Establish the importance of interpreting the analyses of two alcohol use biomarkers, with a different detection window, when monitoring long-term alcohol consumption of an individual.

In many medico-legal cases, such as liver transplantation and traffic incidents, there is a need to provide a proof of alcohol abstinence. Long-term alcohol consumption, particularly proving abstinence, remains a challenge. Therefore, various alcohol consumption biomarkers are used to gain information about the drinking behaviour of an individual. Ethyl glucuronide in hair (hEtG) is a reliable long-term alcohol use biomarker due to its long detection window, depending on the length of hair, allowing for a retrospective evaluation of alcohol use (Crunelle. Drug and Alcohol Dependence 2014:1–11). Phosphatidylethanol 16:0/18:1 (PEth) in whole blood is a direct alcohol biomarker that has gained increasing interest, due to its detection window of two to four weeks. (Ulwelling. Journal of Forensic Science 2018;63(6):1634–1640) This study explores the usefulness of analysis of PEth as a complementary biomarker to hEtG to evaluate alcohol use more accurately over the last weeks to months.

Hair and whole blood samples were obtained over a three-year period in a medico-legal context, from the University Hospital Antwerp and analysed by Toxicological Centre University of Antwerp and the National Institute investigation of Criminalistics and Criminology (NICC), Belgium. Individuals (n=952) were assessed for a 3- or 6- month period of alcohol abstinence. Hair samples were taken from the posterior head region (0–3 or 0–6cm). The sample preparation for hair analysis consisted in solid-phase extraction. At NICC, analysis was performed on an LC-MS/MS (Kummer. Journal of Analytical Toxicology 2015;39(1):17–23). At the Toxicological Centre, the extract was analysed on a GC-MS/MS (Cappelle. Forensic Science International 2015;249:20–24). Whole blood was collected on the same day as the hair sample and analysed for PEth using liquid-liquid extraction. In both laboratories, the analyses were performed on a LC-MS/MS. (Dumitrascu, Drug Testing 2021;13:1219; Van Uytfanghe, Talanta 2021;223:121694).

PEth and hEtG results were classified into three groups, according to thresholds for abstinent/low, moderate, or excessive alcohol consumption (Society of Hair Testing, 2016; Van Uytfanghe. Talanta 2021;223:121694). Spearman correlation test (rs=0.692, P=0.001) showed a significant and moderate correlation between the two biomarkers. It was found that 341 individuals were classified abstinent according to the hair analysis (hEtG<5pg/mg) out of which 82% were also classified abstinent according to the whole blood analysis (Peth<20ng/mL). Furthermore, 329 individuals were classified as heavy drinkers through hair analysis (hEtG>30pg/mg), of which 57% presented a PEth concentration>200ng/mL, confirming the classification. Fifty-two individuals (15%) were categorised as abstinent/low alcohol consumption through hEtG analysis, but as moderate drinkers through PEth analysis (20–200ng/mL). This might be explained by recent, moderate alcohol consumption. While PEth can be detected if alcohol was consumed in the past two to four weeks, EtG had insufficient time to be incorporated into the cut hair sample. Out of 329 individuals classified as heavy drinkers based on hEtG analysis, 45 were categorised abstinent through PEth analysis, suggesting these subjects stopped drinking two to four weeks prior to sample collection. A moderate agreement of Kappa reliability analysis (k=0.444) between PEth and hEtG confirms that, for a more accurate overview of the drinking behaviour of an individual, both biomarkers are needed.

This study demonstrates the necessity of analysing two alcohol use biomarkers with different detection windows, when investigating alcohol use retrospectively. PEth in whole blood is a complementary biomarker to hEtG, as it captures the last two to four weeks of alcohol consumption, which would be missed when only analysing for hEtG.