A high-performance liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for the determination of oxandrolone concentration in human plasma (0.5 mL) was developed and validated according to the 2001 FDA Bioanalytical Guidelines. Oxandrolone is an anabolic steroid used to promote weight gain for cachectic patients with severe burn injuries, HIV/AIDS, hepatitis C and other wasting syndromes. The assay procedure involved a liquid-liquid extraction of oxandrolone and methyltestosterone (the internal standard, IS) from plasma with n-butyl chloride. The organic layer was clarified by centrifugation and evaporated to dryness under a stream of air. The residue was reconstituted in a solution containing 25% methanol and 75% Milli-Q water, and injected onto a Luna C18 reversed-phase HPLC column (30 mm × 2.0 mm, 2 μm). Separation of oxandrolone and methyltestosterone was achieved with a mobile phase starting composition of 55% methanol and 45% ammonium formate buffer at a flow rate of 0.1 mL/min. The total run time was 21 min per sample. Selected reaction monitoring mode was used for quantifying oxandrolone (m/z 307 → 271) and the IS, methyltestosterone (m/z 301 → 149). To the authors' knowledge, this is the first LC-MS-MS method validated for oxandrolone quantification in human plasma. This method can be used in future pharmacokinetic studies involving oxandrolone.
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