DEVELOPMENT OF ANALYTICAL PROCEDURES FOR SCREENING AND QUANTIFICATION OF PSYCHOACTIVE SUBSTANCES IN DIFFERENT BIOLOGICAL MATRICES AND THEIR APPLICATION IN FORENSIC TOXICOLOGY

The forensic toxicology field encompasses several applications, including post mortem and human performance testing, which may have administrative and/or medicolegal consequences and so require accurate and reliable toxicological analysis. Liquid chromatography tandem mass spectrometry (LC-MS/MS) techniques became increasingly important for identification and quantification purposes. LC-MS/MS methods are target screening analysis for molecules with similar pharmacological activity or chemical structure, including thermolabile and non-volatile compounds, achieving high sensibility and specificity. This thesis project focused on the development and validation of LC-MS/MS methods for the detection of psychoactive substances in post mortem investigation and for the quantification of drugs of abuse, especially cannabinoids, in living subjects. Aims of this thesis were: a) the chromatographic and mass spectrometric characterization of the molecules of interest; b) the optimization of the extraction procedures from different biological matrices; c) the full validation of the LC-MS/MS methods developed d) the application to real cases and e) the interpretation of the results.The results obtained proved that LC-MS/MS technique performs reliable and sensitive analysis when used for screening of group of compounds with similar chemical structure or pharmacological activity. During this thesis project, we focused on antidepressants, antipsychotics and benzodiazepines analysis for post mortem investigations. LC-MS/MS target screening methods were developed to identify and quantitate 88 psychoactive substances and metabolites in post mortem blood or hair; both LC-MS/MS methods were fully validated accordingly to international guidelines. The LC-MS/MS analysis, applied to post mortem blood samples, proved to be an accurate and reliable confirmatory method compared to the GC-MS analysis routinely performed and to generally achieve higher sensibility and specificity. This target screening enables the direct identification and quantification of thermolabile and non-volatile compounds as demonstrated in a case of fatal intoxication by duloxetine. An LC-MS/MS method for hair testing was developed and applied to samples form autopsy cases or patients under treatment; the LC-MS/MS method demonstrated to be reliable and accurate in the detection of the psychoactive substances considered, even at very low concentrations. Furthermore, the possibility to perform segmental analysis of hair samples, combined to the high sensibility of the LC-MS/MS technique enable to obtain reliable chronological insight in the medical history of a subject as happened for one of the patient under treatment. The LC-MS/MS analysis fully confirmed the reported drug administration scheme and demonstrated not only the change in therapy but even the suspension period occurred in between.The second part of this project focused on the detection of cannabinoids in urine, which has several forensic applications such as antidoping control, driving under the influence of drugs or workplace drug testing. A LC-MS/MS method for screening and quantification of cannabinoids and metabolites in hydrolyzed urine was fully developed and validated; the screening procedure included molecules not routinely detected in urine, such as cannabigerol, ∆9-tetrahydrocannabivarin and its metabolite 11-nor-9-carboxy-∆9-tetrahydrocannabivarin. Enzymatic hydrolysis with Recombinant E. coli β-glucuronidase was optimized using authentic pooled urine samples collected during a controlled cannabis smoking clinical trial. The extraction procedure was developed using DPX tips and an automated liquid-handling system to reduce processing time and increase reproducibility and throughput of sample preparation. The LC-MS/MS method was fully validated accordingly to SWGTOX guidelines. This method will be applied to search for new urinary markers of recent cannabis intake or chronic frequent smoking habits.