Department of Forensic Medicine, Victorian Institute of Forensic Medicine, Monash University, 57-83 Kavanagh St, Southbank, Melbourne, Victoria 3006, Australia.
Anal Bioanal Chem. 2011 Apr;400(1):189-96. doi: 10.1007/s00216-011-4667-2. Epub 2011 Jan 21.
Detection of the alcohol metabolites ethylglucuronide (EtG) and ethylsulfate (EtS) has become routine in many forensic laboratories over the last few years. Most previously published methods using liquid chromatography coupled with electrospray tandem mass spectrometry require a post-chromatographic addition of solvent and/or extensive sample preparation prior to analysis. The aim of the study was to develop a simplified method. To 20 μL urine, internal standard containing EtG-d5 and EtS-d(5) was added and the mixture was treated with elution buffer internal standard. EtG and EtS were separated using a Shimadzu Prominence high performance liquid chromatography (HPLC) system with a C18 separation column (Restek Ultra Aqueous C18, 4.6 × 150 mm, 5 μm), using isocratic elution with a mobile phase consisting of 10 mM ammonium acetate buffer pH 7 (total run time, 6 min). The compounds were detected using an Applied Biosystems API 5000 liquid chromatography tandem mass spectrometry system (atmospheric pressure chemical ionization, multiple-reaction monitoring mode). The method was fully validated according to international guidelines. The assay was found to be selective for the compounds of interest. It was linear from 0.1 to 10 mg/L for all analytes (R(2) > 0.99). Matrix effects studies showed the presence of a slight but consistent ion enhancement (n = 10 different urine samples) at low concentrations and no effects at higher concentrations. Accuracy data were between 0.75% and 8.1% bias for EtG and between -5.0% and -11.3% bias for EtS. Precision data were between 4.3% and 6.9% relative standard deviations (RSD) for EtG and between 6.0% and 7.5% RSD for EtS. No instability was observed after repeated freezing and thawing. This fast, reliable, and accurate method enables the detection and quantification of alcohol metabolites in urine. The method is easier to use and more sensitive than previously published methods.
在过去的几年中，许多法医实验室已经将酒精代谢物乙基葡萄糖醛酸苷（EtG）和乙基硫酸盐（EtS）的检测常规化。以前发表的大多数使用液相色谱-电喷雾串联质谱法的方法在分析之前需要在后色谱添加溶剂和/或广泛的样品制备。本研究的目的是开发一种简化的方法。向 20 μL 尿液中加入含有 EtG-d5 和 EtS-d(5)的内标，并用洗脱缓冲液内标处理混合物。EtG 和 EtS 采用 Shimadzu Prominence 高效液相色谱（HPLC）系统与 C18 分离柱（Restek Ultra Aqueous C18，4.6×150mm，5μm）分离，采用 10mM 乙酸铵缓冲液 pH7（总运行时间，6 分钟）的等度洗脱。使用应用生物系统 API 5000 液相色谱-串联质谱系统（大气压化学电离，多反应监测模式）检测化合物。该方法根据国际指南进行了全面验证。该测定法对感兴趣的化合物具有选择性。对于所有分析物，其线性范围为 0.1 至 10mg/L（R(2)>0.99）。基质效应研究表明，在低浓度下存在轻微但一致的离子增强（n=10 个不同的尿液样本），而在较高浓度下则没有影响。准确度数据显示，EtG 的偏倚在 0.75%至 8.1%之间，而 EtS 的偏倚在-5.0%至-11.3%之间。精密度数据显示，EtG 的相对标准偏差（RSD）在 4.3%至 6.9%之间，而 EtS 的 RSD 在 6.0%至 7.5%之间。重复冻融后未观察到不稳定。这种快速、可靠、准确的方法能够检测和定量尿液中的酒精代谢物。该方法比以前发表的方法更简单易用，灵敏度更高。
