Ionic liquids as promising solvents for liquid-liquid microextractions in forensic toxicology

Volatile organic solvents (VOSs) are widely consumed in sample preparation, especially in bioanalysis, since clean-up of biological samples is indispensable to avoid quantification errors. With the increased awareness for green techniques, the search for safer and environmentally friendly solvents is trending. In this respect, ionic liquids (ILs) are proposed as promising alternatives for VOSs. Their primary attractive properties are their negligible volatility and low flammability, which limit the IL's environmental pollution and results in less hazardous solvents, compared to VOSs. Furthermore, the physicochemical properties of ILs can be easily tuned to obtain tailored solvents for a specific application; i.e. task-specific ILs (TSILs). This doctoral thesis focuses on introducing ILs in forensic toxicology. With emphasis on multi-analyte method development for frequently detected substances in drug screens, i.e. antidepressants (ADs) and benzodiazepines (BZDs). Moreover, the analysis of postmortem matrices and the contribution of undesired extracted proteins to matrix effects were evaluated. Finally, the possibility of creating a forensically relevant TSIL was investigated, with the aim of increasing extraction yields and improving our understanding of IL extraction behavior. In Chapter III, an IL-based dispersive liquid-liquid microextraction (IL-DLLME) method was optimized for the extraction of 17 BZDs and 2 BZD-like hypnotics. The following extraction parameters were assessed: volume and type of IL, pH adjustment of whole blood, dispersion method, extraction time and dilution factor of the final extract. The optimized 30-min IL-DLLME procedure was coupled to liquid chromatography - tandem mass spectrometry (LC-MS/MS) and validated according to internationally accepted guidelines. A repeatable method was obtained for the quantification of low therapeutic to low toxic concentrations in whole blood samples. Chapter IV, describes a cross comparison study of the validated IL-DLLME-LC-MS/MS method and an established solid-phase extraction (SPE)-LC-MS/MS method, for the quantification of BZDs in whole blood. Bland-Altman analysis was performed based on 11 forensic cases. Both methods showed similar results. However, three deviating results were observed for compounds included in the full-quantitative method. Plausible explanations are the absence of postmortem blood sources during IL-DLLME validation tests or the absence of SPE deuterated internal standards. In Chapter V, an IL-DLLME-LC-MS/MS method was developed for the determination of 18 ADs. Optimization and validation was performed in a similar way as described in Chapter III. The final method was able to quantify ADs at low therapeutic to low toxic concentrations in whole blood samples, in a repeatable and accurate manner. Chapter VI provides insight into the rational design of a forensically relevant TSIL, more specifically, for the extraction of BZDs and BZD-like hypnotics from whole blood. Eleven commercially available ILs were tested and structure-extraction relationships were considered, in order to define favorable IL structural features for BZD extraction. The importance of low IL viscosity, aromatic functionality, absence of arene substitution and a preference for short alkyl substituents was demonstrated. In previous chapters, high ion suppression was observed and could be directly linked to the presence of IL. However, undesirable protein extraction was also mentioned as a potential cause of ion suppression. Chapter VII, compares IL-DLLME, SPE and protein precipitation (PP) extracts based on protein presence, by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Moreover, matrix effects were compared for IL-DLLME extracts of BZDs from water and whole blood samples. No clear contribution of undesired protein to ion suppression was observed. In Chapter VIII, the applicability of ILs in drug screening applications was evaluated. A screening method should be able to detect a wide range of substances with varying polarities. This is a challenging requirement, since previous research showed that ILs co-elute with polar analytes on reversed phase columns, which results in severe analyte suppression. A preliminary evaluation of hydrophilic interaction liquid chromatography (HILIC) was performed for the separation of polar analytes and IL. In conclusion, this doctoral thesis provides a first evaluation of the applicability of ILs as extraction solvents in forensic toxicology. Two fast and simple multi-analyte IL-DLLME-LC-MS/MS methods were successfully validated for BZD and AD analysis. Moreover, its use for the analysis of postmortem blood was proven. No undesired protein extraction was observed and the first steps toward introducing ILs in drug screening have been taken. Future IL-based forensic research should consider the observed electrospray ionization suppression, due to IL presence. Furthermore, BZD-based TSIL design should be further optimized, based on the results obtained so far.status: publishe