Routine forensic analysis of psychoactive substances

Subtitle:in-depth assessment of instrumentation, confirmation and quantification

Toxicological analysis requires broad screening for both therapeutically prescribed and illicit compounds, including their metabolites. This research sought to develop reliable, fast and easy-to-use analytical methods, focussing on three types of psychoactive substances: antidepressants, antipsychotics and benzodiazepines and Z-drugs. They are increasingly prescribed for long-term use, frequently encountered in routine forensic samples and often require close monitoring due to their potential for serious adverse events. The first objective was to develop targeted confirmation methods using triple quadrupole mass spectrometry (QQQ). A liquid-liquid extraction on 200 μL sample yielded sufficient sensitivity for the intended subtherapeutic detection limits. The methods were fully validated according to international guidelines and tested on internal and external quality control samples. These methods allow for unequivocal identification and accurate concentration determination of the substances under investigation in blood. The second objective was to develop screening methods: one using QQQ operated in triggered multiple reaction monitoring mode (tMRM) and another using untargeted, quadrupole time-of-flight mass spectrometry (QTOF). The performance of both methods regarding correct identifications was comparable and within acceptable error margins. The limited number of false negative results predominantly consisted of compounds present at low to sub-ng/mL concentrations. More false positive results were obtained for the QTOF versus the tMRM method, the majority being misidentifications of phenelzine and prothipendyl. Semi-quantitation was feasible for tMRM analysis only, where it resulted in sufficiently accurate concentration determinations at a reduced cost compared to routine methods. The third objective involved the critical assessment of method implementation in routine analyses. The semi-quantitative screening methods performed well and – following continued validation on case samples – could be implemented in routine toxicological analysis, without the need for highly trained, specialised personnel. QTOF screening is recommended over targeted tMRM screening, as it allows for easy expansion of the number of analytes included and for retrospective data analysis, should new information become available. Positive QTOF findings could be verified and semi-quantified by tMRM, without the need for additional sample preparation or further use of, often limited, sample volume. The high accuracy of the semi-quantitative tMRM results may often suffice for interpretation of case findings, particularly given the limitations associated with post-mortem samples. Confirmation of findings and accurate concentration determination may be required depending on the case, for which purpose fully validated, targeted QQQ methods have been developed.

Publication year:2022

Keywords:Doctoral thesis

Accessibility:Open