Quantification of osilodrostat in horse urine using LC/ESI-HRMS to establish an elimination profile for doping control

Abstract

Aim: The use of osilodrostat, developed as a medication for Cushing's disease but categorized as an anabolic agent, is banned in horses by both the International Federation of Horseracing Authorities and the Fédération Equestre Internationale. For doping control purposes, elimination profiles of hydrolyzed osilodrostat in horse urine were established and the detectability of free forms of osilodrostat and its major metabolite, mono-hydroxylated osilodrostat (M1c), was investigated.Materials & methods: Post-administration urine samples obtained from a gelding and three mares were analyzed to establish the elimination profiles of osilodrostat using a validated method involving efficient enzymatic hydrolysis followed by LC/ESI-HRMS analysis.Results: Applying the validated quantification method with an LLOQ of 0.05 ng/ml, hydrolyzed osilodrostat could be quantified in post-administration urine samples from 48 to 72 h post-administration; by contrast, both hydrolyzed osilodrostat and M1c were detected up to 2 weeks. In addition, confirmatory analysis identified the presence of hydrolyzed osilodrostat for up to 72 h post-administration.Conclusion: For doping control purposes, we recommend monitoring both hydrolyzed M1c and osilodrostat because of the greater detectability of M1c and the availability of a reference material of osilodrostat, which is essential for confirmatory analysis.

Keywords: LC/ESI–HRMS; anabolic agent; doping control; elimination profile; equine; osilodrostat; quantitative analysis; urine.

Figure 1.

Brief summary of the metabolic pathway of osilodrostat in equines. Percentages represent approximate relative compositions of osilodrostat and its metabolites in the post-3 h administration samples after 50 mg of osilodrostat was administered to four horses. An entire osilodrostat metabolic pathway including 37 metabolites is available in the authors' previous study [11].

Figure 2.

Typical extracted ion chromatograms of osilodrostat and its IS in horse urine. Left: Osilodrostat; right, IS (TIPB); (A) Spiked sample at 0.05 ng/ml of osilodrostat in urine; (B) Pre-administration urine sample; (C) Post-administration urine sample at 72 h. product ion scan conditions for osilodrostat and IS, respectively: precursor ions, m/z 228.0932, m/z 224.1182; collision energies, 20 eV, 30 eV; product ions, m/z 81.0447, m/z 82.0525.

Figure 3.

Elimination profile of osilodrostat in urine after 50 mg of osilodrostat was orally administered to a gelding and three mares via a nasoesophageal tube. Note that post-54 h and -72 h administration samples for the gelding were unavailable according to the predefined protocol for a pilot study.

Figure 4.

Overlay of extracted ion chromatograms of osilodrostat and mono-hydroxylated osilodrostat with their three diagnostic product ions. Left, osilodrostat; right, mono-hydroxylated osilodrostat (M1c). (A) 50 pg of osilodrostat reference material; (B) 72-h post-administration urine sample; (C) 3-h post-administration urine sample with 100-fold dilution; (D) 240-h post-administration urine sample. Polarity, positive; mass accuracy, 5 ppm; precursor ion of osilodrostat, m/z 228.0932, precursor ion of M1c, m/z 244.0881; product ions of osilodrostat, m/z 81.0447, m/z 54.0338 and m/z 134.0401, product ions of M1c, m/z 97.0396, m/z 95.0604 and m/z 226.0775. RA, relative abundance (%) (ratio of the peak area of the product ion to the peak area of the base peak).