2.7. Hair Analysis for the Detection of Alcohol Consumption (EtG)

EtG was measured in each of the hair segments using a previously published method and re-validated in-house with some analytical improvements [20]. Briefly, aliquots of 25 mg of finely cut hair were extracted after 1 h of incubation at 100 °C in 0.5 mL of an acidic buffered M3 reagent containing 1.5 ng/mL of EtG-d5. Then, the extracted hair was dried under nitrogen flow and re-suspended in 0.1 mL of mobile phase A. After vortex mixing and ultracentrifugation at 10.000 g for ten min, 10 μL of the clear supernatant were injected into UHPLC-MS/MS. Chromatography was carried out using a Luna Omega Polar C18 (100 × 2.1 mm, 1.6 μm) using a linear gradient elution with two solvents: 0.1% formic acid in water (mobile phase A) and methanol (solvent B). Solvent B was maintained at 2.0% for the first 0.5 min. It was increased to 95.0% from 0.5 to 2.0 min and held to 95.0% from 2.0 to 2.5 min. Then it was decreased back to 2.0% from 2.5 to 2–6 min and held to 2.0% from 2.6 to 5 min to re-equilibration. The flow rate was kept constant at 0.3 mL/min during the analysis. EtG and the internal standard (EtG-d5) were detected in negative electrospray ionization mode with the triple quadrupole mass spectrometer operated in multiple reaction monitoring (MRM). Mass spectrometry conditions were the following: capillary voltage of 2.5 kV, desolvation temperature of 650 °C, source temperature of 150 °C, cone gas flow rate of 40 L/h, desolvation gas flow rate of 900 L/h, and collision gas flow rate of 0.07 mL/min. The cone energy voltage was 20 V, and the collision energy voltages were 15 and 18 eV for both EtG and EtG-d5. MRM transitions were: m/z 221.1→75.0, 221.1→85.0 for EtG, and m/z 226.0→75.0, 226.0→85.0 for EtG-d5. Underline transitions were selected for quantification. The limit of quantification was 5 pg/mg.

Both EtG and the internal standard, ethyl glucuronide-d5 (EtG-d5), used in this study were purchased from Cerilliant (Austin, TX, USA). M3 (acidic aqueous buffer) reagent was provided by Comedical S.a.s. (Mattarello, Trento, Italy). HPLC-MS-grade solvents (methanol, acetonitrile, and water) were purchased from Carlo Erba (Milan, Italy). All other chemicals used for experiments were analytical reagents or HPLC-grade from commercial resources.

According to the latest international consensus on the use of alcohol markers in hair for the assessment of abstinence and chronic alcohol consumption [21,22], a concentration lower than or equal to 5 pg/mg EtG in the proximal head hair segment (3–6 cm) does not contradict self-reported abstinence. Conversely, a concentration greater than 5 pg/mg EtG in the proximal head hair segment strongly suggests repeated alcohol consumption, commonly defined as “social drinking”. A concentration greater than or equal to 30 pg/mg EtG in the proximal head hair segment with a length of 3 cm up to 6 cm strongly suggests chronic excessive alcohol consumption.