Applications of LC-MS/MS in forensic toxicology for the analysis of drugs and their metabolites

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