Hegstad Solfrid, Kristoffersen Lena, Liane Veronica H, Spigset Olav
Department of Clinical Pharmacology, St. Olav University Hospital, 7006 Trondheim, Norway.
Division of Forensic Sciences, Norwegian Institute of Public Health, 0403 Oslo, Norway.
J Anal Toxicol. 2017 Mar 1;41(2):107-113. doi: 10.1093/jat/bkw123.
Analytical challenges related to postmortem specimens are well known. The degree of putrefaction of the corpse will influence the quality of the blood samples, and both the efficiency of sample preparation and the subsequent chromatographic performance can be affected. An ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS) method was developed and validated for the determination of ethyl glucuronide (EtG) and ethyl sulfate (EtS) in postmortem whole blood. Sample preparation prior to UPLC-MS-MS analysis consisted of protein precipitation and filtration through a phospholipid removal plate. Chromatography was achieved using an HSS T3 column and gradient elution with formic acid in water in combination with methanol. The injection volume was 0.5 µL. Negative electrospray ionization was performed in the multiple reaction monitoring mode. Two transitions were monitored for the analytes and one for the internal standards. The between-assay relative standard deviations were in the range of 1.7-7.0% and the limits of quantification were 0.025 and 0.009 mg/L for EtG and EtS, respectively. Recovery was 51-55% and matrix effects ranged from 98% to 106% (corrected with internal standard). Blood samples from nine autopsy cases with various extents of putrefaction were analyzed. The sample preparation efficiently removed the phospholipids from the blood specimens. The samples were clean and the analytical quality of the chromatographic performance was satisfactory for both analytes irrespective of the degree of putrefaction.
与尸检样本相关的分析挑战是众所周知的。尸体的腐败程度会影响血样质量，样本制备效率和后续色谱性能都会受到影响。本文开发并验证了一种超高效液相色谱-串联质谱（UPLC-MS-MS）法，用于测定死后全血中的葡萄糖醛酸乙酯（EtG）和硫酸乙酯（EtS）。UPLC-MS-MS分析前的样本制备包括蛋白质沉淀和通过磷脂去除板过滤。使用HSS T3柱进行色谱分离，以水中的甲酸与甲醇进行梯度洗脱。进样体积为0.5μL。在多反应监测模式下进行负离子电喷雾电离。对分析物监测两个跃迁，对内标监测一个跃迁。批间相对标准偏差在1.7-7.0%范围内，EtG和EtS的定量限分别为0.025和0.009mg/L。回收率为51-55%，基质效应范围为98%至106%（用内标校正）。分析了来自9例不同腐败程度尸检病例的血样。样本制备有效地从血样中去除了磷脂。样本干净，无论腐败程度如何，两种分析物的色谱性能分析质量均令人满意。
