Marieke De Boeck

Development and validation of a fast ionic liquid-based dispersive liquid–liquid microextraction procedure combined with LC–MS/MS analysis for the quantification of benzodiazepines and benzodiazepine-like hypnotics in whole blood

To date, thorough clean-up of complex biological samples remains an essential part of the analytical process. The solid phase extraction (SPE) technique is the well-known standard, however, its main weaknesses are the labor-intensive and time-consuming protocols. In this respect, dispersive liquid-liquid microextractions (DLLME) seem to offer less complex and more efficient extraction procedures. Furthermore, ionic liquids (ILs) - liquid salts - are emerging as new promising extraction solvents, thanks to their non-flammable nature, negligible vapor pressure and easily adaptable physiochemical properties. In this study, we investigated whether ILs can be used as an extraction solvent in a DLLME procedure for the extraction of a broad range of benzodiazepines and benzodiazepine-like hypnotics in whole blood samples. 1.0mL whole blood was extracted using an optimized 30-min IL-based DLLME procedure, followed by LC-ESI(+)-MS/MS analysis in scheduled MRM scan mode. The optimized analytical method was successfully validated for 7-aminoflunitrazepam, alprazolam, bromazepam, clobazam, clonazepam, clotiazepam, diazepam, estazolam, ethyl loflazepate, etizolam, flurazepam, lormetazepam, midazolam, oxazepam, prazepam, temazepam, triazolam, zolpidem and zopiclone. The method showed good selectivity for endogenous interferences based on 12 sources of blank whole blood. No benzodiazepine interferences were observed, except for clorazepate and nordiazepam, which were excluded from the quantitative method. Matrix-matched calibration curves were constructed covering the whole therapeutic range, including low toxic plasma concentrations. Accuracy and precision results met the proposed acceptance criteria for the vast majority of compounds, except for brotizolam, chlordiazepoxide, cloxazolam, flunitrazepam, loprazolam, lorazepam and nitrazepam, which can only be determined in a semi-quantitative way. Recoveries were within the range of 24.7%-127.2% and matrix effects were within 20.0%-92.6%. Both parameters were tested using 5 sources of whole blood and coefficients of variance were below 20%. Overall, the applicability of ILs as promising solvents for the extraction of benzodiazepines in whole blood samples has been proven. Moreover, a fast and easy IL-based DLLME procedure was developed for the quantification of 19 benzodiazepines and benzodiazepine-like hypnotics.

Fast and easy extraction of antidepressants from whole blood using ionic liquids as extraction solvent

This study aims to prove that ionic liquids (ILs) can be used as extraction solvents in a liquid-liquid microextraction, coupled to LC-MS/MS, for the quantification of a large group of antidepressants in whole blood samples. The sample preparation procedure consisted of adding 1.0mL aqueous buffer pH 3.0 and 60µL of IL (1-butyl-3-methylimidazolium hexafluorophosphate) to 1.0mL whole blood. Subsequently, a 5-min rotary mixing step was performed followed by centrifugation. The lower IL phase was collected, diluted 1:10 in methanol and 10µL was injected into the LC-MS/MS. The following analytes were included in the full-quantitative method: agomelatine, amitriptyline, bupropion, clomipramine, dosulepin, doxepin, duloxetine, escitalopram, fluoxetine, imipramine, maprotiline, mianserin, mirtazapine, nortriptyline, paroxetine, reboxetine, trazodone and venlafaxine. Selectivity was checked for 10 different whole blood matrices. Additionally, possible interferences of deuterated standards or other antidepressants were evaluated. Overall, no interferences were found. For each analyte a matrix-matched calibration curve was constructed (7 levels, n = 6), covering therapeutic and low toxic concentrations. Accuracy and precision were evaluated over eight days, at three concentration levels (n = 2). Bias, repeatability and intermediate precision results met with the proposed validation criteria, except for fluvoxamine, which was therefore only included in the semi-quantitative method. LOQs were set at the lowest calibrator concentration and LOD values were - for most analytes - within a range of 1-2ng/mL. Recoveries (RE) and matrix effects (ME) were evaluated for five types of donor whole blood, at two concentration levels. RE values were within a range of 53.11-132.98%. ME values were within a range of 61.92-123.24%. In conclusion, this study proves the applicability of ILs as extraction solvents for a large group of antidepressants in complex whole blood matrices.

Ionic Liquid-Based Liquid-Liquid Microextraction for Benzodiazepine Analysis in Postmortem Blood Samples

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.

Evaluation of the suitability of ionic liquid-based liquid-liquid microextractions for blood protein removal

HighlightsProtein comparison of blood extracts using electrophoresis and spectrophotometry.Ionic liquid‐based microextractions are comparable to solid‐phase extractions.Protein precipitation shows a 10‐fold lower protein removal capacity.No significant impact of the whole blood matrix on benzodiazepine matrix effects. Abstract 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, solid‐phase 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 (&agr; = 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.

Evaluation of 11 ionic liquids as potential extraction solvents for benzodiazepines from whole blood using liquid-liquid microextraction combined with LC-MS/MS

Ionic liquids (ILs) are becoming a trending topic in sample preparation technology, due to their low volatility, low flammability and tunable physiochemical properties. The latter affords the opportunity for creating task-specific solvents. In toxicology, these tailored ILs were not introduced yet; however, they can be significant for drug extraction purposes. This study screens 11 commercially available ILs as potential extraction media for benzodiazepines from blood samples via liquid-liquid microextraction, coupled to liquid chromatography - tandem mass spectrometry (LC-MS/MS). Structure-extraction relationships were deducted from recovery and matrix effect results (n = 5), allowing us to devise some of the favorable features in a tailored IL for benzodiazepine extraction. Overall, long alkyl chains are to be avoided, as they may sterically hinder desirable π-stacking interactions. For the same reason, aromatic planar cations, as imidazolium and pyridinium, were more effective in comparison with ammonium and pyrrolidinium. Furthermore, viscosity negatively impacted both recoveries and matrix effects. High viscosities limited mass transfer during dispersion and impeded electrospray ionization processes. In conclusion, this study evaluates which physicochemical features an IL should possess to efficiently extract benzodiazepines from whole blood.