NONTARGETED DISCOVERY OF XENOBIOTICS IN HUMAN URINE BY MALDI-TOF/TOF-MS Dissertation Presented by Yuanyuan Yao To The Bouve’ Graduate School of Health Sciences in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Pharmaceutical Sciences with specialization in Biomedical Sciences NORTHEASTERN UNIVERSITY BOSTON, MASSACHUSETTS April 2018 ABSTRACT In this dissertation research, nontargeted analysis of the urine metabolome, including xenobiotics, was studied using LC (Liquid Chromatography)-MALDI (Matrix Assisted Laser Desorption Ionization) MS (Mass Spectrometry) techniques. MALDI is a sensitive soft ionization MS technique that has been mainly used to analyze large molecules such as peptides, proteins, and nucleic acids. Here, MALDI MS methods were employed for detection of urine metabolites. To increase the recovery of nonpolar metabolites, a novel porous extraction paddle (PEP) was validated with co-workers and used for urine extraction. A method for sample preparation including UHPLC (Ultra High-Performance Liquid Chromatography) was optimized to facilitate detection of nonpolar urine sulfate metabolites by MALDI MS. Using this approach, the detection coverage of such compounds was greatly expanded as compared to prior methods. Detection of 1129 MS precursor ions corresponding to putative sulfate and glucuronide metabolites was achieved. Combining MS and MS/MS experiments, a strategy was developed for tentative identification of the detected metabolites. This led to the first nontargeted analysis of environmental contaminants in urine. It was shown that the detection sensitivity of positive-mode MALDI MS can be enhanced using enzymatic deconjugation and cationic tagging methods. Also, an evaporative derivatization method was developed to increase the sensitivity of negative-mode MALDI MS for detection of phenolic compounds. Overall, through careful method development and optimization, the usefulness of LC-MALDI MS as an analytical platform for the measurement of the urine nonpolar metabolome, including urinary xenobiotics, has been extended. Northeastern University Bouvé College of Health Sciences Dissertation Approval Dissertation title: Nontargeted Discovery of Xenobiotics in Human Urine by MALDI- TOF/TOF-MS Author: Yuanyuan Yao Program: Doctor of Philosophy in Pharmaceutical Sciences with a Specialization in Pharmaceutics Approval for thesis requirements for the Degree of Doctor of Philosophy in: Pharmaceutical Sciences Dissertation Committee (Chair): Signature: _______________________________ Date________________ Printed Name: _______________________________ Other committee members: Signature: _______________________________ Date________________ Printed Name: _______________________________ Signature: _______________________________ Date________________ Printed Name: _______________________________ Signature: _______________________________ Date________________ Printed Name: _______________________________ Signature: _______________________________ Date________________ Printed Name: _______________________________ Associated Dean of Graduate Education, Bouvé College of Health Sciences: Signature: _______________________________ Date________________ Printed Name: _______________________________ ACKNOWLEDGEMENTS Finishing the research and writing the dissertation has been an Odyssey for me. I want to thank my committee for their advices and patience. I appreciate having Dr. Giese as my mentor. He not only taught me about mass spectrometry but also showed me a role model to balance the multiple responsibilities in life. I would not be able to continue and finish my dissertation without Dr. Giese continuous support and encouragement. Also, I am indebted to Dr. Poguang Wang in our lab for his help throughout my research. Dr. Wang always provide me kind guidance with his deep and broad knowledge of chemical analysis and led me through many obstacles in my research. Finally, I am grateful to my daughter and husband who inspired me to persist in overcoming difficulties in life. TABLE OF CONTENTS 1. INTRODUCTION 1-1. The scope of dissertation work………………………………………………………………...1 1-2. Xenobiotics in urine………………………………………………………………....................2 1-2.1. Human urine composition………………………………………………………………....2 1-2.2. Small molecules in urine: endogenous metabolites and xenobiotics…………………........3 1-2.3. Metabolism of xenobiotics…………………………………………………………….......5 1-2.4. Possible correlations between xenobiotics and diseases……………………………….......7 1-3. Analysis of urine xenobiotics………………………………………………………………......8 1-3.1. Metabolomics in general………………………………………………………………......8 1-3.2. Target and Nontargeted analysis .. ………………………………………………………. 