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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 0.25-100 pg/mg for T and 0.5-100 pg/mg for E, with determination coefficient (r²) values0.9987. The limits of detection for T and E were 0.1 pg/mg and 0.25 pg/mg respectively. The accuracy, precision and extraction recovery of the assay were satisfactory for the detection of T and E when ca. 50 mg 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 0.7-11.81 pg/mg and 0.33-6.05 pg/mg and the concentration of E between 0.63-8.27 pg/mg and 0.52-3.88 pg/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, p0.001) 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.

Recent abuse of designer drugs such as mephedrone has presented a requirement for sensitive, reliable and reproducible methods for the detection of these controlled drugs in different matrices. This study focuses on a fully developed validated method for the quantitative analysis of mephedrone and its two metabolites 4-methylephedrine and 4-methylnorephedrine in human hair. The calibration curve was found to be linear in the range 5-100 pg/mg for mephedrone and 10-150 pg/mg for 4-methylephedrine and 4-methylnorephedrine. The method was successfully validated for the intraday precision, interday precision, limit of detection, accuracy and extraction recovery. Five out of 154 hair samples were confirmed to be positive for mephedrone. Due to the structural similarities to other methcathinones and amphetamines, one can propose the metabolism for mephedrone based on a similar pathway that has been previously used for these psychoactive drugs. The outlined method can be valuable for the future detection of mephedrone and its two metabolites in hair.