Doping control for methenolone using hair analysis by gas chromatography–tandem mass spectrometry

A sensitive, specific and reproducible method for the quantitative determination of methenolone in human hair has been developed. The sample preparation involved a decontamination step of the hair with methylene chloride. The hair sample (about 100 mg) was solubilized in 1 ml 1 M NaOH, 15 min at 95°C, in presence of 1 ng testosterone-d3 used as internal standard. The homogenate was neutralized and extracted using consecutively a solid-phase (Isolute C18 eluted with methanol) and a liquid–liquid (pentane) extraction. The residue was derivatized by adding 50 μl MSTFA–NH4I–2-mercaptoethanol (1000:2:5, v/v/v), then incubated for 20 min at 60°C. A 1.5-μl aliquot of the derivatized extract was injected into the column (HP5-MS capillary column, 5% phenyl–95% methylsiloxane, 30 m×0.25 mm I.D., 0.25 μm film thickness) of a Hewlett-Packard (Palo Alto, CA, USA) gas chromatograph (6890 Series). Methenolone was detected by its parent ion at m/z 446 and daughter ions at m/z 208 and 195 through a Finnigan TSQ 700 MS–MS system. The assay was capable of detecting 1 pg/mg of methenolone when approximately 100 mg hair material was processed. Linearity was observed for methenolone concentrations ranging from 2 to 100 pg/mg with a correlation coefficients of 0.965–0.981. Intra-day and between-day precisions at 2, 10 and 25 pg/mg were 10.9–14.1% and 13.7–16.8%, respectively, with an extraction recovery of 97.6%. The analysis of a strand of hair obtained from two bodybuilders, revealed the presence of methenolone at the concentrations of 7.3 and 8.8 pg/mg.