Evaluating Post-Analytical Stability of Blood Ethanol Samples Using Headspace-GC-FID

The analysis and determination of blood alcohol concentration (BAC) is a cornerstone test in forensic toxicology that is well researched. The purpose of this study was to determine the post analytical stability of blood ethanol obtained from re-injected vials via Agilent dual column headspace-GC-FID. Over 6 months and 22 individual batch analyses, 303 samples were prepared and analyzed using Headspace-GC-FID for the presence of blood ethanol using 0.02% n-propanol as an internal standard. Each individual batch was re-injected after storage at room temperature for 72 hours to mimic a typical weekend absence. Ethanol signal response, n-propanol signal response and measured blood ethanol concentration were evaluated for stability in terms of percent change. Sample population consisted of negative human blood fortified with known concentrations of ethanol via certified reference material purchased from accredited vendors (n = 187) in the form of calibrators and controls, as well as unknown post-mortem and ante-mortem blood samples submitted to the Nassau County Medical Examiner's Office Department of Forensic Toxicology (n = 116). All samples were tested using similar conditions and internal standard. Significant differences were observed in the signal response for both n-propanol (p<.001) and ethanol (p<.001) between the two test groups however, there was a non-significant difference in measured BAC between the two groups t(302) = 0.3, p = 0.780, d = 0.016. Results suggest very little effect of re-injection after 72-hour storage at room temperature on measured blood ethanol concentration using headspace GC-FID. While there was significant loss of ethanol and internal standard on reinjection, the calculated ethanol concentration was unaffected due to the similar properties of both volatile measurands as well as the consistency of the response ratio of ethanol:n-propanol in reinjected vials. In addition, the observed differences in measured BAC on re-injection were well within the NCME's measurement uncertainty of 11% at k = 3.