Determination of Ethyl Glucuronide and Ethyl Sulfate in Human Whole Blood and Vitreous Humor by LC-MS-MS and Applications to the Interpretation of Postmortem Ethanol Findings

Abstract

Identifying the source of ethanol in a decedent remained a complicated problem for forensic toxicologists because of postmortem ethanol formation. As ethanol's non-oxidative metabolites, ethyl glucuronide (EtG) and ethyl sulfate (EtS) have the potential to distinguish between antemortem ethanol consumption and postmortem ethanol formation, due to their high sensitivity and selectivity. In the current study, a simple and quick liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed for the determination of EtG and EtS in human whole blood and vitreous humor (VH). A total of 20 μL of the sample was precipitated by methanol, and the analytes were detected by LC-MS/MS in a run of 6 min. This method achieved high sensitivity (limits of detection: 2 ng/mL for both EtG and EtS), with linearity in the range of 5-10,000 ng/mL in both whole blood and VH. Deviations in accuracy, inter- and intra-day precision were all lower than 15% at three quality control levels. Subsequently, this method was applied to 62 real forensic cases. Only blood samples were available in 52 cases. Paired blood and VH samples were present in 10 cases. The concentrations of EtG and EtS in blood were in the range of 0-22,264.8 ng/mL and 0-2,126.0 ng/mL, respectively. In one case with both blood and VH, the blood ethanol concentration was 1.22 mg/mL, with EtG and EtS both below limits of quantification (5 ng/mL) in VH, and no EtG and EtS found in whole blood. The results suggested that EtG and EtS were useful markers for the interpretation of ethanol resource in postmortem blood and VH.