title: Investigation of the presence of endogenous prednisolone in equine urine by high-performance liquid chromatography mass spectrometry and high-resolution mass spectrometry.
abstract: BACKGROUND
  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) MS(2) acquisition with a triple quadrupole (n = 780) and full scan MS(2) and MS(3) (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 MS(2) and MS(3), 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 MS(2) mode and 0.88-1.04 in MS(3) 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.
authors.affiliation: U.N.I.R.E. Lab. S.r.l., Via Gramsci 70, 20019, Settimo Milanese (MI), Italy.
authors.forename: Marco
authors.initials: M
authors.lastname: Fidani
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Pompa
authors.forename: Francesca
authors.initials: F
authors.lastname: Mungiguerra
authors.forename: Alessio
authors.initials: A
authors.lastname: Casati
authors.forename: Maria Letizia
authors.initials: ML
authors.lastname: Fracchiolla
authors.forename: Francesco
authors.initials: F
authors.lastname: Arioli
mesh_terms: D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods* / Q000662:veterinary; D004300:Doping in Sports / Q000517:prevention & control; D006736:Horses; D058573:Performance-Enhancing Substances / Q000652:urine*; D011239:Prednisolone / Q000652:urine*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods* / Q000662:veterinary
chemical_list: D058573:Performance-Enhancing Substances; D011239:Prednisolone