Oxandrolone, a synthetic testosterone analog, is used for the treatment of several diseases associated with weight loss. Unfortunately, oxandrolone is abused by many athletes and bodybuilders due to its strong anabolic effect. We have developed and validated a highly sensitive and rapid on-line SPE-UHPLC-MS/MS method for the determination of oxandrolone and simultaneous identification of its major metabolite 17-epi-oxandrolone in urine matrices. Enrichment of the analytes via an integrated solid-phase extraction was achieved using an Acquity UPLC BEH C18 Column. Subsequently, the chromatographic separation of the on-line preconcentrated sample fraction was achieved using an Acquity HSS T3 C18 Column. For the structural identification of these analytes, a high-resolution mass spectrometer Synapt-G2Si coupled to the Acquity M-class nano-LC system with ionKey source was used. A highly sensitive determination of oxandrolone was achieved using a tandem quadrupole mass spectrometer XEVO TQD. The method was successfully validated in the linear range of oxandrolone from 81.63 pg·mL-1 (limit of quantification, LOQ) to 5000 pg·mL-1 in the human urine matrix. It was applied to the analysis of real urine samples obtained from a healthy volunteer after the oral administration of one dose (10 mg) of oxandrolone. Concentration vs. time dependence was tested in the time interval of 4 h-12 days (after oral administration) to demonstrate the ability of the method to detect the renal elimination of oxandrolone from the human body. Favorable performance parameters along with successful application indicate the usefulness of the proposed method for its routine use in antidoping control labs.

Biomedical Center Martin Jessenius Faculty of Medicine in Martin Comenius University in Bratislava 036 01 Martin Slovakia

Biomedical Research Center of the Slovak Academy of Sciences in Bratislava 84510 Bratislava Slovakia

Department of Analytical Chemistry Faculty of Science Charles University Hlavova 8 128 43 Prague 2 Czech Republic

Department of Chemistry Faculty of Natural Science Matej Bel University in Banska Bystrica 974 09 Banska Bystrica Slovakia

Department of Pharmaceutical Analysis and Nuclear Pharmacy Faculty of Pharmacy Comenius University in Bratislava Odbojarov 10 832 32 Bratislava Slovakia

Department of Pharmacology Jessenius Faculty of Medicine in Martin Comenius University in Bratislava 036 01 Martin Slovakia

Institute of Neuroimmunology Slovak Academy of Sciences Dubravska cesta 9 84510 Bratislava Slovakia

Toxicological and Antidoping Center Faculty of Pharmacy Comenius University in Bratislava Odbojarov 10 832 32 Bratislava Slovakia