Carbon isotopic characterization of prednisolone and prednisone pharmaceutical formulations: Implications in antidoping analysis

Twenty-two pharmaceutical formulations containing prednisolone or prednisone commercially available in Italy, Belgium, Spain, Brazil, and India were analyzed through a specific gas chromatography combustion isotope ratio mass spectrometry (GC-C-IRMS) method. All of them showed typical non-endogenous delta C-13 values, except for the Belgian nasal spray, Sofrasolone (R), with a less depleted(13)C content (-17.84 +/- 0.18 parts per thousand). Observational studies were performed on two volunteers in therapy with Sofrasolone (R) to confirm the applicability of the method and to suggest adequate interpretation criteria also in the case of drugs with less negative delta C-13 values. Urine samples were collected before, during, and within the 36 hours after the administration of the spray. Both delta C-13 values and urinary concentrations of prednisolone and prednisone were evaluated. All samples were subjected to an adequate pre-treatment (enzymatic hydrolysis, liquid/liquid extraction, and two sequential HPLC steps) before injection to the GC-C-IRMS instrument, according to the method recently developed and validated in our laboratory. Pregnanediol (PD), tetrahydro-11-deoxycortisol (THS), and pregnanetriol (PT) were selected as endogenous reference compounds (ERC). The excretion profile was estimated through liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) method used routinely for the quali-quantitative detection of glucocorticoids. delta C-13 values and urinary levels of prednisolone and prednisone were also determined after the intake of one single vial of Sintredius (R), a prednisolone oral formulation with a conventional more negative delta C-13 value (-29.28 +/- 0.25 parts per thousand). Finally, the potential masking effect that combined therapy with Sofrasolone (R) and Sintredius (R) could induce on the IRMS findings was investigated.