Doping control for metandienone using hair analyzed by gas chromatography–tandem mass spectrometry

A sensitive, specific and reproducible method for the quantitative determination of the anabolic metandienone in human hair has been developed. The preparation involved a decontamination step with methylene chloride. The hair sample (about 50 mg) was solubilised in 1 ml 1 M NaOH, 10 min at 95 °C, in presence of 2 ng of nandrolone-d3 used as internal standard. The homogenate was neutralized and extracted using consecutively a solid-phase extraction (Isolute C18 eluted with methanol) and a liquid–liquid extraction with pentane. The residue was derivatized by adding 5 μl MSTFA/NH4I/2-mercaptoethanol (250 μl; 5 mg; 15 μl) and 45 μl MSTFA, then incubated for 20 min at 60 °C. A 1 μl aliquot of derivatized extract was injected into the column (HP5-MS capillary column, 5% phenyl–95% methylsiloxane, 30 m × 0.32 mm i.d., 0.25 μm film thickness) of a Hewlett Packard (Palo Alto, CA, USA) gas chromatograph (6890 Series). Metandienone was identified using three transitions (its daughter ions at m/z 339 and 206 for the parent 444 and 191 for 206) using a Waters Quattro Micro MS–MS system. The transition m/z 444 to 206 has been used as quantification transition and the others as identification transitions. The assay was capable of detecting 2 pg/mg of metandienone when approximately 50 mg of hair material was processed. Linearity was observed for metandienone concentrations ranging from 2 to 500 pg/mg with a correlation coefficient of 0.9997. Intra-day and between-day precisions at 50 pg/mg were 13.4–16.5% and 22.0%, respectively, with an extraction recovery of 48%. The analysis of hair, cut into four segments, obtained from an athlete, revealed the presence of metandienone at the concentrations of 78, 7, 10 and 108 pg/mg in each segment of hair (0–1, 1–2, 2–3 and 3 cm to the tip).