Development and validation of a method to confirm the exogenous origin of prednisone and prednisolone by GC‐C‐IRMS

Prednisone and prednisolone are two anti‐inflammatory steroidal drugs listed by the World Anti‐Doping Agency (WADA) within the class of glucocorticoids, which are prohibited “in competition” and when administered systemically. Their presence in collected urine samples may be attributed, if no exogenous administration has occurred, to an in situ microbial formation from endogenous steroids. In this work, a gas chromatography coupled to carbon isotope ratio mass spectrometry (GC‐C‐IRMS) method was developed and validated to distinguish an exogenous origin from an endogenous one. Eight prednisone/prednisolone pharmaceutical preparations commercially available in Italy were analysed to establish an exogenous δ¹³C value reference range (?28.96 ± 0.39‰). No more than 25 mL of urine was processed and no derivatization nor intentional steroids structure modifications were performed before the GC‐C‐IRMS analysis. A first HPLC purification step was set up to isolate the three endogenous reference compounds (ERCs) selected (tetrahydro‐11‐deoxycortisol (THS), pregnanediol (PD), and pregnanetriol (PT)), while a second LC purification was necessary to separate prednisone from prednisolone. In the GC‐C‐IRMS analysis, two different GC run methods were set up to guarantee better sensitivity and selectivity for each compound. Both prednisone and prednisolone showed signals (m/z 44) with amplitudes within the method linearity range to a lower urinary concentration of 20 ng/mL (< WADA reporting level, 30 ng/mL). The method was fully validated according to WADA requirements. As a proof of concept, urine samples collected from two excretion studies in healthy male volunteers, after a prednisone or prednisolone administration, were analysed by the proposed method, demonstrating its applicability for the analysis of real samples.