Sample Pretreatment for Confirmation of 19 – Norsteroids by GC – C – IRMS in Doping Control

Abstract

Confirmation of the use of 19-norsteroids requires the identification of the exogenous origin of the main metabolite, 19-norandrosterone (1g-NA), when found in the urine at concentrations between 2.5 ng/ml and 15 ng/ml. An effective pretreatment procedure is necessary prior to the IRMS measurement. Two HPLC clean up steps were employed to isolate the fractions of 19-NA, '19-noretiocholanolone (19-NE) and two endogenous reference compounds (ERCs), pregnandiol (PD) and androsterone (Andro). Validation followed the WADA requirements as specified in TD2017NA and TD2016|RMS. Blank urine, 20-30 mL, spiked with 19-NA and 19- NE at 2.5-15 ng/ml, were used for method validation-regarding intra and inter-assay precision. The accuracy and isotopic fractionation were evaluated using standard 19-NA, 19-NE and certified standard USADA34-1 spiked in steroid stripfed urine. The results show no interference peak close to the target compounds. The difference between the 613C (%d of pure certified standard and spiked steroid stripped urine of less than 0.5 %. verified the accuracy of 613C (%.) measurement and the absence of isotopic fractionation during sample prep.aratipn. This method is fit for the determination of 6l3C (%.) of 19-NA and 19-NE for anti-doping purposes.