Postmortem ethanol formation is a well-known problem in forensic toxicology. Ethyl glucuronide (EtG) and ethyl sulfate (EtS) are ethanol metabolites that can be used to distinguish antemortem alcohol intake from postmortem formation of ethanol and in addition can be a helpful tool in assessment of the hip-flask defense. To an aliquot of 100 µL whole blood, internal standard (IS) and water was added before protein precipitation treatment (PPT) with ice-cold acetonitrile (ACN). The supernatants were filtered through a 96-well phospholipid removal plate, evaporated to dryness and reconstituted in 150 µL water/ACN/formic acid (FA). Identification of compounds was performed using multiple reaction monitoring (MRM) in negative mode. Gradient elution was performed on a C18 column with methanol (MeOH) and 0.1% FA. The run time was 4.5 min, and 0.5 µL was injected on an ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS-MS) instrument. Linearity was achieved (coefficient of determination (R²) ≥ 0.999) for EtG in the range of 0.089 to 22 mg/L (0.40–100 µM) and EtS 0.025 to 6.3 mg/L (0.20–50 µM). The limit of quantification (LOQ) was 0.067 mg/L (0.30 µM) for EtG and 0.019 mg/L (0.15 µM) for EtS. Between assay accuracy was –15% to 8% and precision reported as relative standard deviation (RSD) was ≤ 4.5%. Precision, estimated as the RSD of the concentration difference between results from two independent analyses of authentic whole blood samples, was ≤ 6.7%. Recovery was ≥ 61% for EtG and ≥ 77% for EtS and matrix effects (ME) were 99% to 103%. Method comparison was carried out with a previously used UHPLC–MS-MS method, and satisfactory agreement was achieved, and external proficiency testing control samples had z-score < ± 1. The method has been used in routine work for more than 4 years analyzing about 6,000 antemortem and postmortem whole blood samples and has proven to be robust and reliable.

中文翻译：

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磷脂去除96孔板和UHPLC-MS-MS定量测定死后和死前全血中的乙醛酸乙酯和硫酸乙酯

死后乙醇的形成是法医毒理学中的一个众所周知的问题。乙醛糖醛酸乙酯（EtG）和硫酸乙酯（EtS）是乙醇代谢物，可用于区分事前乙醇摄入与事后乙醇形成之间的区别，此外，还可作为评估臀部烧瓶防御能力的有用工具。在用冰冷的乙腈（ACN）进行蛋白质沉淀处理（PPT）之前，向等份的100 µL全血中加入内标（IS）和水。将上清液通过96孔磷脂去除板过滤，蒸发至干，然后在150 µL水/ ACN /甲酸（FA）中复溶。使用负反应模式的多反应监测（MRM）进行化合物鉴定。在C18色谱柱上用甲醇（MeOH）和0.1％FA进行梯度洗脱。运行时间是4.5分钟，并且是0。在超高效液相色谱-串联质谱（UHPLC-MS-MS）仪器上进样5 µL。达到线性（测定系数（R^2个）≥0.999）的EtG在0.089至22 mg / L（0.40–100 µM）和EtS 0.025至6.3 mg / L（0.20–50 µM）的范围内。EtG的定量限（LOQ）为0.067 mg / L（0.30 µM），EtS的定量限为0.019 mg / L（0.15 µM）。分析之间的准确度为–15％至8％，报告为相对标准偏差（RSD）的准确度为≤4.5％。准确度估计为≤6.7％，其为两次独立的真实全血样品分析结果之间的浓度差的RSD。EtG的回收率≥61％，EtS的回收率≥77％，基质效应（ME）为99％至103％。使用先前使用的UHPLC-MS-MS方法进行方法比较，取得了令人满意的一致性，并且外部能力测试对照样品的z得分<±1。该方法已用于常规工作超过4年，进行了分析大约6