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Download Thesis This electronic thesis or dissertation has been downloaded from the King’s Research Portal at https://kclpure.kcl.ac.uk/portal/ The Role of Liquid Chromatography-High Resolution Mass Spectrometry in the Diagnosis and Treatment of Substance Misuse Murch, Sarah Louise Awarding institution: King's College London The copyright of this thesis rests with the author and no quotation from it or information derived from it may be published without proper acknowledgement. END USER LICENCE AGREEMENT Unless another licence is stated on the immediately following page this work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International licence. https://creativecommons.org/licenses/by-nc-nd/4.0/ You are free to copy, distribute and transmit the work Under the following conditions: Attribution: You must attribute the work in the manner specified by the author (but not in any way that suggests that they endorse you or your use of the work). Non Commercial: You may not use this work for commercial purposes. No Derivative Works - You may not alter, transform, or build upon this work. Any of these conditions can be waived if you receive permission from the author. Your fair dealings and other rights are in no way affected by the above. Take down policy If you believe that this document breaches copyright please contact [email protected] providing details, and we will remove access to the work immediately and investigate your claim. Download date: 25. Sep. 2021 THE ROLE OF LIQUID CHROMATOGRAPHY- HIGH RESOLUTION MASS SPECTROMETRY IN THE DIAGNOSIS AND TREATMENT OF SUBSTANCE MISUSE Sarah Louise Murch (nee Belsey) Institute of Pharmaceutical Science King’s College London A thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy Abstract Substance misuse remains problematic with current concerns being the rise in acute poisoning deaths, particularly opioid-associated, and the ever-widening range of drugs available. Strategies for tackling opioid addiction and opioid related-deaths include researching alternative routes of therapeutic agent administration. Initial urine screening for substance misuse has traditionally employed immunoassays, with confirmation of specific analytes by chromatographic methods. Liquid chromatography-high resolution mass spectrometry (LC-HRMS) offers untargeted analysis without compromising selectivity, and enables users to ascertain putative elemental compositions of an analyte, retrospectively interrogate data, and to incorporate novel analytes easily. These features enable screening and confirmation of drugs in a single method, and may be advantageous for detecting novel psychoactive substances (NPS). This thesis aims to investigate the role of LC-HRMS in drug analysis in the clinical setting. A simple system was developed that is capable of detecting a wide range of commonly-encountered drugs and metabolites. Non-selective sample preparation was used to enable detection of as many compounds as possible, but significant matrix effects were observed. Additional information regarding selected NPS was ascertained through retrospective identification of mephedrone metabolites in patient urines, and through later incorporation of ethylphenidate, methylphenidate, and ritalinic acid, into the method. A separate quantitative LC-HRMS method was developed to facilitate pharmacokinetic studies of naloxone and naltrexone administered through alternative routes. The method was also applied to urine samples, with naloxone-3-glucuronide identified as a potential marker to differentiate between Subutex and Suboxone use. LC-HRMS has advantages in drug detection, particularly in regard to NPS, and in method development. However, application in the clinical setting is restricted by requirements for high throughput, timely results, and operation to accepted ‘cutoff’ values that introduce awkward compromises in system operation. LC-HRMS may have greater application in the forensic setting where more time is available for the analysis of a single sample. ii Table of Contents 1 INTRODUCTION ........................................................................................... 1 1.1 Substance Misuse .............................................................................................. 2 1.1.1 Legal Classification of Drugs ............................................................................ 2 1.1.2 Prevalence of Substance Misuse ........................................................................ 4 1.1.3 Opioid Receptors and Pharmacology ................................................................. 7 1.1.4 The Changing Drug Scene ............................................................................... 12 1.2 The Role of the Clinical Toxicology Laboratory in Drug Testing ..................... 20 1.2.1 Poisoning ........................................................................................................ 20 1.2.2 Identification of Unknown Substances ............................................................. 20 1.2.3 Treatment of Drug Addiction ........................................................................... 21 1.2.4 Analytical Methods ......................................................................................... 24 1.3 Aims and Objectives of This Work .................................................................. 32 2 QUALITATIVE DRUG ANALYSIS BY LC-HRMS: APPLICATION TO ROUTINE CLINICAL SCREENING FOR DRUGS IN URINE SAMPLES ...................................................................................................... 33 2.1 Introduction ..................................................................................................... 34 2.1.1 Biological Matrices ......................................................................................... 35 2.1.2 Qualitative Measurement of Drugs .................................................................. 36 2.1.3 Sample Preparation .......................................................................................... 37 2.1.4 Analytes in a Routine Drug Screen .................................................................. 40 2.1.5 Aims ............................................................................................................... 41 2.1.6 Ethics Considerations ...................................................................................... 41 2.2 Materials and Methods .................................................................................... 42 2.2.1 Chemicals and Reagents .................................................................................. 42 2.2.2 Instrumentation ............................................................................................... 42 2.2.3 Liquid Chromatography ................................................................................... 43 2.2.4 Mass Spectrometry .......................................................................................... 43 2.2.5 Assay Calibration and Acceptance Criteria ...................................................... 43 iii 2.2.6 Preparation of Calibration and Internal Quality Control Solutions.................... 44 2.2.7 Internal Standard Solution ............................................................................... 46 2.2.8 Immunoassay Procedures ................................................................................ 46 2.2.9 Sample Preparation .......................................................................................... 46 2.2.10 Method Validation Protocol ............................................................................ 46 2.2.11 Patient Samples ............................................................................................... 47 2.3 Results and Discussion .................................................................................... 48 2.3.1 Analyte Selection ............................................................................................ 48 2.3.2 Liquid Chromatography Method Development ................................................ 49 2.3.3 Mass Spectrometry Method Development ....................................................... 52 2.3.4 Selection of ‘cutoff’ concentrations ................................................................. 60 2.3.5 Selection of Internal Standards ........................................................................ 63 2.4 Sample Preparation .......................................................................................... 65 2.5 Practical Considerations .................................................................................. 66 2.5.1 Benzodiazepines .............................................................................................. 66 2.5.2 Cannabis.......................................................................................................... 68 2.6 Method Validation ........................................................................................... 72 2.6.1 Isobaric Interferences ...................................................................................... 83 2.7 Patient Sample Comparison ............................................................................. 85 2.7.1 Immunoassay False-Positives .......................................................................... 85 2.7.2 Immunoassay False-Negatives ......................................................................... 87 2.7.3 Differences in Analyte
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