EtG and EtS in Autopsy Blood Samples With and Without Putrefaction Using UPLC-MS-MS

Area of Science:

• Forensic Toxicology
• Analytical Chemistry
• Biomarker Analysis

Background:

• Postmortem blood analysis presents significant challenges due to sample degradation and putrefaction.
• Accurate quantification of biomarkers like EtG and EtS is crucial for postmortem investigations.
• Existing analytical methods may be compromised by the condition of postmortem specimens.

Purpose of the Study:

• To develop and validate an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS) method for determining ethyl glucuronide (EtG) and ethyl sulfate (EtS) in postmortem whole blood.
• To assess the method's robustness and reliability across varying degrees of sample putrefaction.
• To ensure efficient sample preparation that mitigates matrix effects in challenging postmortem samples.

Main Methods:

• Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS) was employed for sensitive and specific detection.
• Sample preparation involved protein precipitation and phospholipid removal via filtration.
• HSS T3 column with gradient elution using formic acid/water and methanol was utilized for chromatography.

Main Results:

• The validated UPLC-MS-MS method demonstrated excellent performance with low between-assay relative standard deviations (1.7-7.0%).
• Limits of quantification were as low as 0.025 mg/L for EtG and 0.009 mg/L for EtS.
• The sample preparation effectively removed phospholipids, yielding clean samples and satisfactory chromatographic performance regardless of putrefaction extent.

Conclusions:

• The developed UPLC-MS-MS method is suitable for the reliable quantification of EtG and EtS in postmortem whole blood.
• The method's robustness against putrefaction ensures its utility in forensic toxicology.
• This analytical approach overcomes common challenges associated with analyzing degraded biological specimens.