Improved Forensic Hair Evidence for Drugs of Abuse by Mass Spectrometry

Improved Forensic Hair Evidence for Drugs of Abuse by Mass Spectrometry Wilco F. Duvivier Thesis committee Promotor Prof. Dr M.W.F. Nielen Professor of Analytical Chemistry, with special emphasis for the detection of chemical food contaminants Wageningen University Co-promotor Dr T.A. van Beek Assistant professor, Laboratory of Organic Chemistry Wageningen University Other members Prof. Dr H.A. Schols, Wageningen University Prof. Dr R.M.A. Heeren, Maastricht University Prof. Dr A.C. van Asten, University of Amsterdam Dr F. Drijfhout, Keele University, Staffordshire, United Kingdom This research was conducted under the auspices of the Graduate School VLAG (Advanced studies in Food Technology, Agrobiotechnology, Nutrition and Health Sciences). Improved Forensic Hair Evidence for Drugs of Abuse by Mass Spectrometry Wilco F. Duvivier Thesis submitted in fulfilment of the requirements for the degree of doctor at Wageningen University by the authority of the Rector Magnificus Prof. Dr A.P.J. Mol, in the presence of the Thesis Committee appointed by the Academic Board to be defended in public on Tuesday 5 July 2016 at 4 p.m. in the Aula. Wilco F. Duvivier Improved Forensic Hair Evidence for Drugs of Abuse by Mass Spectrometry 194 pages. PhD thesis, Wageningen University, Wageningen, NL (2016) With references, with summaries in English and Dutch ISBN 978-94-6257-815-9 DOI 10.18174/381180 Table of contents List of abbreviations 7 Chapter 1: General Introduction 9 Chapter 2: Evidence Based Decontamination Protocols for the Removal of 33 External Δ9-Tetrahydrocannabinol from Contaminated Hair Chapter 3: Rapid Analysis of Δ 9-Tetrahydrocannabinol in Hair using Direct 63 Analysis in Real Time Ambient Ionization Orbitrap Mass Spectrometry Chapter 4: (Un)targeted Scanning of Locks of Hair for Drugs of Abuse by 83 Direct Analysis in Real Time − High-Resolution Mass Spectrometry Chapter 5: Critical Comparison of Mass Analyzers for Forensic Hair 115 Analysis by Ambient Ionization Mass Spectrometry Chapter 6: Ultratrace LC–MS/MS Analysis of Segmented Calf Hair for 139 Retrospective Assessment of Time of Clenbuterol Administration in Agriforensics Chapter 7: General Discussion and Future Perspectives 157 Summary 175 Samenvatting 179 Acknowledgments 183 Curriculum Vitae 187 List of Publications 189 Overview of Completed Training Activities 191 List of abbreviations APCI – atmospheric pressure chemical ionization APPI – atmospheric pressure photoionization ASE – accelerated solvent extraction BZE – benzoylecgonine CBD – cannabidiol CBN – cannabinol CE – capillary electrophoresis CID – collision-induced dissociation DAPCI – desorption atmospheric pressure chemical ionization DART – direct analysis in real time DCBI – desorption corona beam ionization DESI – desorption electrospray ionization EIC – extracted ion chronogram FWHM – full width at half maximum GC – gas chromatography GHB – gamma -hydroxybutyric acid HCD – higher–energy collisional dissociation HRMS – high resolution mass spectrometry LAESI – laser ablation electrospray ionization LC – liquid chromatography LLE – liquid-liquid extraction LOD – limit of detection LTP – low-temperature plasma MALDI – matrix-assisted laser desorption/ionization MDMA – 3,4-methylenedioxymethamphetamine 7 MetA–SIMS – metal assisted-secondary ion mass spectrometry MMS – matrix-matched standards MRM – multiple reaction monitoring MS – mass spectrometry MS/MS – tandem mass spectrometry NCE – normalized collision energy PADI – plasma assisted desorption ionization PMMA – p-methoxymethylamphetamine RSD – relative standard deviation SDS – sodium dodecyl sulfate SEM – scanning electron microscopy SFE – supercritical fluid extraction SoHT – Society of Hair Testing SPE – solid phase extraction SRM – selected reaction monitoring THCA–A – Δ9-tetrahydrocannabinolic acid THC – Δ9-tetrahydrocannabinol THC–COOH – 11-nor-9-carboxy-Δ9-tetrahydrocannabinol TIC – total ion current TLC – thin layer chromatography TOF – time-of-flight TWIM – travelling wave ion mobility U(H)PLC – ultra(high)-performance liquid chromatography 8 Chapter 1 General Introduction 9 Introduction Analytical chemistry plays an important role in forensic toxicology. Blood, urine and other body fluids are very useful sources of toxicological information on both intentional and unintentional abuse of substances. However, when a longer time frame of drug use is of interest, e.g., in drug abuse cases, the detection window provided by body fluids can be insufficient. Cocaine, for example, can be detected in body fluids for only several days after use. [1] As an alternative to body fluids, hair can provide such an extended detection window, allowing retrospective monitoring of drug use for up to several months to even years before sample