9 1-3.3. Special aspects of urine xenobiotic analysis……………………………………………...11 1-3.4. Current development of xenobiotics analysis of urine……………………………………13 1-4. MS methods for chemical analysis of biological samples……………………………………15 1-4.1. A new analytical platform for urine metabolic analysis based on MALDI-TOF-MS…….15 1-4.2. Soft ionization techniques for MS spectrometry………………………………………….17 1-4.3. MS analyzers……………………………………………………………………………..24 1-4.4. Tandem MS………………………………………………………………………………29 1-4.5. Application of MS techniques in urine metabolomics……………………………………35 1-4.6. Use of MALDI for small molecule analysis……………………………………………...36 1-5. Sample preparation methods for MS analysis………………………………………………..37 1-5.1. Urine sample collection …………………………………………………………………37 1-5.2. Chemical derivatization of xenobiotics for MS analysis…………………………………39 1-5.3. Solid phase exaction of biological samples………………………………………………43 1-5.4. HPLC of complex biological samples for MS analysis………………………………….47 I 2. MATERIALS AND METHODS 2-1. Evaporative derivatization reaction with sulfobenzoic anhydride……………………………48 2-2. Preparation of PEP bags for exaction of urine metabolites…………………………………49 2-3. Urine sample collections……………………………………………………………………...51 2-4. Direct MALDI-MS analysis of urine metabolites extracted by MP-PEP……………………...52 2-5. Optimized sample preparation method for UHPLC-MALDI MS analysis…………………... 53 2-6. HPLC and UHPLC ………………………………………………………………………….54 2-7. MALDI TOF MS and TOF/TOF tandem MS ………………………………………………. 55 2-8. Enzymatic deconjugation and cationic tagging of urine……………………………………... 56 3. RESULTS AND DISCUSSION 3-1. Evaporative derivatization of phenols with sulfobenzoic anhydride for negative-mode MALDI- MS …………………………………………………………………………………………... 56 3-1.1. SBA evaporative derivatization reaction and MALDI MS analysis of a model phenol (4- phenyl-phenol)……………………………………………………………………………… 57 3-1.2. Anionic Tagging and MALDI MS analysis of a 15-phenol mixture……………………...68 3-2. Development of Porous Extraction Paddle (PEP) for urine metabolite extraction………….73 3-2.1. The PEP with solid phase extraction adsorbents………………………………………….73 3-2.2. Evaluation of extraction efficiency of various PEP with a standard dye………………….75 3-2.3. PEP extraction of urine samples for qualitative analysis of metabolites………………… 76 3-2.4. PEP extraction of urine samples for quantitative analysis of targeted metabolites………..83 II 3-3. Optimized procedure for UHPLC-MALDI MS analysis of urine metabolites………………..86 3-3.1. Our urine sample preparation strategy for LC/MALDI MS analysis……………………. 87 3-3.2. High reproducibility of our sample preparation strategy…………………………………89 3-4. UHPLC-MALDI analysis of sulfatome and glucuronidome of urine samples……………….91 3-4.1. UHPLC-UV spectra of 6 urine samples…………………………………………………. 91 3-4.2. Sulfatome and glucuronidome of six urine samples detected by UHPLC-MALDI MS… 94 3-4.3. Tentative assignment of the sulfate conjugates by matching METLIN database…………96 3-4.4. Examples of sulfated metabolites discovered in urine samples by MALDI MS MS/MS. 100 3-4.5. Candidate pollutants in the 6 urine samples……………………………………………. 104 3-5. Deconjugation and cationic tagging of urine samples for UHPLC-MALDI analysis………..107 3-5.1. Optimization of conditions for enzymatic deconjugation of urine …………………….108 3-5.2. Cationic tagging of the deconjugated urine sample…………………………………….. 111 3-5.3. UHPLC MALDI MS analysis of cationic tagged urine sample………………………… 113 3-5.4. MALDI-MS spectra of a cationic tagged deconjugated urine sample………………….. 115 4. SUMMARY AND CONCLUSSION…………… ………………………………………..117 5. PUBLICATIONS IN THIS STUDY………………………….…………………………. 118 6. REFERENCES……………………………………………………………………….… ...119 III LIST OF TABLES Table 1. Common conjugations of xenobiotics (denoted as X in the table) in the phase II metabolism….11 Table 2. Examples of matrix molecules for UV and IR MALDI……………………………………….…19 Table 3. Silylating and acylating reagents for chemical derivatization of acids, alcohols and amines for GC- MS…………………………………………………………………………………………………………32 Table 4. Cationic tagging reagents for positive mode LC-MS chemical analysis………………………….35 Table 5. Examples of ion exchange adsorbents…………………………………………………………….38 Table 6. Yield of solution phase derivatization reactions of 4-phenylphenol at different reaction conditions………………………………………………………………………………………………….54 Table 7. Yield of derivatization reaction in solid phase at room temperature at different time lengths. The reactants are from concentrating of 50-uL of 0.05M 4-phenylphenol, 0.5M SBA, and DMAP (2% w/w of 4-phenylphenol) …………………………………………………………………………………………..55 Table 8. Yield of solid phase derivatization reaction at different temperature for 2 hours. The reactants are from concentrating of 50-μL of 0.05M 4-phenylphenol, 0.5M SBA, and DMAP (2% w/w of 4- phenylphenol)……………………………………………………………………………………………..55