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

Abstract

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.

Elimination profiles of prednisone and prednisolone after different administration routes: Evaluation of the reporting level and washout periods to ensure safe therapeutic administrations

“…It is important to highlight that the proposed reporting levels will solve problems of results interpretation due to the endogenous presence of PRED and PSONE in urine. The production of low concentrations of PRED and PSONE from cortisol and cortisone, respectively, due to the microbial activity has been described in urine samples, and sophisticated confirmation procedures, including IRMS analysis, need to be performed when these compounds are detected in urine at concentrations up to 60 ng/ml 28,29 . With the new proposed reporting levels, the application of these confirmation procedures will be not needed.…”

A novel approach to improve detection of glucocorticoid doping in sport with new guidance for physicians prescribing for athletes

“…The new RLs will avoid problems of results interpretation of some specific GCs. In particular, the problem of the endogenous production of PRED and PSONE at low concentrations due to microbial activity in the urine samples which involved sophisticated confirmatory procedures including isotope-ratio mass spectrometer (IRMS) analysis 82 is solved with the new RLs.…”

Annual banned‐substance review: Analytical approaches in human sports drug testing 2019/2020

“…This proposal is of particular relevance in the light of WADA's Technical Letter on the in situ formation of prednisone and prednisolone, where GC/C/IRMS confirmation of the exogenous origin of these analytes is recommended, when urinary concentrations between 30 and 60 ng/ml are observed 168 . An analytical approach addressing this question was presented by Iannella et al, which included a urine sample preparation protocol composed of enzymatic hydrolysis, LLE, and two subsequent LC fractionation steps employing a C‐18 as well as a C‐18 amide semi‐preparative column (both 250 × 4.6 mm, 5 μm particle size) 169 . As ERCs, the analytes PD, pregnanetriol, and tetrahydro‐11‐deoxycortisol were selected, and dedicated GC programs were developed for optimized prednisone and prednisolone peak purity, employing a 5%‐phenyl‐methylpolysiloxane capillary column (30 m × 0.25 mm, 0.25 μm film thickness).…”