Oslo University Hospital, Division of Laboratory Medicine, Department of Forensic Sciences, Norway.
Oslo University Hospital, Division of Laboratory Medicine, Department of Forensic Sciences, Norway.
J Chromatogr B Analyt Technol Biomed Life Sci. 2018 Sep 1;1093-1094:8-23. doi: 10.1016/j.jchromb.2018.06.050. Epub 2018 Jun 25.
A high-throughput UHPLC-MS/MS method for the most frequently found compounds; tetrahydrocannabinol (THC), amphetamine, methamphetamine, MDMA, clonazepam, diazepam, nordiazepam, oxazepam, alprazolam, nitrazepam, morphine, and codeine, in driving under the influence of drugs (DUID) cases in whole blood, is presented. Automated sample preparation by 96-well supported liquid extraction (SLE) plates with ethyl acetate + heptane (80 + 20, v/v) as organic solvent was carried out on a Freedom Evo 200 platform from Tecan. An aliquot of 100 μL whole blood was used. Sample preparation time for 96 samples was 1.5 h. Compounds were separated with gradient elution on a C column (50 × 2.1 mm, 1.7 μm) with a mobile phase consisting of 5 mM pH 10.2 ammonium formate and methanol. The run time was 4.5 min and 1 μL was injected on an Acquity UPLC I-Class system with a Xevo TQS tandem-quadrupole mass spectrometer in multiple-reaction monitoring mode (MRM) from Waters. Isotope labelled, C, internal standards (ISs) were used for all compounds except for alprazolam and morphine, which had deuterated analogs. Quantification was carried out with calibrators without whole blood matrix. Full validation was carried out according to international guidelines, and a new approach for evaluation of process efficiency (PE) has been presented. Linear or quadratic weighted (1/x) calibration curves were used with R ≥ 0.999. The method showed satisfactory deviations ±16% when compared to the existing methods, and satisfactory agreement with proficiency testing control samples (z-score -1.6 to 1.8, n = 16 samples). The precision, estimated as the relative standard deviation (RSD) of the concentration difference between results from two independent analyses of authentic whole blood samples, was ≤7.2% in antemortem and ≤9.3% in postmortem samples. Recovery was ≥85% for all the compounds, except morphine ≥62% and THC ≥ 50%. PE was satisfactory for all the compounds with low variation in IS response, RSD ≤ 16% (THC 27%) in antemortem samples and ≤34% (THC 66%) in postmortem samples. To the best of our knowledge, this is the first automated 96-well SLE UHPLC-MS/MS method developed for the simultaneous determination of these 12 compounds in whole blood covering the concentration ranges found in forensic samples. The method has been used in routine work during the last ten months, analysing about 9900 antemortem and 1000 postmortem whole blood samples, and has proven to be robust and reliable.
本文介绍了一种高通量 UHPLC-MS/MS 方法，用于检测全血中最常见的化合物，包括四氢大麻酚（THC）、苯丙胺、甲基苯丙胺、摇头丸、氯硝西泮、地西泮、去甲西泮、奥沙西泮、阿普唑仑、硝西泮、吗啡和可待因，这些化合物均与吸毒后驾车（DUID）有关。采用 Tecan 的 Freedom Evo 200 平台，通过 96 孔支持液体萃取（SLE）板用乙酸乙酯+庚烷（80+20，v/v）作为有机溶剂进行自动化样品制备。使用 100μL 全血作为分析样本。96 个样本的样品制备时间为 1.5 小时。采用 C 柱（50×2.1mm，1.7μm）进行梯度洗脱，流动相由 5mM pH10.2 甲酸铵和甲醇组成。运行时间为 4.5 分钟，在 Waters 的 Acquity UPLC I-Class 系统上以 1μL 注入，采用 Xevo TQS 串联四极杆质谱仪以多反应监测模式（MRM）进行分析。除了阿普唑仑和吗啡，所有化合物都使用了同位素标记、C 内标（ISs），而阿普唑仑和吗啡则使用了氘代类似物。定量分析采用不含有全血基质的校准器进行。根据国际指南进行了全面验证，并提出了一种新的过程效率（PE）评估方法。采用线性或二次加权（1/x）校准曲线，相关系数（R）≥0.999。与现有方法相比，该方法的偏差符合±16%的要求，与能力验证控制样品的一致性良好（z 值-1.6 至 1.8，n=16 个样本）。在法医样本中，该方法的精密度以两个独立分析真实全血样本浓度差的相对标准偏差（RSD）表示，生前样本的 RSD 不超过 7.2%，死后样本的 RSD 不超过 9.3%。除了吗啡≥62%和 THC≥50%外，所有化合物的回收率均≥85%。除 THC 的 IS 响应变异率为 27%（生前样本）和 66%（死后样本）外，所有化合物的 PE 均令人满意，RSD≤16%（生前样本）和≤34%（死后样本）。据我们所知，这是第一个针对全血中这 12 种化合物的自动化 96 孔 SLE UHPLC-MS/MS 同步检测方法，涵盖了法医样本中发现的浓度范围。该方法已在过去十个月的常规工作中使用，分析了约 9900 份生前和 1000 份死后全血样本，结果证明该方法稳健可靠。
