The author(s) shown below used Federal funding provided by the U.S. Department of Justice to prepare the following resource: Document Title: Metabolism and Analysis of Desomorphine Author(s): Jessica N. Winborn Document Number: 254755 Date Received: May 2020 Award Number: 2015-R2-CX-0031 This resource has not been published by the U.S. Department of Justice. This resource is being made publically available through the Office of Justice Programs’ National Criminal Justice Reference Service. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice. METABOLISM AND ANALYSIS OF DESOMORPHINE _____________ A Dissertation Presented to The Faculty of the Department of Forensic Science Sam Houston State University _____________ In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy _____________ by Jessica N. Winborn December, 2018 This resource was prepared by the author(s) using Federal funds provided by the U.S. Department of Justice. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice. METABOLISM AND ANALYSIS OF DESOMORPHINE by Jessica N. Winborn ______________ APPROVED: Sarah Kerrigan, PhD Dissertation Co-Director Donovan Haines, PhD Dissertation Co-Director Madeline Swortwood, PhD Committee Member Chi-Chung (Jorn) Yu, PhD Committee Member Phillip Lyons, PhD Dean, College of Criminal Justice This resource was prepared by the author(s) using Federal funds provided by the U.S. Department of Justice. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice. DEDICATION “You must do the things you think you cannot do.” -Eleanor Roosevelt To my advisor, Dr. Sarah Kerrigan, for her unwavering faith in me and for all of her words of advice and encouragement. To my co-advisor, Dr. Donovan Haines, for helping me look at problems from a different perspective. To my PhD cohort, for all of their encouragement and the laughter when I needed it most. To my parents, for their continued support and belief in me. To my grandparents, who saw me start this journey but couldn’t be here to see me finish it. iii This resource was prepared by the author(s) using Federal funds provided by the U.S. Department of Justice. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice. ABSTRACT Winborn, Jessica N., Metabolism and analysis of desomorphine. Doctor of Philosophy (Forensic Science), December, 2018, Sam Houston State University, Huntsville, Texas. Desomorphine is a semi-synthetic opioid that is ten times more potent than morphine, with a faster onset but shorter duration of action. It is a major component of the drug referred to as “Krokodil”, which is used as a heroin substitute. Its prevalence is difficult to estimate due to a lack of analytically confirmed cases, which may in part be due to the limited studies regarding its pharmacology or methodology to detect the drug in biological specimens. This research seeks to further the understanding of both desomorphine’s metabolism and its detection in biological specimens, to facilitate its identification in clinical and forensic toxicology laboratories. Six commercially available enzyme-linked immunosorbent assays were evaluated to determine their effectiveness with respect to desomorphine detection. Cross-reactivities were highly variable between assays, ranging from <2.5-77%. Recombinant human cytochrome P450 enzymes (rCYPs) and recombinant uridine 5'-diphospho- glucuronosyltransferases (rUGTs) were used to investigate the biotransformational pathways involved in desomorphine metabolism. Phase I metabolism could be attributed to seven rCYPs (rCYP2B6, rCYP2C8, rCYP2C9, rCYP2C18, rCYP2C19, rCYP2D6 and rCYP3A4), producing a total of nine phase I metabolites (nordesomorphine, desomorphine-N-oxide, two norhydroxydesomorphine isomers, and five hydroxylated isomers). During phase II metabolism, desomorphine-glucuronide was produced by nine rUGTs (rUGT1A1, rUGT1A3, rUGT1A8, rUGT1A9, rUGT1A10, rUGT2B4, rUGT2B7, rUGT2B15, and rUGT2B17). iv This resource was prepared by the author(s) using Federal funds provided by the U.S. Department of Justice. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice. Chemical and enzymatic hydrolysis of conjugated metabolites were investigated using desomorphine-glucuronide generated in situ using rUGT enzyme. Acid hydrolysis ™ ™ and five β-glucuronidase sources (BGTurbo , IMCSzyme , Escherichia coli, Helix pomatia and Patella vulgata) were evaluated. Acid hydrolysis produced complete hydrolysis of desomorphine-glucuronide, and under optimal conditions, each enzyme ™ ™ produced complete or near complete hydrolysis (≥96%), with BGTurbo and IMCSzyme offering the shortest incubation times. Under simulated challenging conditions, P. vulgata was the most effective enzyme evaluated. Desomorphine was analyzed in blood and urine samples using gas chromatography-mass spectrometry (GC-MS), and urine samples were additionally analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and liquid chromatography-quadrupole/time of flight-mass spectrometry (LC-Q/TOF-MS). Each method was validated in accordance with published guidelines for forensic use. Additionally, LC-Q/TOF-MS was used to analyze desomorphine metabolites, which in the absence of commercially available reference material or authentic urine specimens, were generated in-vitro. KEY WORDS: Desomorphine, Immunoassays, Metabolism, Gas Chromatography, Liquid Chromatography, High Resolution Mass Spectrometry v This resource was prepared by the author(s) using Federal funds provided by the U.S. Department of Justice. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice. ACKNOWLEDGEMENTS This work was supported by Award No. 2015-R2-CX-0031 awarded by the National Institute of Justice, Office of Justice Programs, U.S. Department of Justice. The opinions, findings, and conclusions or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect those of the Department of Justice. vi This resource was prepared by the author(s) using Federal funds provided by the U.S. Department of Justice. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice. TABLE OF CONTENTS Page DEDICATION ................................................................................................................... iii ABSTRACT ....................................................................................................................... iv ACKNOWLEDGEMENTS ............................................................................................... vi TABLE OF CONTENTS .................................................................................................. vii LIST OF TABLES ............................................................................................................ xii LIST OF FIGURES .......................................................................................................... xv I INTRODUCTION .................................................................................................. 1 Background ............................................................................................................. 1 History of desomorphine......................................................................................... 2 Chemistry of desomorphine .................................................................................... 9 Pharmacology ....................................................................................................... 20 Experimental approaches to drug metabolism ...................................................... 27 Forensic toxicology ............................................................................................... 33 Analytical methodology for desomorphine .......................................................... 52 Statement of the problem ...................................................................................... 58 References ............................................................................................................. 60 II DESOMORPHINE SCREENING USING COMMERCIAL ENZYME- 1 LINKED IMMUNOSORBENT ASSAYS .......................................................... 77 Abstract ................................................................................................................. 78 Introduction ........................................................................................................... 79 Materials and methods .......................................................................................... 83 vii This resource was prepared by the author(s) using Federal funds provided by the U.S. Department of Justice. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice. Results