Polarising Poison – Synthesis, characterization and development of rapid, sensitive methods for the detection of potent synthetic opiates. NICOLAS GILBERT PhD 2020 Polarising Poison – Synthesis, characterization and development of rapid, sensitive methods for the detection of potent synthetic opiates. NICOLAS GILBERT A thesis submitted in partial fulfilment of the requirements of Manchester Metropolitan University for the degree of Doctor of Philosophy Department of Natural Sciences Manchester Metropolitan University 2020 ACKNOWLEDGEMENTS I first wish to thank Dr. Oliver Sutcliffe for his supervision, his constant support and his guidance throughout this project. Dr. Ryan Mewis has been equally important in the realisation of this thesis, through his invaluable advice and continued involvement. Both of them contributed to make this doctorate a successful experience and made me a better researcher. I also want to thank the other members of the Sucliffe group, especially Matt and Lysbeth, who have been great colleagues, teachers and friends. Thanks also to the other Ph.D. students met at MMU, but especially Jade, Mike, Olly, Antoine, Isabella and Matthieu, who made these three years enjoyable. Their friendship and understanding were, at times, all I needed to keep going. Je veux aussi remercier mes parents, ma sœur, et toute ma famille, de m’avoir supporté depuis tout petit à poursuivre mon éducation. Dans tout ce que je fais, vous êtes au creux de moi. Thanks to Chris for putting up with me for three years now, and hopefully more. Finally, I must thank the Fonds de Recherche du Québec – Nature et Technologies and the National Research Council of Canada for their doctorate funding, without which none of this would have been possible. ABSTRACT The occurrence of synthetic opioid fentanyl and its derivatives has grown significantly in forensic casework in recent years. This poses a threat for public health, as it has been reported that minute quantities of fentanyl are enough to induce an overdose. Approximately 32,400 people died because of synthetic opioids in the United States in 2018, and the current situation has been described as an “opioid epidemic”. The increase in fentanyl abuse has been associated with the emergence of a large variety of fentanyl analogues. These novel compounds are more difficult to identify by forensic chemists and may not be detected when they emerge, because they have yet to be characterised and added to mass spectral databases. Another insidious phenomenon has been contributing to the increase in overdoses: fentanyl and its analogues have often been mixed into or sold as other illicit drugs. Most commonly, fentanyl has been found in heroin samples, a practice which may occur unbeknown to drug users and increase the risk of overdose. This thesis aimed to solve these challenges associated with fentanyl detection, to aid in forensic casework and harm reduction. In Chapter II, eighteen common fentanyl analogues were synthesised, fully characterised, and submitted to presumptive colour tests, TLC and FT-IR analysis. A GC-MS method was developed to separate target analogues and allow their detection and quantification in seized heroin samples. Chapter III focused on fluorofentanyl regioisomers, which could not be separated by GC-MS. An orthogonal benchtop 19F NMR method was developed to differentiate and quantify these analogues. Chapter IV focused on the eosin Y colour test, which can be used to detect fentanyl analogues. The mechanism of the reaction between eosin Y and heroin/fentanyl was investigated by NMR. UV-Vis spectrophotometry and RGB detection were used to develop a quantitative version of the test. Finally, in Chapter V, a principal component analysis (PCA) model based on MS data from fentanyl analogues was developed. The model was able to group analogues based on their structural class and identify structural modifications in novel compounds. Table of Contents ACKNOWLEDGEMENTS ............................................................................ 2 ABSTRACT ................................................................................................. 3 LIST OF TABLES ........................................................................................ 9 LIST OF FIGURES .................................................................................... 13 ABBREVIATIONS AND ACRONYMS........................................................ 20 CHAPTER I .................................................................................................. 22 INTRODUCTION ....................................................................................... 22 1.1 Fentanyl ........................................................................................ 22 1.2 Fentanyl detection ........................................................................ 32 1.3 Objectives ..................................................................................... 41 CHAPTER II ................................................................................................. 42 SYNTHESIS AND DETECTION OF AMIDE-CHAIN FENTANYL DERIVATIVES ........................................................................................... 42 2.1 Overview....................................................................................... 42 2.2 Synthesis ...................................................................................... 46 2.3 Presumptive colour tests .............................................................. 50 2.4 Thin-layer chromatography (TLC) ................................................. 53 2.5 Infrared spectroscopy ................................................................... 56 2.6 Electron ionisation mass spectrometry ......................................... 57 2.7 Qualitative GC-MS analysis .......................................................... 63 2.8 Quantitative GC-MS analysis ........................................................ 70 2.9 Analysis of seized heroin samples ................................................ 77 2.10 Conclusion ................................................................................ 91 CHAPTER III ................................................................................................ 93 SYNTHESIS AND DETECTION OF FLUORINATED FENTANYL REGIOISOMERS ...................................................................................... 93 3.1 Overview....................................................................................... 93 3.2 Synthesis ...................................................................................... 95 3.3 Presumptive colour tests ............................................................ 101 3.4 Thin-layer chromatography (TLC) ............................................... 102 3.5 Infrared spectroscopy ................................................................. 103 3.6 Electron ionisation mass spectrometry ....................................... 104 3.7 Qualitative GC-MS analysis ........................................................ 107 3.8 Quantitative GC-MS analysis ...................................................... 109 3.9 Qualitative NMR analysis............................................................ 116 3.10 Quantitative low-field 19F NMR analysis .................................. 120 3.11 Conclusion .............................................................................. 124 CHAPTER IV ............................................................................................. 125 INVESTIGATION OF THE EOSIN Y COLORIMETRIC TEST ................. 125 4.1 Overview..................................................................................... 125 4.2 Detection of fentalogues ............................................................. 126 4.3 Investigation of the reaction mechanism .................................... 127 4.4 Standardisation of colorimetric tests ........................................... 155 4.5 Conclusion .................................................................................. 169 CHAPTER V .............................................................................................. 171 PRINCIPAL COMPONENT ANALYSIS MODEL FOR FENTANYL ANALOGUE CLASSIFICATION .............................................................. 171 5.1 Overview..................................................................................... 171 5.2 Data handling ............................................................................. 173 5.3 Hierarchical clustering ................................................................ 185 5.4 Model evaluation ........................................................................ 215 5.5 Conclusion .................................................................................. 220 CHAPTER VI ............................................................................................. 221 CONCLUSION AND FUTURE WORK ..................................................... 221 CHAPTER VII ............................................................................................ 225 EXPERIMENTAL SECTION .................................................................... 225 7.1 Presumptive colour tests ............................................................ 225 7.2 Thin-layer chromatography ......................................................... 226 7.3 Infrared spectroscopy ................................................................. 226 7.4 GC-MS analysis .........................................................................