MSACL 2019 EU Abstract

Prednisone and Prednisolone Detection in Urine Samples: A GC-C-IRMS Method to Discriminate Exogenous or Endogenous Origin

Loredana Iannella (1,2), Cristiana Colamonici (1), Davide Curcio (1), Francesco Botrè (1,3), Xavier de la Torre (1)
(1) Laboratorio Antidoping, Federazione Medico Sportiva Italiana, Rome, Italy (2) Dipartimento di Chimica e Tecnologie del Farmaco, “Sapienza” Università di Roma, Rome, Italy (3) Dipartimento di Medicina Sperimentale, “Sapienza” Università di Roma, Rome, Italy

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Loredana Iannella (Presenter) Laboratorio Antidoping, FMSI >> POSTER (PDF)

Presenter Bio: Loredana Iannella is a scientific researcher at the WADA accredited Anti-doping Laboratory of Rome since 2016. She obtained her master degree in Pharmaceutical Chemistry and Technology at the “Sapienza” University of Rome; her master project was focused on the development and application of flow cytofluorimetry based techniques to detect liposome encapsulated hemoglobins in human biological fluids. She studied and characterized different drug delivery systems carrying peptide hormones relevant to anti-doping purpose. She is currently a PhD student in Pharmaceutical Sciences at the “Sapienza” University. Her work focuses specifically on the development, the validation and the optimization of GC-C-IRMS analytical methods to detect pseudo-endogenous steroids in doping controls, also evaluating potential confounding factors on the 13C/12C ratio of the selected target compounds.

Relevant Financial Disclosures (within past 24 months) No relevant financial relationship(s) to disclose. Conflict mitigation NOT REQUIRED.

Introduction: Prednisolone and its prodrug, prednisone, are two glucocorticoids banned “in competition” by the World Anti-Doping Agency (WADA) when administered by intravenous, intramuscular or rectal routes. They could be found in human urine even if no exogenous administration occurred, because of an ex vivo Δ1-steroid-hydrogenation of cortisol and cortisone physiologically excreted in urine. WADA has established that an additional confirmatory analysis should be performed on samples in which the prednisone and prednisolone concentrations are between 30 and 60 ng/mL to discriminate their exogenous origin from the ex vivo endogenous one.
Objective: A new method based on gas chromatography coupled to carbon isotope ratio mass spectrometry (GC-C-IRMS) was developed and fully validated.
Method: Urine samples (max volume: 25 mL) were processed without any derivatization or chemical structure modification according to the method routinely used in our laboratory for the confirmation analysis of pseudo-endogenous steroids. Two LC purification steps were developed before the GC-C-IRMS instrumental analysis to remove potential interferences in the determination of the δ13C (‰) values. Tetrahydro-11-deoxycortisol (THS), pregnanediol (PD) and pregnanetriol (PT) were selected as endogenous reference compounds (ERCs). Two different GC methods were implemented to guarantee an adequate sensitivity for each compound. Linearity, selectivity, limit of quantification (LOQ), recovery, repeatability and measurement uncertainties were evaluated. Sixteen different prednisone and prednisolone pharmaceutical formulations commercially available in Europe were analysed to determine their carbon isotope composition. The method was assessed to be fit for purpose by the analysis of urine samples from excretion studies on healthy volunteers.
Results: Prednisone showed a linear response (r2 = 0.999) within 300 to 3200 mV and a δ13C (‰) average value of -28.94 ± 0.33 ‰, whereas prednisolone exhibited a linear response (r2 = 0.997) between 250 and 2860 mV and a δ13C (‰) average value of -28.70 ± 0.27 ‰. In both methods, no interfering peaks were detected at the expected retention time of the two target compounds and the LOQ was established at 20 ng/mL. The δ13C (‰) of samples deviated less than 0.5 ‰ from the reference materials delta values. The uncertainty of the method was estimated at 0.3 ‰ for both compounds. All the pharmaceutical formulations showed typically exogenous δ13C (‰) values, except for one Belgian product (-16.26 ± 0.24 ‰). The δ13C (‰) values obtained from the excretion studies agreed with those previously determined from the pharmaceutical preparations.
Conclusions: The set-up conditions allowed to obtain reproducible and reliable delta values within the linearity range down to the concentration of 20 ng/mL. The method can be applied, as required by WADA, to identify the exogenous or the ex vivo origin of prednisone and prednisolone.