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

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.