Determination of Ethyl Glucuronide and Ethyl Sulfate in Dried Blood Spots by UHPLC-MS-MS: Method Validation and Assessment of Ethanol Exposure in Postmortem Samples from Road Traffic Victims

The determination of ethyl glucuronide (EtG) and ethyl sulfate (EtS) in blood has been proposed in clinical and forensic applications to identify recent alcohol consumption. Also, there is a growing interest on the use of dried blood spots (DBS) in toxicological analysis, allowing increased stability of the analytes and simplifying sample transportation and storage. This study presents the development and validation of a method for quantifying EtG and EtS in DBS using ultra-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS-MS). The DBS samples were extracted with a mixture of methanol and acetonitrile (80:20 v/v) and analyzed using UHPLC-MS-MS with electrospray source in negative mode, after separation with a fluoro-phenyl stationary phase. Validation was performed according to the Scientific Working Group for Forensic Toxicology (SWGTOX) guidelines, with calibrations ranging from 0.10 to 18 mu g/mL for EtG and 0.02 to 6 mu g/mL for EtS. The analytes were stable in DBS stored from -20 to 45 degrees C for 21 days. The method was successfully applied to capillary and venous DBS samples from 20 volunteers after ethanol ingestion and to DBS samples from 99 fatal victims of road traffic injuries. Capillary DBS was comparable to venous DBS and fresh whole blood in Passing-Bablok and Bland-Altman analysis, with correlation coefficients >0.91 (P < 0.001) for all comparisons. In postmortem application, the DBS EtG and EtS analysis indicated positive exposure to ethanol in 72.7% of the cases (EtG: 0.10-24.0 mu g/mL and EtS: 0.03-4.11 mu g/mL). The identification of ethanol consumption from blood alcohol concentrations (BACs) and EtG/EtS in DBS was in agreement in 98.6% of positive and 96.3% of negative cases (kappa 0.877, P < 0.001), indicating a high level of concordance with BAC in assessing alcohol use in postmortem samples.