Postmortem correlation of ethanol and minor metabolites ethylglucuronide and ethylsulfate in blood, vitreous humor and bile.

Accurate determination of ethanol use in postmortem cases is crucial in forensic investigations. While vitreous humor (VH) is considered a stable matrix that lacks endogenous alcohol production and serves as a reliable confirmatory sample, challenges arise when VH is unavailable or only blood samples are present. In such cases, interpreting the presence of ethanol becomes difficult due to the potential forpostmortem ethanol production. Ethyl glucuronide (EtG) and ethyl sulfate (EtS), minor non-oxidative metabolites of ethanol, have been proposed as alternative biomarkers. However, the significance of their levels, especially when only one metabolite is detected, remains uncertain. This study analyzed ethanol, EtG, and EtS levels in blood, VH, and bile samples from 40 postmortem cases with blood ethanol levels ranging from 0.11 to 4.35 g/L. Ethanol concentrations were measured using HS-GC-FID, while EtG and EtS were quantified via LC-ESI-MS/MS. The highest ethanol level was found in blood (4.35 g/L), while bile contained the highest concentrations of EtG (4.98 mg/L) and EtS (0.76 mg/L). Very strong to moderate correlations were observed for ethanol and its minor metabolites across all matrices, as well as between blood and bile EtG, indicating that bile EtG is a reliable surrogate biomarker for antemortem ethanol assessment. There were no weak or non-significant correlations present. These findings suggest that ethanol and its minor metabolites in VH, blood, and bile can be reliably used in postmortem assessments. Moreover, combined analysis of EtG and EtS may enhance the interpretation of antemortem alcohol consumption in forensic cases.