Detecting the abuse of 19-norsteroids in doping controls: A new gas chromatography coupled to isotope ratio mass spectrometry method for the analysis of 19-norandrosterone and 19-noretiocholanolone

The detection of 19-norsteroids abuse in doping controls currently relies on the determination of 19-norandrosterone (19-NA) by gas chromatography-tandem mass spectrometry (GC-MS/MS). An additional confirmatory analysis by gas chromatography coupled to isotope ratio mass spectrometry (GC-C-IRMS) is performed on samples showing 19-NA concentrations between 2.5 and 15 ng/ml and not originated from pregnant female athletes or female treated with 19-norethisterone. 19-Noretiocholanolone (19-NE) is typically produced to a lesser extent as a secondary metabolite. The aim of this work was to improve the GC-C-IRMS confirmation procedure for the detection of 19-norsteroids misuse. Both 19-NA and 19-NE were analyzed as target compounds (TCs), whereas androsterone (A), pregnanediol (PD), and pregnanetriol (PT) were selected as endogenous reference compounds (ERCs). The method was validated and applied to urine samples collected by three male volunteers after the administration of nandrolone-based formulations. Before the instrumental analysis, urine samples (<25 ml) were hydrolyzed with beta-glucuronidase from Escherichia coli and extracted with n-pentane. Compounds of interest were purified through a single (for PT) or double (for 19-NE, 19-NA, A, and PD) liquid chromatographic step, to reduce the background noise and eliminate interferences that could have affect the accuracy of delta C-13 values. The limit of quantification (LOQ) of 2 ng/ml was ensured for both 19-NA and 19-NE. The 19-NE determination could be helpful in case of unstable urine samples, in late excretion phases or when coadministration with 5 alpha-reductase inhibitors occur.

Automated summary

The current method for detecting 19-norsteroids abuse relies on GC-MS/MS analysis of 19-NA, with confirmatory analysis by GC-C-IRMS. This study aimed to improve the confirmation procedure using GC-C-IRMS and validated the method using urine samples from male volunteers administered nandrolone-based formulations. Both 19-NA and 19-NE were analyzed as target compounds, with A, PD, and PT chosen as endogenous reference compounds for validation.