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The aim of this project was to evaluate the use of LC-MS in the forensic toxicology setting. To investigate if its use could solve problems encountered while using other instrumentation including downtime, limits of detection, chromatography and specificity. Benzodiazepines as a class, are known for their undesirable chromatographic behaviour when using GC. Using LC-MS two models were developed for the analysis of these drugs in whole blood. Increasing awareness of drug facilitated sexual assault, has led to an increase in the number of cases to be analysed for drugs such as Rohypnol®. This prompted the development and validation of a sensitive and specific method for its detection in blood using solid phase extraction and liquid chromatography – mass spectrometry. To enhance the qualitative data and cope with degraded samples, the use of LC-MS was used in the analysis of diazepam and its three metabolites. A method was developed and validated and is now used in the routine analysis of blood samples in the laboratory. The LC-MS proved invaluable in the analysis of sildenafil, Viagra®, in a post-mortem blood sample received in the laboratory. A single quadrupole mass spectrometer was used to develop and validate a method to determine if the dose was therapeutic or toxic. The use of oral fluid as an alternative specimen to blood or urine was investigated. This was through the British Roadside Impairment Test Evaluation (BRITE) project. LC-MS in combination with GC-MS was used to screen for over 50 licit and illicit drugs in 1mL of oral fluid. LC-MS-MS was then used to identify and quantitated 21 drugs of abuse and their metabolites using a similar sample size. The use of one extraction and the reliability of LC-MS proved invaluable in these projects.

This thesis studied opioids and alcohol in forensic toxicology by LC-MS/MS, which avoids time-consuming procedures involving hydrolysis, extraction and derivatisation. Initially, a method was validated for quantification of opioids and unhydrolysed polar metabolites in autopsy specimens and was used to develop procedures for interpretation of forensic toxicology results. The LC-MS/MS method developed has been validated for the simultaneous determination of 24 opioids in human whole blood, including, for the first time in human whole blood, naloxone-3-glucuronide. Although a large number of drugs of interest were included in the method, acceptance criteria for linearity, precision, and recovery for all analytes were achieved. The method was found useful for differentiating between users of heroin and other opioids, such as codeine and morphine, and for determining the survival time in deaths attributed to heroin use. Subsequently, the efficiencies of hydrolytic and non-hydrolytic methods for opioid analysis were compared for buprenorphine (BUP) analysis. The aims were to develop and validate a method for the direct determination (DM) of buprenorphine (BUP), norbuprenorphine (NBUB), buprenorphine-3-glucuronide (B3G) and norbuprenorphine-3-glucuronide (NBUP3G). This method was compared with an in house enzymatic hydrolysis method (HM) for the determination of total buprenorphine (TBUP) and norbuprenorphine (TNBUP), using real positive BUP urine case samples. A comparison between the drug and metabolite concentrations obtained by direct and hydrolysis methods was reported for the first time in this work. LC-MS analysis was also applied to paediatric plasma specimens obtained from a clinical pharmacokinetic study of intravenous and intranasal administration of diamorphine. This work was aimed at obtaining pharmacokinetic data for diamorphine and its metabolites in children following intravenous (IVDIM) and intranasal (INDIM) administration in a blind study. It was intended that the concentrations of active metabolites would be used to evaluate whether or not IN-DIM can deliver rapid and efficient analgesia in children comparable to that obtained with IV-DIM. The pharmacokinetics of DIM and its metabolites following INDIM and IVDIM administration in children have been compared for the first time in this study, which confirmed that INDIM can achieve therapeutic plasma concentrations of active metabolites, although these were lower than those obtained with IVDIM and occur at later times after administration. In Scotland, the number of prescriptions for oxycodone has risen by 430% since prescribing began in 2002. Blood samples from fatalities in the West of Scotland involving oxycodone were analysed using an LC-ESI-MS/MS method developed for the determination of oxycodone and its metabolites in post-mortem specimens. To the author’s knowledge, this is the first report of blood and urine concentrations of noroxycodone and oxymorphone in acute oxycodone overdoses. Also, it is the first LC-MS/MS application to be reported with oxycodone related fatalities cases in forensic toxicology as most of previous reports used GC or HPLC applications. Moreover, this work reported for the first time vitreous humour levels of noroxycodone following oxycodone intoxication. Ten oxycodone-related deaths were identified in the short period of this study in the Strathclyde region of Scotland alone, highlighting the importance of including this drug in routine laboratory screening and confirmation procedures. Polar alcohol metabolites ethyl glucuronide and ethyl sulfate are biomarkers of ante-mortem alcohol consumption and are used to test for post-mortem artefactual formation of alcohol. An LC-MS method for these metabolites using a novel hydrophilic interaction liquid chromatography column was validated and applied to routine forensic casework. Ninety urine case samples were divided into three groups depending on the ethanol concentration found in blood and analysed by the developed method: group A with post-mortem blood ethanol higher than 200 mg/100 mL; group B with ethanol concentration in the range 80 to 200 mg/100 mL and group C with ethanol concentration less than 80 mg/100 mL. It was concluded that the risk of false positive ethanol results increased in the low ethanol concentration group as several cases tested negative for both biomarkers. ETG was detected at low concentrations in some cases for which ETS tested negative, suggesting that either ETG may have a longer half-life in urine or else ETS is unstable. The data was compared with previous studies and confirmed that both ethanol biomarkers should be determined in heavily putrefied cases and when the ethanol level in post-mortem blood is low, suggesting the production of ethanol after death. To the authors’ knowledge, this is the first report of the determination of ETS using an LC-ESI-ion trap-MS/MS method, and of a HILIC-ESI-ion trap-MS/MS method for the simultaneous determination of ETG and ETS in post-mortem urine samples.

