Systematic optimisation of ethyl glucuronide extraction conditions from scalp hair by design of experiments and its potential effect on cut‐off values appraisal

Abstract

The quantitative determination of ethyl glucuronide (EtG) in hair samples is consistently used throughout the world to assess chronic excessive alcohol consumption. For administrative and legal purposes, the analytical results are compared with cut-off values recognised by regulatory authorities and scientific societies. However, it has been recently recognised that the analytical results depend on the hair sample pretreatment procedures, including the crumbling and extraction conditions. A systematic evaluation of the EtG extraction conditions from pulverised scalp hair was conducted by Design of Experiments (DoE) considering the extraction time, temperature, pH, and solvent composition as potential influencing factors. It was concluded that an overnight extraction at 60°C with pure water at neutral pH represents the most effective conditions to achieve high extraction yields. The absence of differential degradation of the internal standard (isotopically-labelled EtG) under such conditions was confirmed and the overall analytical method was validated according to SGWTOX and ISO17025 criteria. Twenty real hair samples with different EtG content were analysed with 3 commonly accepted procedures: (a) hair manually cut in snippets and extracted at room temperature; (b) pulverised hair extracted at room temperature; (c) hair treated with the optimised method. Average increments of EtG concentration around 69% (from a to c) and 29% (from b to c) were recorded. In light of these results, the authors urge the scientific community to undertake an inter-laboratory study with the aim of defining more in detail the optimal hair EtG detection method and verifying the corresponding cut-off level for legal enforcements.

Evidence of seasonal variation of ethyl glucuronide in hair: Modeling a seven‐year data series

“…The first result highlighted by comparing our large data‐series is that changing the pre‐analytical treatment of hair from cutting to milling resulted in an increase of the measured EtG mean concentration of about 38%. This confirms the previously reported findings, where the same average increase was established when the two crumbling techniques were applied to the same hair specimens, suggesting that a more exhaustive extraction of EtG is obtained when the hair matrix is pulverized with a metal ball mill rather than cut into small snippets with scissors. This procedural change within the same analytical framework proved to have a significant impact on the final EtG result.…”

“…On one hand, analytical evidence is delivered that the cut‐off values should be interconnected with the sample's pre‐treatment procedure and the corresponding EtG extraction yield. This evidence confirms the conclusions contained in various studies . On the other hand, the climatic effect reported in Salomone et al is expanded and approached by a rigorous time series analysis protocol.…”

“…The method was internally validated and accredited in accordance with ISO/IEC 17025:2005 rules . Further details on the most recent validation procedures for the analytical method are extensively discussed elsewhere . Operational (ie, fully guaranteed under routine conditions throughout the investigated period) limits of detection (LOD) and quantification (LOQ) were conventionally set at 3 and 10 pg/mg, respectively, even if the actual LOQ was calculated as 1.7 pg/mg and experimentally verified at 2.0 pg/mg .…”

“…Further details on the most recent validation procedures for the analytical method are extensively discussed elsewhere . Operational (ie, fully guaranteed under routine conditions throughout the investigated period) limits of detection (LOD) and quantification (LOQ) were conventionally set at 3 and 10 pg/mg, respectively, even if the actual LOQ was calculated as 1.7 pg/mg and experimentally verified at 2.0 pg/mg . The laboratory performance in hair EtG analysis was constantly monitored through regular participation to inter‐laboratory proficiency tests organized by SoHT, the Gesellschaft für Toxikologische und Forensische Chemie (Jena University Hospital, Germany), and the Centre Universitaire Romand de Médecine Légale (University Hospital of Geneva, Switzerland).…”

“…This evidence confirms the conclusions contained in various studies. [25][26][27] On the other hand, the climatic effect reported in Salomone et al 28 is expanded and approached by a rigorous time series analysis protocol. These two sources of potential variability are addressed in the present work and approached by meeting the two main goals of time series analysis that include the identification of the nature of the presented phenomenon as well as the task of predicting future values of the time series variable (the EtG concentration).…”