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.

Keywords: 17-epi-oxandrolone; human urine; on-line SPE extraction; oxandrolone; tandem mass spectrometry; ultra-high performance liquid chromatography.

Figure 1

Chemical structure of (A) oxandrolone, (B) epi-oxandrolone, and (C) the methandienone as IS.

Figure 2

LC-QTOF identification of oxandrolone (OXA) and its metabolite. The MS2 spectrum of (A) standard OXA, (B) OXA in a urine sample, (C) OXA metabolite 17-epi-oxandrolone in a urine sample, and (D) the structures of molecular and daughters ions used for identification and quantification.

Figure 3

Elimination of urine matrix interferents in the on-line SPE sample pretreatment. Influence of the loading time of the washing solution (30% CAN) on the sample clean-up. MRM chromatogram of oxandrolone (OXA) and methandienone as IS: (A) urine spiked with the standard of OXA and IS with the loading time of the washing solution to be 1.5 min, (B) urine spiked with the standard of OXA and IS with the loading time of the washing solution to be 3.0 min.

Figure 4

Representative chromatograms of oxandrolone (OXA) and methandienone as IS illustrating a real biomedical application of the SPE-UHPLC-MS/MS method. (A) Blank urine spiked with IS; (B) blank urine spiked with standard OXA (100 pg·mL⁻¹) and IS, (C) urine sample taken 48 h after administration of one tablet (Oxandrix (DMX laboratories), 10 mg declared content per tablet) (OXA, Rt = 5.22 min; 17-epi-OXA, Rt = 5.70 min), (D) urine sample taken 9 days after the administration (OXA, Rt = 5.20 min). MRM transition 307.3 → 271.2 for OXA and 17-epi-oxandrolone, and 301.3 → 149.2 for IS were used.

Figure 5

Time vs. concentration profiles of OXA eliminated from the body in urine. OXA was monitored in the urine samples taken from a healthy volunteer after administration of a 10 mg dose of OXA (one tablet Oxandrix). Each point in the profiles corresponds to a mean obtained from 3 consecutive measurements. (A) Time vs. concentration profile of OXA, (B) time vs. concentration profile of OXA employing quantitative OXA data normalized to creatinine.

Figure 6

Scheme of SPE enrichment process in SPE-UHPLC-MS/MS method. Diagrams illustrating two positions of switching valve: (A)—load position, (B)—elute position. Enrichment column: Acquity UPLC BEH C18 Column (1.7 µm, 2.1 × 50 mm) (Waters), loading solution: ammonium formate (10 mM; pH = 6.2)/ACN (30%), elution solution: ammonium formate (10 mmol/L; pH = 6.2)/ACN (30–90%) gradient.