Quantitative Estimation of Alcohol Marker Ethyl Glucuronide (EtG) in Human Hair by LC-MS/MS: An Application Towards Doping Control & Forensic Science

Background: Testing of hair samples for drugs of abuse is advantageous as compared to other complementary matrices like urine or blood, due to longer detection window, non-invasive, and easy storage conditions. Ethyl glucuronide (EtG) is a promising biomarker for the identification of alcohol abuse in doping control or forensic analysis. Objective: This paper introduces a fast and sensitive method for determination of EtG in human hair using liquid chromatography-tandem mass spectrometry (LC–MS/MS) as per ISO:IEC 17025/2005 guidelines. Methods: Hair strands (50 mg) were washed with dichloromethane for 5 minutes followed by methanol for 2 minutes. The extraction was performed using an ultrasonic incubation for 2 h. These samples were incubated overnight at ambient temperature. After incubation, again ultrasonicated for 2 minutes and then centrifuged at 3500 rpm for ten minutes. The supernatant was decanted & dried under nitrogen (N2). The obtained residue was reconstituted in 40 μl of 0.1% formic acid out of which, 20 μl of extract was injected into the LC-MS/MS system. Results: The best separation was achieved using a C-18 column and a mobile phase comprising of 0.1% formic acid: acetonitrile in a gradient mode, with flow rate and temperature being 0.6 ml/min and 30ºC, respectively. The run time of the developed method is 6 minutes. The LOD was obtained at 3 pg/mg and LOQ at 10 pg/mg. The linearity for quantification analysis was established from 10- 200 pg/mg. The coefficients of variation in intra- and inter-assay precision were always lower than 15%. The method was successfully applied and qualified for the quantitative determination of EtG in proficiency test (PT) samples received from the Society of Hair Testing (SoHT) for the year 2015 & 2016. Conclusion: This method proved to be fast and sensitive to detect EtG in human hair and would be useful in the field of doping control and forensic toxicology.