A simplified procedure for GC/C/IRMS analysis of underivatized 19-norandrosterone in urine following HPLC purification

Nandrolone and/or its precursors are included in the World Anti-doping Agency (WADA) list of forbidden substances and methods and as such their use is banned in sport. 19-Norandrosterone (19-NA) the main metabolite of these compounds can also be produced endogenously. The need to establish the origin of 19-NA in human urine samples obliges the antidoping laboratories to use isotope ratio mass spectrometry (IRMS) coupled to gas chromatography (GC/C/IRMS). In this work a simple liquid chromatographic method without any additional derivatization step is proposed, allowing to drastically simplify the urine pretreatment procedure, leading to extracts free of interferences permitting precise and accurate IRMS analysis. The purity of the extracts was verified by parallel analysis by gas chromatography coupled to mass spectrometry with GC conditions identical to those of the GC/C/IRMS assay. The method has been validated according to ISO17025 requirements (within assay precision of ±0.3‰ and between assay precision of ±0.4‰). The method has been tested with samples obtained after the administration of synthetic 19-norandrostenediol and samples collected during pregnancy where 19-NA is known to be produced endogenously. Twelve drugs and synthetic standards able to produce through metabolism 19-NA have shown to present δ13C values around −29‰ being quite homogeneous (−28.8 ± 1.5; mean ± standard deviation) while endogenously produced 19-NA has shown values comparable to other endogenous produced steroids in the range −21 to −24‰ as already reported. The efficacy of the method was tested on real samples from routine antidoping analyses.

Research highlights
► We developed a method based on HPLC prepurification and GC-IRMS analysis to discriminate the endogenous from the exogenous origin of 19-norandrosterone in human urine.
► Samples obtained after the administration of synthetic 19-norandrostenediol (exogenous origin) and samples collected during pregnancy (endogenous origin) were preliminarily assayed by this method to confirm the validity of the approach.
► The method has been validated according to ISO17025 requirements (within assay precision of ±0.3‰ and between assay precision of ±0.4‰), fullfilling the criteria of the World Antidoping Agency for the accredited laboratories and it is currently applied for the analysis of real samples in routine antidoping analyses.