Detection of testosterone and epitestosterone in human hair using liquid chromatography–tandem mass spectrometry

Abstract: The feasibility of using hair analysis as a complimentary test in doping control has received increased attention in the scientific community. The aim of the study was to take a step forward to this goal and develop a method that, for the first time, is able to detect testosterone (T) and epitestosterone (E) in human hair, using liquid chromatography–tandem mass spectrometry (LC–MS/MS) and alkali digestion followed by extraction using pentane. The method was linear within the quantification range of 0.25–100pg/mg for T and 0.5–100pg/mg for E, with determination coefficient (r 2) values >0.9987. The limits of detection for T and E were 0.1pg/mg and 0.25pg/mg respectively. The accuracy, precision and extraction recovery of the assay were satisfactory for the detection of T and E when ca. 50mg hair was processed. The validated method was successfully applied for the analysis of 75 hair samples collected from healthy volunteers (65.3% males), with the concentration of T between 0.7–11.81pg/mg and 0.33–6.05pg/mg and the concentration of E between 0.63–8.27pg/mg and 0.52–3.88pg/mg in males and in females respectively. In males, the T levels were significantly higher (p =0.020) but there was no difference in the E levels (p =0.359). However, E was not detectable in 34 samples (of which 19 were females). The T and E levels showed linear correlation (r =0.698, p <0.001) with average T/E ratio of 1.32±0.7. The newly developed analytical method was rapid, facile, sensitive, selective, reproducible and reliable for determining the levels of T and E in hair and thus for calculating the T/E ratio in hair. [Copyright &y& Elsevier]