Ethyl glucuronide and ethyl sulfate: a review of their roles in forensic toxicology analysis of alcohol postmortem.

PURPOSE: This review presents the current methods used for determining ethyl glucuronide (EtG) and ethyl sulfate (EtS) concentrations in postmortem specimens, including sample preparation, analysis and the role of EtG and EtS in the postmortem assessment of the extent of alcohol abuse. METHODS: Papers pertaining to postmortem investigation were collected from scientific databases and reviewed. The papers were published between January 2006 and October 2020. RESULTS: Most of the analyses involved postmortem blood and urine samples, with a few reports using other bodily specimens and tissues. The method validation was not conducted for all applications. These reports were mostly intended to present data rather than interpret them, and the lack of effort in relating these ethanol biomarkers with the cause of death and/or determination of the time of deaths due to ethanol intoxication might decrease the applicability of these makers after a promising start between 2006 and 2010. Nevertheless, by the beginning of 2020, papers investigating ethanol biomarkers were still increasing. A considerable number of methods used liquid chromatography coupled with mass spectrometry (LC-MS) techniques that require less sample preparation (e.g., protein precipitation extraction, dilution, filtration, and centrifugation). Although solid-phase extraction can be applied, only three applications were reported. CONCLUSIONS: Matrix effects can be a substantial challenge in analytical methods based on LC-MS because they directly affect the ionization of analytes. However, these problems can be avoided due to the high cutoff values used to identify positive results for these ethanol biomarkers, which are often above 0.1-1 mg/L, and using internal standards. Research on using tissue specimens is recommended as most of the reported results on this type of specimen were promising.

Effects of postmortem interval, putrefaction, diabetes, and location of death on the analysis of ethyl glucuronide and ethyl sulfate as ethanol biomarkers of antemortem alcohol consumption.

This study evaluated the forensic value of ethanol biomarkers ethyl glucuronide (EtG) and ethyl sulfate (EtS) under different conditions, including diabetes mellitus, drug abuse, and advanced decomposition. In addition, we explored whether ethanol, EtG, or EtS formation occurred in patients who died as a result of diabetes mellitus. Fifty-two routine postmortem cases were divided into three groups. Group 1 included only the post-mortem cases in which at least blood samples were available (n=47). Group 2 included all cases with positive BAC (n=28). Group 3 included the cases with negative BAC while information surrounding the cases suspected antemortem alcohol consumption and cases that tested negative for ethanol but positive for EtG and EtS. We analyzed multiple bodily fluid specimens, including the vitreous humor, for ethanol biomarker analysis and accurately identified antemortem ethanol consumption or postmortem ethanol synthesis. We also determined the utility of urine samples for analyzing ethanol and its metabolites in putrefaction cases. If no urine sample was available at autopsy due to urination before death or diabetes-associated glucosuria, vitreous humor samples were an appropriate alternative for ethanol biomarker testing. We observed postmortem ethanol synthesis in diabetic individuals even with a short postmortem interval (PMI), however, glucose did not increase postmortem ethanol production in individuals with diabetes under appropriate preservation. The shorter the PMI, the better the ethanol source can be determined. Postmortem ethanol production occurred in all body fluid specimens analyzed herein, including the vitreous humor. EtG and EtS levels were stable and provided accurate insight into ethanol sources, even in cases of postmortem ethanol production. While the present study focused on the use of vitreous humor for the analysis, it is expected such samples may not be available in cases of advanced decay. In cases where no other bodily fluid specimens are available, solid tissue specimens are highly preferred.

The role of ethanol in fatalities in Jeddah, Saudi Arabia.

In Saudi Arabia, alcohol consumption is prohibited by law, but interpreting postmortem ethanol can be complicated by its postmortem production. This study developed and validated a method using headspace gas chromatography with flame ionization detection and liquid chromatography tandem mass spectroscopy to detect ethanol and its polar metabolites (ethyl glucuronide [EtG] and ethyl sulfate [EtS]) in postmortem blood and urine specimens, respectively. All calibration curves were linear with coefficients of determination greater than 0.999. The limits of detection ranged 4.5-5.0mg/dL for ethanol and 0.05-0.06mg/L for EtG and EtS. The limits of quantification were 10.0mg/dL for ethanol and 0.075mg/L for EtG and EtS. Within-run precision was less than 11% for all analytes of interest. Matrix effects for EtG and EtS ranged 3-47%. After excluding matrix effects, analytical recoveries ranged 72-100%. This validated method was then used for routine postmortem forensic toxicology analyses in 592 routine postmortem cases to distinguish between antemortem ethanol consumption and its postmortem microbial formation. Among them, 98 blood samples (17%) were positive for ethanol or its polar metabolites. Thirty-two of these cases (33%) were positive for EtG and EtS and therefore due to antemortem ethanol consumption. The remaining 66 (67%) cases were negative for both EtG and EtS and therefore due to postmortem ethanol synthesis. Because this is the first study to report the problem of alcohol consumption in Saudi Arabia, further studies are essential for validating these findings.