De Boeck Marieke, Missotten Sophie, Dehaen Wim, Tytgat Jan, Cuypers Eva
Department of Pharmaceutical and Pharmacological Sciences, Toxicology and Pharmacology, University of Leuven (KU Leuven), Campus Gasthuisberg, O&N II, P.O. Box 922, Herestraat 49, 3000 Leuven, Belgium.
Department of Chemistry, Molecular Design and Synthesis, University of Leuven (KU Leuven), Campus Arenberg, P.O. Box 2404, Celestijnenlaan 200F, 3001 Leuven, Belgium.
Forensic Sci Int. 2017 May;274:44-54. doi: 10.1016/j.forsciint.2016.12.026. Epub 2016 Dec 26.
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.
迄今为止，对复杂生物样品进行彻底净化仍是分析过程的重要组成部分。固相萃取（SPE）技术是众所周知的标准方法，然而，其主要缺点是操作繁琐且耗时。在这方面，分散液液微萃取（DLLME）似乎提供了更简单、更高效的萃取程序。此外，离子液体（ILs）——液态盐——正作为一种新的有前景的萃取溶剂出现，这得益于其不可燃的性质、可忽略不计的蒸气压以及易于调节的物理化学性质。在本研究中，我们研究了离子液体是否可作为萃取溶剂用于分散液液微萃取程序，以萃取全血样品中的多种苯二氮䓬类药物和苯二氮䓬类催眠药。使用优化的基于离子液体的30分钟分散液液微萃取程序萃取1.0mL全血，随后在定时多反应监测扫描模式下进行液相色谱 - 电喷雾电离（+） - 串联质谱（LC - ESI(+) - MS/MS）分析。该优化分析方法已成功验证可用于7 - 氨基氟硝西泮、阿普唑仑、溴西泮、氯巴占、氯硝西泮、氯噻西泮、地西泮、艾司唑仑、依替唑仑、氟西泮、氯美扎酮、咪达唑仑、奥沙西泮、普拉西泮、替马西泮、三唑仑、唑吡坦和佐匹克隆的检测。基于12份空白全血样本，该方法对内源性干扰显示出良好的选择性。除了氯氮䓬和去甲西泮（这两种物质被排除在定量方法之外），未观察到苯二氮䓬类干扰。构建了涵盖整个治疗范围（包括低毒血浆浓度）的基质匹配校准曲线。除了溴替唑仑、氯氮卓、氯恶唑仑、氟硝西泮、洛拉卓仑、劳拉西泮和硝西泮只能进行半定量测定外，绝大多数化合物的准确度和精密度结果均符合提议的验收标准。回收率在24.7% - 127.2%范围内，基质效应在20.0% - 92.6%范围内。这两个参数均使用5份全血样本进行测试，变异系数低于20%。总体而言，已证明离子液体作为全血样品中苯二氮䓬类药物萃取的有前景溶剂的适用性。此外，还开发了一种快速简便的基于离子液体的分散液液微萃取程序，用于定量测定19种苯二氮䓬类药物和苯二氮䓬类催眠药。
