Added value evidences of a simultaneous determination of two phosphatidylethanol isoforms and ethylglucuronide in dried blood spots in forensic contexts

Abstract

In clinical or postmortem forensic medicine, acute or chronical alcohol abuse are of main concern and direct/indirect biomarkers are widely use in order to assess recent or retrospective alcohol abuse. We aim to illustrate that a simultaneous determination of 3 directs ethanol biomarkers [two phosphatidylethanol isoforms, 16:0/18:1 (PEth18:1) and 16:0/20:4 (PEth20:4), and ethylglucuronide (EtGB)] using dried blood spot (DBS) can be a useful additional tool in many situations. (Case#1) Sexual assault of a 17-year-old girl with negative urine and blood ethanol concentration (BAC) (but urine EtG concentration was 10 mg/L) sampled 19 hours after the facts. (Case#2) Only blood sampling (negative BAC) was performed 14 hours after a 36-year-old man (with heavy psychiatric disorders) tried to kill 3 persons. He was reported drunk at the time of the attack by witnesses. (Case#3) A 27-year-old man was implicated in a car-related fatality that occurred 11 days before sampling. Blood and hair sampling were performed in order to investigate his alcoholic status: hair EtG concentration was < 5 pg/mg. (Case#4) The alcoholic status of a 43-year-old woman killed in a car accident had to be established. BAC was 140 mg/L in the postmortem peripheral blood (only available biological sample; sampled less than one hour after death and kept refrigered until time of analysis, 2 days later). (Case#5) A low BAC (70 mg/L) was found in postmortem cardiac blood (only sample available) sampled on the corps of a 42-year-old man found submerged in the sea. (Case#6) Peripheral blood sampling (for BAC determination) and direct DBS sampling (MitraTM) were performed at the time of discovery of the 36-year-old man corpse, allegedly heavy drinker according to the testimonies (BAC was 2810 mg/L). 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; HemaxisTM, MitraTM). These DBS are analyzed using a LC-MS/MS (routinely used in clinical toxicology) method (LLOQ: 2 μg/L for the 3 analytes). Results (μg/L; PEth18:1, PEth20:4 and EtGB, respectively) were: – (Case#1) 22, 2 and 300, in coherence with both a recent alcohol intake hypothesis [EtGB above the 45 μg/L threshold supported an alcohol consumption [> 2 standard units (SU)] in the last 24 to 48 hours] and that this girl was not an excessive drinker [owing to the low PEths concentrations]; – (Case#2) 98, 103 and 71, consistent with an alcohol intake hypothesis arround the time of the attack by this man who had a significant consumption of alcohol in the last 10 days as PEth 20:4 concentration was similar to the PEth18:1 one; – (Case#3) 9, < 2 and < 2, and did not contradict the negative EtG hair result as these low values were, at most, compatible with an “occasional” or “social drinker” status (< 14 SU per week); – (Case#4) 817, 44 and 130. EtGB positive result supported a recent alcohol consumption and the very high PEth18:1 value strongly suggested chronic excessive alcohol consumption; – (Case#5) negative (< 2 μg/L) for the 3 analytes in postmortem cardiac blood, in agreement with both the hypothesis of a light postmortem ethanol production, and a probable “abstinent” or “occasional consumer” status of the victim; – (Case#6) 3470, 1030 and 13,400, supporting the massive alcohol intoxication hypothesis close to the death of a heavy drinker. This analytical tool is of high interest when only blood samples are available, although postmortem use (due to PEths neoformation issues) should be limited to cases with negative BAC, or when blood sampling is performed close to death using a VAMS or a refrigerated preserved (< 4 days) tube.