAYS AND SAMPLE PREPARATION METHODS FOR THE BIOLOGICAL SAMPLE ANALYSIS SUJATHA CHILAKALA ABSTRACT The area of biosample analysis encompass a very broad range of assays which support the clinical and nonclinical studies. Biosample analysis is used to provide a quantitative or qualitative measure of the active drug and/or its metabolite(s) in the biological matrix for the purpose of pharmacokinetics, toxicokinetics, bioequivalence, and exposure–response (pharmacokinetics /pharmacodynamics) studies. Due to the significance of pharmacological analysis, sensitive, reproducible and robust analytical methods are critically needed for pharmacological studies of the biosamples. A bioanalytical method mainly contains two components I) Sample preparation II) detection of the compound. Therefore, the main aims of this thesis, development of quantitative and qualitative analytical methods for the target compounds using LC-MS(/MS) and development of accelerated sample preparation for high throughput sample analysis for V DNA and proteins. In this dissertation, a brief review on the method rationale, workflow of the method development, sample preparation methods, instrumentations and analytical method validation, are discussed in Chapter 1. Also, research projects were discussed and the techniques used in the experiments for this thesis were reviewed. As so, chapter II and III were mainly focused on the accelerated sample preparation methods for the high throughput sample analysis of DNA and proteins respectively, where the sample preparation time was significantly reduced from hours to minutes, which are suitable for qualitative and quantitative analysis of DNA and proteins. In Chapter IV, a systematic study on the structural characterization of the model glycoprotein Human IgG was described. In chapter V successful development of LC-MS method was developed for the determination of Oxygen -18 isotope enrichment in the phosphate samples in the positional isotope exchange reactions to study the reversibility of certain enzymatic reactions was described. Successful development and validation of a new and sensitive analytical LC- MS/MS method for the determination and quantitation of incorporation rates of decitabine, an anti-cancer drug which can be applied to determine the sensitivity and responsiveness in patients treated with decitabine was described in Chapter VI. VI TABLE OF CONTENTS ABSTRACT………………………………...………………………………………........V LIST OF TABLES...........................................................................................................XV LIST OF FIGURES.........................................................................................................XVI CHAPTER I. INTRODUCTION TO BIOLOGICAL SAMPLE ANALYSIS AND ANALYTICAL METHOD DEVELOPMENT 1.1 General introduction and research objectives ……...………………….…..…….....1 1.2 Analytical methods for biosample analysis………………………………..……….6 1.2.1 Mass spectrometry………………..……………………………..……….7 1.2.1.1 Ionization methods………………………………………..……...10 1.2.1.2 Mass analyzers……....…………………………………...………13 1.2.1.3 Modes of detection …………………………………………....…20 1.3 Method validation. …………………………….……………………………...…..21 1.4 Conclusion ...………………..……………………………………………….....….26 1.5 References….…………….………………………………………………… ..…….27 VII CHAPTER II. MICROWAVE-ASSISTED ENZYMATIC HYDROLYSIS OF DNA FOR MASS SPECTROMETRIC ANALYSIS: A NEW APPROACH TO DNA HYDROLYSIS IN MINUTES……………………………………………………....29 2.1 Introduction ……………………………….……...………………………………30 2.2 Experimental………………..……………………………………….……….…...32 2.2.1 Chemicals and materials…...………………....…...………......………….....32 2.2.2 Solutions…………………………………………………………………….33 2.2.3 Instrumentation………………..…………………………………………….33 2.2.3.1 Microwave digestion system ……………. …………………………33 2.2.3.2 LC-MS/MS system………..…………………………………………34 2.2.3.3 Cell culture and DNA extraction……...…… …………………….....35 2.2.4 Tetra-enzyme digestion mix ………… ………………………...………… 36 2.2.5 DNA digestion………………………………….…………………………..36 2.2.5.1 Microwave-assisted digestion…….………...………..…….……….37 2.2.5.2 Conventional digestion………..……………………………………39 2.2.6 Extraction of dNs from enzyme digest…………………….……………….39 2.2.7 Application to other DNA samples ……..………………….....…… …….39 2.3 Results and Discussion……………………………..……………………….. .......40 VIII 2.3.1 Microwave Technology………..……………………………………..……...40 2.3.2 Optimization of parameters for microwave digestion ……..………...….…....41 2.3.3 Tetra–enzyme digestion mix…………………………………..……………...41 2.3.4 Microwave assisted hydrolysis…………….…..……………………………..42 2.3.5 Application of the method to the digestion of DNA extracted from cell lines…………………………………………………………………………...................49 2.4 Conclusion…………………………………………………………….…………...51 2.5 References……………………………………………………………………….....52 CHAPTER III. AN ACCELERATED PROTEIN SAMPLE PREPARATION METHOD AND SELECTIVE EXTRACTION OF PEPTIDES FROM PLASMA FOR LC-MS-BASED PROTEOMICS……………………………………………….55 3.1 Introduction ………………………………………….……...………………….....56 3.2 Experimental…………………………..……………………………………….…...61 3.2.1 Chemicals and materials…………………….………….......…..………...…..61 3.2.2 Optimization of protein reduction and alkylation time……………………...61 3.2.3 Instrumentation……………………..………………………………………...65 3.2.3.1 Chromatography separation and mass spectrometric conditions……..65 3.2.3.2 Microwave digestion system ………..………………………………..66 IX 3.2.4 Data processing………..………………………...………………….………..66 3.2.5 Peptide extraction…………………………………………………………….67 3.2.5.1 Preparation of spiked plasma samples………………………………..67 3.2.5.2 Peptide extraction protocols….………………………………………68 3.2.6 Instrumentation………..……………………………………………………..69 3.2.7 Data processing……………………………………………………………….70 3.3 Results and Discussion…..…………………..……………………….…………….70 3.3.1 Optimization of sample preparation time…………………….……………..70 3.3.1.2 Application of optimized method on mixture of proteins and serum proteins.77 3.3.3 Comparison of extraction methods……………………………………………81 3.4 Conclusion………………..………………………..……………………………….83 3.5 References…………………..………………………………………………………84
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