collection. Over the past decades, hair analysis has advanced from early usage like the detection of arsenic in Napoleon’s hair [2] towards a valuable tool with applications ranging from obtaining retrospective timelines of drug administration in drug-facilitated crime evidence to doping monitoring. Other examples can be found in workplace drug testing, investigating exposure of children to drugs of abuse in child protection cases, and in veterinary control. [3-9] Forensic hair evidence and analysis Hair is an interesting matrix in forensics not only because of the prolonged detection window compared to body fluids, but also because sampling is non-invasive and hair samples can be easily stored. [10] Furthermore, hair can provide a chronological timeline of exposure to substances by the analysis of longitudinal hair segments. Hair anatomy and physiology Hair is a complex structure, consisting of several layers of different material. The hair shaft consists of overlapping cuticle cells covering the core of the hair containing the cortex and the central medulla (Figure 1A). The cuticle protects the inner regions of the hair, but can be damaged by outside sources like mechanical friction, heat or chemicals. Most of the hair is constituted by the cortex composed of long spindle-shaped, keratinized cells, which also contain pigment granules responsible for the color of hair with melanin as the most common type of pigment molecules. [4,11,12] The core of the cortex contains condensed cells, which after dehydration leave vacuoles forming the central medulla. Thick animal hair generally has a bigger medulla, while the medulla only compromises a small percentage of, thinner, human hair. [11] Hair growth is induced from the follicle, a bulb-like skin organ surrounded by a capillary system that provides metabolic material. [13] The outer root sheath, a part of the epidermis, surrounds the inner root sheath region of the follicle. Division of cells at the bottom of the 10 Chapter 1 follicle bulb, just above the papilla, induces an upward movement of vertically aligned cells to the keratogenous zone where they synthesize melanin, keratinize and, after gradually dying, form a horn-like mass (Figure 1B). [11-14] Sulphur-rich keratin proteins formed during keratinization bind together through disulfide bonds and cross-linking with other proteins resulting in long fibers. [14] Figure 1. A) Structure and constituents of the human hair shaft. Reprinted from [13] , with permission from Elsevier. B) Formation of hair in a follicle from matrix cells on the basement membrane to the mature hair shaft. Drug incorporation from blood should occur in a 1.2 – 1.5 mm zone before completion of keratinization. Reprinted from [13] , with permission from Elsevier. C) Different phases of the hair growth cycle. Reprinted from [12] , with permission from John Wiley and Sons. The growth cycle of hair consists of three different stages: the anagen (growth), catagen (regression) and telogen (resting) phase (Figure 1C). Scalp hairs grow for 2-6 years during the anagen phase, in which rapid division of cells takes place in the follicle causing growth of the hair. [14] In the following regression phase, cell division stops causing the hair shaft to become fully keratinized and the follicle to become shorter. [11,14] During the 2-3 weeks in the catagen phase, hair starts degenerating and the club hair, a brush-like keratinized structure, is formed at the base of the hair shaft. In the following telogen phase, hair stops growing completely and only the root of the hair is anchored in the follicle. After the hair falls out, a new hair in the anagen phase starts to grow. [11,12,14] The different growth phases occur simultaneously in different hairs, i.e., not all hairs grow synchronous. 80-90% of the scalps hairs are in the anagen phase, 2% in the catagen phase and the remaining 18-20% in the telogen phase. [11,14] Although different hair growth rates have been reported, generally ranging from 0.6 to 1.5 cm/month, [7] the use of a hair growth rate of 1.0 cm/month is recommended by the Society of Hair Testing (SoHT). [15] 11 Introduction Drug incorporation On a molecular level, hair consist of 65-95% protein, 15-35% water and 1-9% lipids, mainly originating from sebum and secretions of apocrine glands, and 0.25-0.95% minerals. Next to this, hair may contain compounds, such as the drugs of abuse shown in Table 1, which are incorporated into the hair matrix via different routes. Although the incorporation of compounds is not fully understood, the main incorporation model is considered to be via passive diffusion from the bloodstream into the growing hair (Figure 2A). [13] Administered drugs travel through the body after absorption into the bloodstream and reach the capillary

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