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Kapendra Sahu

Kapendra Sahu

National Dope Testing Laboratory, India

Title: A simple, rapid & cost effective method for the determination of EtG in hair by using liquid chromatography–tandem mass spectrometry (LC–MS/MS)

Biography

Biography: Kapendra Sahu

Abstract

Background:       In recent years, hair has become an alternative biological specimen for drug testing within the field of doping in sports, forensic toxicology & workplace testing. Hair testing can complement technique for conventional blood and urine analysis because it prolongs the detection window and by segmental analysis, permits discrimination between long-term therapeutic use and acute exposure.

Aims: The present study containing a simple, rapid & cost effective method for the determination of EtG in hair was developed using liquid chromatography–tandem mass spectrometry (LC–MS/MS).

Methods:Hair was successively washed with dichloromethane for 5 minutes and then with methanol for 2 minutes. The hair was then dried at room temperature and cut into fine segments with a scissor. Ten nanograms of internal standard (EtG-d5) and 2 ml of distilled water were added to 50 mg hair fine segments. 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 3500rpm for ten minutes. The supernatant was decanted & dried under nitrogen (N2). The obtained residue was reconstituted in 40µl of 0.1% formic acid. Twenty microliters of extract were injected into the LC–MS/MS system.

Result & Discussion: The best separation was achieved using a C-18 column (Inertsil, 50mm x 4.6mm x 5µ) 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 LOQ and LOD were found to be 10 pg/mg & 3 pg/mg respectively.

Conclusions: The method allows the determination of Ethyl Glucuronide (EtG) ie., metabolite of alcohol  in hair by LC-ESI-MS/MS in concentrations from 10 pg/mg (quantitatively) and 3pg/mg (Quantitatively), respectively, with an extremely reduced total time of analysis of 6 minutes. This validated method was successfully applied and qualified for the quantitative determination of EtG on proficiency test (PT) samples receives from Society of Hair Testing (SoHT).