This thesis examines the use of nail as an alternative biological specimen in forensic toxicology. Nail is a difficult analytical matrix from which to extract drugs because of its tough physical composition, based on keratin. The initial part of the project investigated the use of a cryogenic grinding method for fingernail clippings. Grinding at liquid nitrogen temperatures was found to be an effective procedure and the conditions were optimised to a two or three cycle programme of freezing and grinding. Small particle sizes were obtained of approximate size 1µm. It was established that drugs could be extracted directly from nail powder with a range of solvents without the need for alkaline hydrolysis. Methanol was found to be the most effective extraction solvent, which also gave the lowest number of co-extracted interfering compounds. This procedure was subsequently used with nail samples from different types of forensic cases, including cannabis, heroin and steroid abusers. Cryogenic grinding of nail was evaluated as an extraction method for cannabinoids in nail clippings from chronic cannabis smokers. This method was also compared to the alkaline hydrolysis method. Fingernail clippings were collected with prior informed consent from volunteers attending the Edinburgh Drug Addiction Study (EDAS) clinic. The collected nail clippings were decontaminated and divided into two groups: the first group was extracted with methanol after pulverisation in a liquid nitrogen cryogenic mill; the second was extracted with ethyl acetate after hydrolysis in sodium hydroxide. In both groups deuterated cannabinoids were used as internal standards. Both sets of extracts were derivatised with BSTFA before being analysed by gas chromatography – mass spectrometry (GC-MS). Cannabidiol, ?9-tetrahydrocannabinol and 11-hydroxy-?9-tetrahydrocannibinol were quantified in both sets of extracts. 11-nor-?9 –tetrahydrocannabinol-9-carboxylic acid was only identified and quantified in the extracts resulting from the cryogenic grinding method. Cannabinoid concentrations were very low, in the range 0-4 ng/mg. These results strongly support the use of nail as a biological specimen for the detection and quantification of past exposure to cannabis, and secondly, they indicate that grinding with a cryogenic mill is a useful procedure, which yields simultaneous results for the primary psychoactive cannabinoid and its metabolites. Cryogenic grinding was then evaluated for the extraction of opioids in nail in comparison with the conventional alkaline hydrolysis method. Finger and toe nails were collected from donors with informed consent.

The aim of this study was to investigate new methods of analysis which might be applied to forensic toxicology problems including those resulting from pesticides, particularly the quaternary ammonium herbicide group, and from drugs, particularly the benzodiazepine group. In the first part of this study, an efficient method for the determination of quaternary ammonium (QA) compounds (pesticides and drugs) in human whole blood was developed. The second part of this study concerned the development of a novel sorbents for solid phase extraction using molecularly imprinted polymers (MIPs). The approach adopted was initially to synthesise a known MIP using diazepam as template then to prepare novel MIPs using other benzodiazepines and analogues of QA compounds as templates. In the first of these stages, an anti-diazepam MIP was synthesized using methacrylate acid (MAA) as the monomer and ethylene glycol dimethacrylate (EGDMA) as the cross-liner and was then ground and prepared for use as an SPE sorbent by packing it into SPE cartridges. These cartridges were used to clean up extracts of diazepam and other benzodiazepine drugs made from hair samples via a molecularly imprinted solid-phase extraction (MISPE) protocol. The MISPE method was also found to be applicable to the analysis of diazepam metabolites and other benzodiazepine drugs in addition to diazepam itself. The application of the extraction method to post-mortem hair samples yielded results that were in good agreement with ELISA data (from blood samples) and data arising from the analysis of the same blood samples using a validated in-house SPE-LC-MS-MS method. The MISPE procedure was also compared with a conventional SPE method for analysis of benzodiazepines in hair samples. The results from MISPE protocol showed better selectivity, specificity and accuracy toward diazepam (template molecule) and other benzodiazepines that display a similar resemblance to diazepam in terms of molecular structure. The MISPE procedure was found to be simpler and to offer cleaner extracts compared to a conventional SPE method.