Validation of a New Salt-Assisted HS-GC-FID Method for the Determination of Ethanol in Vitreous Humor.

Headspace gas chromatography with a flame ionization detector (HS-GC-FID) is a well-established approach for determining blood alcohol concentration (BAC), including in cadaveric specimens. Although the integrity of blood specimens can be adequately guaranteed after the sampling, the quantification of ethanol in cadaveric blood can be affected by postmortem fermentative phenomena occurring between the time since death and the sampling of biofluids. Vitreous humor is less affected by putrefactive phenomena allowing compound determination and its use as an alternative biological matrix. The present work aimed to develop and validate a method using the salting-out effect and based on HS-GC-FID for the ethanol determination in vitreous humor. The reported analytical method is based on a simple VH pre-treatment consisting of a dilution (1:9) with a solution of 2.5 mol/L K2CO3 and 0.0012 mol/L tert-butanol (internal standard). After 1 minute of incubation, part of the specimen evaporated in the headspace (2000 µL) is injected into the chromatographic system and analyzed in isothermal mode (40°C), with a chromatographic time of 1.6 minutes. The method was validated in terms of selectivity, lowest limit of detection, intraday and total imprecision, and trueness (bias). The determination of ethanol in vitreous humor and blood was carried out in 75 cases. The correlation between the two matrices was confirmed in 61 cases. However, 14 vitreous humor specimens showed lower ethanol concentrations, and in the related blood specimens, it was possible to identify the signal of n-propanol, a typical product of postmortem microbial fermentation, that justifies the excess of ethanol in the blood specimens.