An evaluation of the stability and prevalence of alcohol and related biomarkers in biological matrices with applications to the interpretation of medico-legal cases

Ethanol is a poison that adversely affects the health of individuals and is detrimental to society as a whole. Analysis of ethanol in biological matrices is the most frequent test carried out in forensic toxicology laboratories with application across a range of cases types including fatalities, road traffic accidents, criminal investigations and workplace drug testing. The interpretation of ethanol concentrations in post mortem samples can be challenging in relation to medico-legal investigations. The source of ethanol can be as a result of the ingestion of an alcoholic beverage or it may have been formed after death. The stability of alcohol is also adversely affected by the presence of bacteria, high temperatures and unsuitable storage containers. A robust and sensitive method was developed for the analysis of common volatiles such as ethanol, methanol, isopropanol, acetone and n-propanol using headspace gas chromatography coupled with a flame ionization detector (HS-GC-FID). The method was validated in accordance with ISO/IEC 17025, and was used to investigate the stability of volatiles in blood when stored under different conditions, and to investigate the prevalence of volatiles in different biological matrices collected post-mortem. Storage of blood samples in the freezer within sample vials containing preservative and antioxidant improved the stability of all volatiles with the exception of methanol which remained stable under all conditions investigated. The identification of ethanol or acetone in vitreous humour was found to be a suitable alternative matrix in cases where femoral blood was unavailable or ethanol production was suspected. The concentration of ethanol in bile correlated well with femoral blood but to a lesser extent than vitreous humour. Urine was not a suitable matrix for estimating blood ethanol concentrations. Alcohol biomarkers, Beta-Hydroxybutyrate (BHB) and fatty acid ethyl esters (FAEEs) have been reported as useful markers for, investigating the role of alcoholic ketoacidosis (AKA) in post-mortem cases, and foetal exposure to chronic maternal alcohol consumption, respectively. Methods were developed and validated in accordance with ISO/IEC 17025 for BHB in blood and urine using gas chromatography - mass spectrometry (GC/MS), and for FAEEs in meconium by liquid chromatography - tandem mass spectrometry (LC/MS/MS). Post-mortem case samples submitted to the Forensic Medicine and Science (FMS) toxicology laboratory for routine tests were analysed for BHB using the validated method. The findings highlighted the importance of measuring BHB in blood in all cases where the deceased has a history of alcohol misuse and where the cause of death cannot be determined following the post-mortem. The role of alcoholic ketoacidosis in the cases analysed in this study was significantly under-reported. Meconium samples collected from infants born at the Princess Royal Maternity Hospital, Glasgow, were analysed for FAEEs, using the validated method to investigate the prevalence of each FAEE (ethyl laurate, ethyl myristate, ethyl palmitate, ethyl stearate, ethyl oleate, ethyl linoleate, ethyl linolenate, and ethyl arachidonate). The study found evidence of chronic maternal alcohol consumption in approximately one third of the cases tested, in contrast to self-reported use and highlights the importance of screening for the presence of FAEEs in meconium. The identification of suitable biomarkers of excessive maternal alcohol consumption during pregnancy, carried out as part of screening program, in addition to clinical evaluation would help to diagnose and support newborns with Foetal Alcohol Spectrum Disorder (FASD). The method developed for the analysis of BHB in blood and urine was successfully adapted and validated for analysis of structurally similar drugs such as Beta-hydroxy-Beta-methylbutyrate (HMB), a legal dietary supplement, in plasma and urine collected from 8 subjects, pre- and post-administration of a 3g dose of HMB. A significant increase was observed in urine and plasma following administration of of HMB. The method was then applied to the analysis of post-mortem blood and urine to investigate the concentrations of exogenous and endogenous levels of γ-hydroxybutyrate (GHB).