Investigation of the presence of endogenous prednisolone in equine urine by high-performance liquid chromatography mass spectrometry and high-resolution mass spectrometry

RATIONALE: After the detection of low concentrations of prednisolone in racehorse urine samples collected at Italian racetracks, a study was initiated to investigate the accuracy of the analytical protocol used and the possible endogenous origin of detected prednisolone. METHODS: Multiple reaction monitoring (MRM) MS2 acquisition with a triple quadrupole (n = 780) and full scan MS2 and MS3 (n = 180) acquisition with a linear ion trap were checked. As a further confirmation, ten urine samples were analysed by high-resolution mass spectrometry (HRMS). RESULTS: The study showed the difficulty of identifying prednisolone, probably due to interfering compounds with the same molecular weight (360 Da) present in the matrix. The characteristic transitions for prednisolone were identified, both in MS2 and MS3, as the ions 187 and 280; the ion 295 was also used for identification. The concentrations detected with the triple quadrupole and the linear ion trap were not statistically different. The exact mass of prednisolone formiate (the adduct acting as a molecular ion) was identified by HRMS. CONCLUSIONS: The very high frequency of prednisolone detection in the samples (78.5%), the low concentration of this steroid and, importantly, the narrow range of the 95% confidence limits (0.97–1.05 in MS2 mode and 0.88–1.04 in MS3 mode), could represent evidence that its presence is endogenous. In the light of these results, this hypothesis seems the most probable, even if further studies are required to confirm it. Furthermore, a microbiological origin (i.e. fermentation of cortisol after sample collection) could not be disregarded.