Validation of a direct injection-GC-MS method for the quantification of volatile alcohols in blood and vitreous humor

Volatile compounds such as ethanol, methanol, isopropanol, and acetone are frequently encountered in forensic toxicology, particularly in postmortem investigations, poisoning cases, and incidents involving impaired driving. While headspace gas chromatography (HS-GC) remains the gold standard for their analysis, its limited availability in many laboratories highlights the need for more accessible alternatives. This study aimed to develop and validate a direct injection gas chromatography-mass spectrometry (DI-GC-MS) method for the simultaneous quantification of ethanol, methanol, isopropanol, and acetone in human blood and vitreous humor (VH). The method was developed in accordance with the Scientific Working Group for Forensic Toxicology (SWGTOX) guidelines. Analytical standards were prepared in both blood and VH. Method validation included assessment of selectivity, linearity, limits of detection (LOD) and quantification (LOQ), accuracy, precision, recovery, and stability. The DI-GC-MS method demonstrated excellent selectivity and linearity (R² >0.99) for all target analytes. The LODs were 0.01 mg/mL VH and 0.05 mg/mL in blood, while LOQs were 0.05 mg/mL and 0.2 mg/mL, respectively. Accuracy and precision were within acceptable limits. Recoveries exceeded 85% across all tested conditions. The method was successfully applied to 30 real forensic cases. Ethanol was detected in four cases, with blood alcohol concentrations (BAC) ranging from 0.40 to 2.30 mg/mL. The validated DI-GC-MS method provides a reliable, sensitive, cost-effective, and reproducible alternative to HS-GC for the quantification of volatile alcohols in postmortem blood and VH. It is particularly well-suited for forensic laboratories with limited access to specialized instrumentation.