Blood simultaneous determination of PEth 16:0/18:1, PEth 16:0/20:4 and ethylglucuronide using DBS: Application to a clinical study of patients hospitalized for withdrawal

Abstract

This study aims to present a LC-MS/MS method for simultaneous determination of phosphatidyléthanol (PEth)16:0/18:1, PEth16:0/20:4 and ethylglucuronide (EtG) using dried blood spots (DBS) and to report preliminary results of PETHOXICO, a monocentric non-interventional non-randomized cross-sectional clinical study (ClinicalTrials.gov NCT04557631 ) in a population of patients hospitalized for withdrawal. Blood samples can alternatively consist in 10 μL from usual blood sampling tubes (e.g. fluoride preserved) transferred on DBS card, or direct DBS sampling using Volumetric Absorptive MicroSampling devices (VAMS; Hemaxis™, Neoteryx™). For PETHOXICO, PEth16:0/18:1, PEth16:0/20:4 and EtG were analyzed simultaneously together with urinary EtG and blood alcohol concentration (BAC), determined using immunoassay and GC-FID, respectively. The fully validated LC-MS/MS method exhibits a LLOQ of 2 μg/L for the 3 analytes. A total of 37 chronic and excessive ethanol consumers (ethanol per week consumptions ranging from 560 to 1750 g) were included in PETHOXICO [sex ratio 11/26 (F/M); 30 to 67 YO with a median at 46 YO]. At D0, the mean concentration of PEth16:0/18:1 was 1180 μg/L (range: 53–3889 μg/L) with 95% of participants above the 210 μg/L threshold. PEth16:0/20:4-to-PEth16:0/18:1 mean ratio was 0.46 with PEth16:0/20:4 concentrations ranging from 9 to 2353 μg/L. The mean BAC was 1060 mg/L (range: undetected–3540 mg/L) and for 78% of participants, urinary EtG was above the 500 μg/L threshold. At D7, no BAC nor EtG (urine and blood) were found as expected. The mean concentration of PEth16:0/18:1 dropped to 527 μg/L (range: 16–1655 μg/L) with still 78% of participants above the 210 μg/L threshold. PEth16:0/20:4 concentrations dropped more significantly (range: undetected–923 μg/L) with a mean PEth16:0/20:4-to-PEth16:0/18:1 ratio of 0.26. This decreased ratio (0.46 at D0 and 0.26 at D7) is coherent with the expectation of a shortened detection window for PEth16:0/20:4. Blood elimination half-life of PEths were calculated with the assumption of a single elimination phase: mean half-life were 6.9 days (range: 2.2–18.6 days) and 4.6 days (range: 1.3–9.3 days) for PEth16:0/18:1 and PEth16:0/20:4, respectively. PEth16:0/18:1 observed half-life is consistent with literature data (around 7 days with a significant inter-individual variability). Observed PEth16:0/20:4 half-life is higher than the very scarce data from literature (2 days). In this study, the use of an analytical method combining 3 direct metabolites of ethanol allows, in one run, to discriminate a very recent consumption (< 48 h) from a recent consumption (< 2 weeks) and an older consumption (2–4 weeks). PEth16:0/18:1 sensitivity to detect chronic and excessive alcohol consumers was 95% with a 210 μg/L threshold. However, these preliminary results need yet to be confronted to detailed clinical data.