Detection of stanozolol in hair by negative ion chemical ionization mass spectrometry

Stanozolol is an anabolic androgenic steroid occasionally abused by athletes. A sensitive, specific, and reproducible method for the quantitative determination of stanozolol in hair has been developed. After the addition of stanozolol-d3 as the internal standard, hair samples (10-25 mg) were digested with 2 mL of 1N NaOH at 65°C for at least 2 h. Digest solutions were then extracted using solid-phase extraction. The eluents were evaporated, a mixture of N-methyl-N-trimethylsilylheptafluorobutryamide (MSHFBA) and trimethylsilylimidazole (TSIM) (1000:20, v/v) was added, and the mixture heated at 80° C for 5 minutes. After cooling to room temperature, N- methyl-bis-heptafluorobutyramide (MBHFBA) was added and the mixture healed at 80°C for 30 min. The derivatized extracts were analyzed on a Finnigan MAT(TM) 4500 mass spectrometer in the negative chemical ionization mode. Chromatographic separation was achieved with helium carrier gas on a HP-1 capillary column (15 m x 0.2-mm i.d.; 33-μm film thickness). The assay was capable of reliably quantitating 50 pg/mg of stanozolol and was linear to 2500 pg/mg. Intra-assay precision was 13.2% at 50 pg/mg and 6.6% at 2500 pg/mg. Interassay precision was 13.7% at 50 pg/mg and 6.1% at 2500 pg/mg. This method has been applied to the analysis of stanozolol incorporated into rat hair. Male Long-Evans rats were given stanozolol 20 mg/kg intraperitoneally once daily for 3 days. The mean concentrations of stanozolol in the rat hair collected on day 14 were 362.4 ± 332.4 pg/mg in pigmented hair and 90.0 ± 46.9 pg/mg in nonpigmented hair. These data demonstrate that stanozolol is incorporated preferentially into pigmented hair.