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O'connor, Lauren (2020) Detection of Designer Benzodiazepines in Scottish Sub-Populations

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O'connor, Lauren (2020) Detection of Designer Benzodiazepines in Scottish Sub-Populations O'Connor, Lauren (2020) Detection of designer Benzodiazepines in Scottish sub-populations. PhD thesis. http://theses.gla.ac.uk/81840/ Copyright and moral rights for this work are retained by the author A copy can be downloaded for personal non-commercial research or study, without prior permission or charge This work cannot be reproduced or quoted extensively from without first obtaining permission in writing from the author The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the author When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given Enlighten: Theses https://theses.gla.ac.uk/ [email protected] Detection of Designer Benzodiazepines in Scottish Sub-populations Thesis Submitted in Accordance with the Requirements of the University of Glasgow for the Degree of Doctor of Philosophy By Lauren O‘Connor BSc Hons MRes Forensic Medicine and Science School of Medicine, Dentistry and Nursing November 2020 i Acknowledgments I would like to thank my supervisors Dr Hazel Torrance and Denise McKeown for their guidance and expertise throughout this research, you both gave me the opportunity to obtain the samples to make all this possible, and were never short of ideas to improve this research. I would also like to thank Dr Gail Cooper for employing me at FMS and giving me the opportunity to undertake a PhD, Dr Hilary Hamnett for always being supportive and Dr Peter Maskell for his thesis writing advice. I am very thankful to have had Alice Seywright as a fellow PhD student and colleague; she really has been the best at listening and understanding. Do we have time to learn how the telephone works? Just like Alice, Claire Parks has listened, supported and helped me every step of the way. Thank you. I am also very grateful to the FMS technicians who have provided more help and support than they know. I especially thank Nikki Kellichan who doesn‘t get to escape any ridiculous thought that crosses my mind at any time of the day or night. You make me laugh everyday. WE WANT ANSWERS. My life would not be the same without Craig. Throughout this journey you bought a flat with me and put a lovely sparkly ring on my finger. Thank you for last decade of adventures and for reading through this thesis with me for hours. God only knows what I‘d be without you. My mum and dad are a constant support throughout my life, they have always encouraged me in whatever I have wanted to do. Thank you for always being there and always believing in me. Thanks also go to my sister, Holly who never fails to make me laugh and keep me entertained. Finally, my best fluffy pal Riley has kept me company with his cuddles throughout the write up of this thesis and he often brightened up the end of a long day by running to the door to welcome me home. ii Author‘s Declaration ―I declare that, except where explicit reference is made to the contribution of others, that this thesis is the result of my own work and has not been submitted for any other degree at the University of Glasgow or any other institution‖ Signature ________________________ Printed name ________________________ iii Table of Contents 1. Introduction to Benzodiazepines and Designer Benzodiazepines ........................ 1 1.1. Introduction ....................................................................................................... 1 1.2. History of benzodiazepines ............................................................................... 2 1.2.1. The emergence of designer benzodiazepines ............................................... 3 1.3. Prescribing benzodiazepines ............................................................................ 6 1.4. Abuse of benzodiazepines ................................................................................ 7 1.5. Structure .......................................................................................................... 10 1.5.1. Designer benzodiazepines structure............................................................ 11 1.6. Mechanism of action ....................................................................................... 14 1.7. Metabolism ...................................................................................................... 15 1.7.1. Designer benzodiazepines metabolism ....................................................... 17 1.8. Legislation ....................................................................................................... 20 1.9. Benzodiazepines in drug related deaths ......................................................... 21 1.9.1. Designer benzodiazepines role in drug related deaths ................................ 24 1.9.2. Designer benzodiazepine prevalence .......................................................... 25 1.10. Analytical testing of benzodiazepines .......................................................... 29 1.10.1. Electrospray ionisation ................................................................................. 33 1.10.2. (Atmospheric Pressure) Chemical ionisation ............................................... 34 1.10.3. Mass analysers ............................................................................................ 35 1.11. Aims and objectives ..................................................................................... 37 2. ELISA cross-reactivity determination of phenazepam, etizolam, diclazepam, delorazepam, pyrazolam and flubromazepam ........................................................... 38 2.1. Introduction ..................................................................................................... 38 2.1.1. Immunoassay .............................................................................................. 38 2.1.2. Immunoassays in forensic toxicology .......................................................... 38 iv 2.2. Aim .................................................................................................................. 43 2.3. Materials and Methods .................................................................................... 43 2.3.1. Materials ...................................................................................................... 43 2.4. Solution preparation ........................................................................................ 44 2.4.1. Stock solutions ............................................................................................ 44 2.4.2. Working solutions ........................................................................................ 44 2.4.3. Calibrator and spiked sample preparation ................................................... 44 2.4.4. Instrumentation ............................................................................................ 45 2.4.5. Immunoassay procedure ............................................................................. 45 2.4.6. Cross-reactivity determination ..................................................................... 46 2.4.7. Data handling and statistical analysis .......................................................... 46 2.5. Results and discussion.................................................................................... 47 2.5.1. Limitations ................................................................................................... 50 2.5.2. Case study ................................................................................................... 51 2.6. Conclusion ...................................................................................................... 51 3. Validation of a LC-MS/MS method for the quantitation of diclazepam, delorazepam, lormetazepam, flubromazepam and pyrazolam in blood ..................... 53 3.1. Introduction ..................................................................................................... 53 3.2. Aim .................................................................................................................. 54 3.3. Materials and Methods .................................................................................... 54 3.3.1. Materials ...................................................................................................... 54 3.3.2. Reagent preparation .................................................................................... 55 3.3.3. Calibrator, QC and Internal Standard Preparation ....................................... 57 3.3.4. LC-MS/MS Instrumentation and parameters ............................................... 58 3.3.5. Sample extraction. ....................................................................................... 59 3.3.6. Data handling and statistical analysis .......................................................... 60 v 3.4. Method validation experimental ....................................................................... 61 3.4.1. Selectivity .................................................................................................... 61 3.4.2. Specificity .................................................................................................... 61 3.4.3. Linearity ....................................................................................................... 62 3.4.4. Limit of detection ......................................................................................... 63 3.4.5. Precision and bias ......................................................................................
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