Department of Forensic Medicine and Toxicology, Faculty of Medicine, School of Health Sciences, University of Ioannina, University Campus, Ioannina 45500, Greece.
Victorian Institute of Forensic Medicine-Toxicology, 65 Kavanagh St, Southbank, Melbourne, Victoria 3006, Australia; Department of Forensic Medicine, Monash University, Melbourne, Victoria 3006, Australia.
J Chromatogr B Analyt Technol Biomed Life Sci. 2024 Oct 15;1247:124323. doi: 10.1016/j.jchromb.2024.124323. Epub 2024 Sep 18.
Liquid chromatography coupled with mass spectrometry (LC-MS) has been tremendously used for screening purposes in forensic toxicology, because of their great adaptability and reasonable time/resource consumption. Herein, a fully validated method based on liquid-liquid extraction (LLE) in human whole blood, by a multiple reaction monitoring (MRM) analysis through LC-MS/MS, is described. The proposed method simultaneously detects 100 analytes (plus three deuterated internal standard compounds) belonging to many different classes, including drugs of abuse, prescription and over-the-counter drugs commonly involved in poisoning and medical malpractice cases in our territory, as well as certain new psychoactive substances (NPS) and toxic substances potentially associated with adverse effects. The optimised LLE employs one extraction step of 200 μL blood using 0.1 M HCl methyl-tert-butyl-ether (MTBE) (acidified with concentrated HCl) proved to be suitable for the extraction of basic and neutral substances; as a reconstitution solvent a mixture of 88:12v/v, 0.1 % formic acid in 10 mM aqueous ammonium acetate, pH 3.5: 0.1 % formic acid in acetonitrile was used, yielding satisfactory recoveries for all analytes. The method was sensitive, showing low LOD/ LOQ for all substances ranging from 0.01 to 5/ 0.05-20 ng/mL, respectively. Linearity ranged between 0.05-500 ng/mL (R = 0.9811-0.9995), and the inter- and intra-day precisions ranged between 3-15 % and 7-18 %, respectively. Accuracy was evaluated in terms of percentage recovery, lying within acceptable range. The matrix effect expressed as ion suppression/enhancement of each analyte was in the range ±25 % for all analytes. Post-preparative stability of analytes was higher than 85 %, while no carryover between runs was observed. The developed method has been successfully applied in routine toxicological analyses for the analysis of biological samples from clinical and autopsy cases.
液相色谱-质谱联用（LC-MS）在法医毒理学的筛查中得到了广泛应用，因为它具有很强的适应性和合理的时间/资源消耗。本文描述了一种基于液液萃取（LLE）的、在人全血中进行的、通过 LC-MS/MS 进行多反应监测（MRM）分析的完全验证方法。该方法同时检测了 100 种分析物（加 3 种氘代内标化合物），这些分析物属于许多不同的类别，包括药物滥用、处方和非处方药物，这些药物通常与我们地区的中毒和医疗事故案件有关，以及某些新的精神活性物质（NPS）和可能与不良反应有关的有毒物质。优化的 LLE 采用一步提取 200 μL 血液，使用 0.1 M HCl 甲基叔丁基醚（MTBE）（用浓 HCl 酸化），证明适合提取碱性和中性物质；作为复溶溶剂，使用 88:12v/v、0.1%甲酸在 10 mM 水溶液中铵乙酸盐、pH3.5:0.1%甲酸在乙腈的混合物，得到所有分析物的满意回收率。该方法具有较高的灵敏度，所有物质的最低检测限/定量下限（LOD/LOQ）均为 0.01-5/0.05-20ng/mL。线性范围在 0.05-500ng/mL 之间（R=0.9811-0.9995），日内和日间精密度分别在 3-15%和 7-18%之间。准确度以回收率的百分比表示，在可接受的范围内。以每个分析物的离子抑制/增强表示的基质效应在所有分析物的±25%范围内。分析物的提取后稳定性高于 85%，并且没有观察到运行之间的交叉污染。该方法已成功应用于临床和尸检病例生物样本的常规毒理学分析。
