Direct determination of ethyl glucuronide and ethyl sulfate in postmortem urine specimens using hydrophilic interaction liquid chromatography-electrospray ionization-tandem mass spectrometry

Abstract

This work was aimed at developing and validating a hydrophilic interaction chromatography (HILIC)-electrospray ionization (ESI)-ion trap-tandem mass spectrometric method for identification and quantification of ethyl glucuronide (ETG) and ethyl sulfate (ETS) as ethanol biomarkers and at employing this method for analysis of postmortem urine samples. Analytes of interest were separated on a ZIC®-HILIC column (150 × 2.1 mm, 3.5 μm) connected to a Thermo Finnigan LCQ Deca Plus liquid chromatographic-tandem mass spectrometric instrument operated in the ESI-selected reaction monitoring mode. Seventy-nine urine case samples were divided into three groups depending on the ethanol concentration found in blood and analyzed by the developed method: group A with postmortem blood ethanol concentrations higher than 200 mg/100 mL; group B with ethanol concentrations in the range 80-200 mg/100 mL; and group C with ethanol concentrations in the range 10-80 mg/100 mL. ETG and ETS had high recoveries of 98-99%, and the HILIC column produced fine, sharp peak shapes and achieved baseline separation in less than 7 min. Both ethanol markers were detected in all groups with overall median concentrations of 100 and 23 mg/L for ETG and ETS, respectively. It can be concluded that the potential for postmortem production of alcohol increased in the low ethanol concentration group as several cases tested negative for both biomarkers in group C. ETG was detected at low concentrations in some cases for which ETS tested negative. Although ETS is stable after being subjected to many stability conditions, the use of ETS as sole evidence of alcohol ingestion may lead to a false-negative result, as we noticed in groups A and C in the present study. The use of ETG is a more reliable ethanol biomarker. Both ethanol biomarkers should be determined in heavily putrefied cases and when the ethanol concentration in postmortem blood is low.