Fast and Sensitive Screening of Oxandrolone and Its Major Metabolite 17-Epi-Oxandrolone in Human Urine by UHPLC—MS/MS with On-Line SPE Sample Pretreatment

Abstract

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.

Fast and Sensitive Quantification of AccQ-Tag Derivatized Amino Acids and Biogenic Amines by UHPLC-UV Analysis from Complex Biological Samples

“…The developed method was further validated based on the work of Galba et al and Grey et al [ 26 , 27 ] in accordance with the FDA guidelines. Calibration curves of 0.25–30 µmol/L range were registered in MilliQ water, serum and tear matrices and the linear range, limit of detection (LOD) and limit of quantification (LOQ) were calculated based on the results obtained by LC ( Table 1 and Table S1 ) and by SRM analysis ( Table S2 ).…”

“…The UHPLC-UV method coupled with SRM analysis was validated for linearity, accuracy, intra- and inter-day variability, recovery, matrix effect, limit of detection (LOD), limit of quantification (LOQ) and stability according to the FDA guidelines [ 58 ] as described by Galba et al and Grey et al [ 26 , 27 ].…”