ONLINE BIOANALYTICAL MASS SPECTROMETRY METHODS FOR ANALYSIS OF SMALL AND LARGE BIOMOLECULES USING ONE- AND TWO-DIMENSIONAL LIQUID CHROMATOGRAPHY by Yehia Zakaria Baghdady DISSERTATION Submitted to the Graduate School of The University of Texas at Arlington in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY December 2018 Supervising Committee: Professor Kevin Schug, Chair Professor Daniel Armstrong Professor Krishnan Rajeshwar Associate Professor Kayunta Johnson-Winters Copyright © by Yehia Baghdady 2018 All Rights Reserved Acknowledgments Thank you is never enough to express how grateful I am to all who have supported me to reach this major stage of my life. Thanks to Allah, the most gracious and the most merciful, for surrounding me with those gorgeous people who have positive impact on my whole life starting with my research advisor, Dr. Kevin A. Schug. He trusted my capabilities, accepted me into his wonderful group and gave me the freedom to grow as a scientist and to try new ideas for all new challenges which I have faced in my research projects under his continuous guidance and support. I could not have imagined having a better mentor and research advisor for my Ph.D. degree. I would like to give special thanks to Dr. Armstrong, Dr. Rajeshwar and Dr. Johnson- Winters for being my committee members, their interest in my research, their invaluable suggestions and letting my defense be an enjoyable experience. I would also like to thank all our lab group members. I was very fortunate to be surrounded with smart and friendly lab mates. I would never forget to thank Restek Corporation for funding my research, providing their newest column technologies and providing me with an enriching industrial collaboration through their frequent visits and educational presentations. I would also like to thank the Shimadzu team for their training, industrial collaboration and instrumentation support. Finally, I would like to acknowledge my family, thanks from the bottom of my heart to my parents, sister and brother for always being there for me and for putting moral values which have guided me throughout my whole life. Thanks to my wife Omnia who sacrificed her job as a dentist to travel with me from Egypt to USA and to surround our family with love, care and support. I would also like to thank my beloved children, Adam and Omar, for always cheering me up with their amazing smiles and for encouraging me to be the father whom they will always be proud of. iii Abstract Online Bioanalytical Mass Spectrometry Methods for Analysis of Small and Large Biomolecules Using One- and Two-Dimensional Liquid Chromatography Yehia Baghdady, Ph.D. The University of Texas at Arlington, 2018 Supervising Professor: Kevin A. Schug In recent years, the innovation in biomarkers and biotherapeutics has increased the demand for state-of-the-art analytical platforms to achieve reliable and accurate discoveries and quantitative analysis methods. The challenges in their determinations are not limited to the large number of small and large biomolecules but also involve the great heterogeneity in chemical properties and abundance of those analytes. Liquid chromatography and mass spectrometry have advanced significantly to be the most common platform for analyzing biological samples. However, there are still many unmet challenges that that require application of novel bioanalytical methods to fill many gaps in online sample preparation, chromatographic separation, and mass spectrometric detection of different analyte sets. Frontal chromatography was used to investigate the trapping efficiency and capacity of online restricted access media trap columns to streamline sample preparation for online enrichment of small molecule analytes in complex biological samples. To enhance the sensitivity and selectivity for determination of 14 biomarkers of exposure to BTEX in urine, we developed a novel one pot charge reversal derivatization kit, optimized its performance through multivariate analysis using central composite design, and then validated its performance by using isotope dilution mass spectrometry. We further developed a novel high pH iv reversed phase chromatographic method for orthogonal separation of intact proteins using mass spectrometry compatible buffers. Finally, we adopted this method as the first dimension in a comprehensive two-dimensional reversed phase x reversed phase-mass spectrometry platform for intact protein separations. v Contents Acknowledgments....................................................................................................................... iii Abstract ....................................................................................................................................... iv CHAPTER ONE: INTRODUCTION TO DISSERTATION ...................................................... 1 1.1 Introduction .......................................................................................................................... 1 1.2 References ............................................................................................................................ 3 CHAPTER TWO: IN SITU DERIVATIZATION TECHNIQUES FOR ENHANCED BIOANALYSIS USING LIQUID CHROMATOGRAPHY WITH MASS SPECTROMETRY ................................................................................................................. 5 2.1 Abstract ................................................................................................................................ 7 2.2 Introduction .......................................................................................................................... 8 2.3 Aldehydes and ketones ....................................................................................................... 11 2.4 Alcohols, phenols and other hydroxyl-bearing compounds ............................................... 14 2.5 Amines ................................................................................................................................ 18 2.6 Carboxylic acids ................................................................................................................. 23 2.7 Thiols .................................................................................................................................. 26 2.8 Conclusion .......................................................................................................................... 29 2.9 References .......................................................................................................................... 30 CHAPTER THREE: EVALUATION OF EFFICIENCY AND TRAPPING CAPACITY OF RESTRICTED ACCESS MEDIA TRAP COLUMNS FOR ONLINE TRAPPING OF SMALL MOLECULES ........................................................................................................ 37 3.1 Abstract .............................................................................................................................. 39 3.2 Introduction ........................................................................................................................ 40 3.3 Frontal Analysis Theory ..................................................................................................... 42 3.4 Materials and methods ........................................................................................................ 45 3.4.1 Chemicals ..................................................................................................................... 45 3.4.2 Apparatus ...................................................................................................................... 45 3.4.3 Experimental conditions ............................................................................................... 45 3.5 Results and discussion ........................................................................................................ 47 3.5.1 Effect of pH of the mobile phase .................................................................................. 47 3.5.2 The effect of different flow rates .................................................................................. 52 3.5.3 The Effect of different restricted access media phases ................................................. 54 3.6 Conclusion .......................................................................................................................... 56 vi 3.7 References .......................................................................................................................... 58 CHAPTER FOUR: A NOVEL DIAGNOSTIC IN SITU DERIVATIZATION KIT FOR THE SIMULTANEOUS DETERMINATION OF 14 BIOMARKERS OF EXPOSURE TO BENZENE, TOLUENE, ETHYL BENZENE AND XYLENES IN HUMAN URINE BY ISOTOPE DILUTION LIQUID CHROMATOGRAPHY TANDEM MASS SPECTROMETRY AND KIT OPTIMIZATION USING RESPONSE SURFACE METHODOLOGY ................................................................................................................ 63 4.1 Abstract .............................................................................................................................. 65 4.2 Introduction ........................................................................................................................ 66 4.3 Experimental ...................................................................................................................... 69 4.3.1 Reagents and chemicals ...............................................................................................