Alfazil, Abdulkareem Abdulwahab (2009) Stability of drugs and pesticides of forensic toxicological interest and their metabolites in biological samples. PhD thesis http://theses.gla.ac.uk/1309/ Copyright and moral rights for this thesis are retained by the author A copy can be downloaded for personal non-commercial research or study, without prior permission or charge This thesis 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. Glasgow Theses Service http://theses.gla.ac.uk/ [email protected] STABILITY OF DRUGS AND PESTICIDES OF FORENSIC TOXICOLOGICAL INTEREST AND THEIR METABOLITES IN BIOLOGICAL SAMPLES Thesis submitted in Accordance with Requirements of the University of Glasgow for the Degree of Doctor of Philosophy by Abdulkareem Abdulwahab Alfazil Department of Forensic Medicine and Science May 2009 © Abdulkareem A. Alfazil ii Acknowledgement Initially I would like to thank Almighty God for helping and guiding me throughout my life and specifically for accomplishing this thesis. A special thanks to Dr Robert Anderson for his support and guidance throughout this research. Many thanks to Dr Hazel Torrance and Dr Fiona Wiely for their help and advice during my study. I would also like to thank all of staff and students in department of forensic medicine and science for the good study environment they provided. A massive thanks to my parents, my brothers and my sisters for their encouragement and support. Finally I would like to thank my government, particularly ministry of interior for supporting me financially and sponsorship my PhD study. iii Table of Contents Acknowledgement......................................................................... ii List of Tables ............................................................................. viii List of Figures .............................................................................xii Summary .................................................................................. xiv Abbreviations............................................................................ xvii 1 Introduction ........................................................................... 1 1.1.1 Toxicology................................................................... 1 1.1.2. Forensic toxicology: an introduction.......................................... 2 1.1.2.1. Post-mortem forensic toxicology............................................ 2 1.1.2.2. Human performance toxicology ............................................. 3 1.1.2.3. Forensic drug testing ......................................................... 4 1.2 Drugs and poisons – trends ..................................................... 4 1.3 Pharmacology .................................................................... 7 1.3.1 Pharmacodynamics......................................................... 7 1.3.2 Pharmacokinetics .......................................................... 8 1.4. Samples used.................................................................... 12 1.5 Forensic toxicology problems .................................................... 14 1.6 Aims .............................................................................. 15 2 Stability of Drugs and Poisons: Literature Review............................16 2.1 Introduction ..................................................................... 16 2.2 Drugs.............................................................................. 17 2.3 Pesticides ........................................................................ 25 2.3.1 Organophosphates......................................................... 25 2.4 Stabilisation of samples........................................................ 26 2.4.1 Additives.................................................................... 26 2.4.2 Filter paper ................................................................ 28 2.4.3 Freezing .................................................................... 29 2.4.4 Acidification ............................................................... 29 2.4.5 Antioxidants................................................................ 30 3 General Experimental Procedures Used in this Project .....................46 3.1 Extraction........................................................................ 46 3.1.1 Solid phase extraction.................................................... 46 3.2 Derivatization ................................................................... 49 iv 3.2.1 Silylation.................................................................... 49 3.3 Gas Chromatography/Mass Spectrometry (GC/MS) ........................ 51 3.4 Liquid chromatography - tandem mass spectrometry (LC/MS/MS)...... 53 3.5 Method validation .............................................................. 55 3.5.1 Linearity .................................................................... 55 3.5.2 Limit of Detection and Lower Limit of Quantitation................. 55 3.5.3 Recovery.................................................................... 56 3.5.4 Precision.................................................................... 56 3.6 Statistical evaluation of stability of substances during storage ......... 57 4 Stability of Benzodiazepines in Whole Blood under Different Storage Conditions..................................................................................58 4.1 Introduction ..................................................................... 58 4.2 Benzodiazepines included in the present study............................ 59 4.3 Aims .............................................................................. 60 4.4 Experimental Section .......................................................... 61 4.4.1 Chemical and material: .................................................. 61 4.4.2 Solutions:................................................................... 61 4.4.3 Preparation of blood samples: .......................................... 62 4.4.4 Sample storage and time for analysis .................................. 63 4.4.5 Analytical method......................................................... 64 4.5 Results............................................................................ 68 4.5.1 Validation results.......................................................... 68 4.5.2 Stability results............................................................ 70 4.5.3 Summary of benzodiazepine stability in blood samples ............. 86 4.6 Discussion ........................................................................ 87 4.7 Conclusions ...................................................................... 90 5 Stability of Opiate Glucuronides in Blood and Urine .........................91 5.1 Introduction ..................................................................... 91 5.2 Aims .............................................................................. 95 5.3 Experimental Section .......................................................... 95 5.3.1 Chemical and material: .................................................. 95 5.3.2 Solutions:................................................................... 95 5.3.3 Preparation of blood and urine samples: .............................. 96 5.3.4 Sample storage and times of analysis .................................. 97 5.3.5 Analysis ..................................................................... 98 5.3.6 Instrumentation ........................................................... 98 v 5.4 Stability results ................................................................100 5.4.1 Stability of M3G in blood and urine....................................100 5.4.2 Stability of M6G in blood and urine....................................103 5.4.3 Stability of C6G in blood and urine ....................................106 5.4.4 Summary of M3G, M6G, and C6G stability in blood and urine samples.. .............................................................................109 5.5 Discussion .......................................................................110 5.6 Conclusion ......................................................................113 6 Stability of Cocaine and Benzodiazepines in Dried Blood Spots ......... 114 6.1 Introduction ....................................................................114 6.2 Experimental Section .........................................................118 6.2.1 Chemical and material: .................................................118 6.2.2 Solutions:..................................................................118 6.2.3 Preparation of DBS samples:............................................119 6.2.4 Extraction procedure ....................................................120 6.2.5 Instrumentation ..........................................................123 Internal standards .....................................................................124 6.3 Validation of analytical method .............................................125 6.4 Results...........................................................................125 6.4.1 Method validation results ...............................................125 6.4.2 Stability