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Sample preparation is rapidly improving to fulfill the need for faster and more environmentally friendly alternatives. In this respect, ionic liquid-based dispersive liquid-liquid microextraction (IL-DLLME) is an interesting technique. However, it has not yet been evaluated for the analysis of postmortem samples, which are frequently analyzed in forensic toxicology. This study investigates the applicability of IL-DLLME coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS), for the analysis of benzodiazepines in postmortem blood of 11 forensic cases. The method was compared with a validated solid-phase extraction (SPE) method. Bland-Altman analysis was performed on 24 benzodiazepine measurements. Both methods gave comparable results, except for flurazepam and temazepam (>55% difference). A feasible explanation is high postmortem matrix variability that was not considered during IL-DLLME validation experiments. Another issue could be the use of a single nondeuterated SPE internal standard. Overall, IL-DLLME has proven its usability for the analysis of postmortem blood.

The analysis of biological samples, such as whole blood, comes with several sample preparation challenges. Biological matrices often contain a variety of endogenous components that can interfere with the determination of xenobiotics. Especially blood plasma proteins (e.g. serum albumin) are known to interfere with electrospray ionization and result in analyte ion suppression. Sample preparation techniques should guarantee adequate removal of these biomolecules. The current study aims to determine to which extent proteins are removed from whole blood samples, using ionic liquid-based dispersive liquid-liquid microextraction (IL-DLLME). A qualitative comparison of the protein presence in extracts of IL-DLLME, solidphase extraction (SPE) and protein precipitation (PP) was performed, using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Additionally, UV/VIS spectrophotometry was used to determine the protein content of a whole blood sample and IL-DLLME, SPE and PP extracts of the same sample. Finally, a quantitative comparison of matrix effects of benzodiazepines present in both whole blood and water samples. SDS-PAGE results showed that IL-DLLME extracts still contained proteins (i.e. albumin, hemoglobin); however, band intensities were comparable to SPE extracts. Spectrophotometric tests showed a total protein content of approximately 2 mg/mL in the final extracts. PP showed the highest protein extraction rate (19 mg/mL). Quantitative ME results showed no significant differences (α = 0.05) between blood and water IL-DLLME extracts. Overall, this is the first study to conclude that IL-DLLME is able to sufficiently remove blood proteins from whole blood samples, in order to avoid significant ion suppression.