Isolation and enrichment of Boldenone and its main metabolite in urinary samples to further determine the 13C/12C ratios by gas chromatography / combustion / isotope ratio mass spectrometry

Abstract

Gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) can be used to detect the synthetic forms of endogenous anabolic androgenic steroids (EAAS) by comparing the 13C/12C ratios of the endogenous reference compound to that of the target compound. Isolation and enrichment of the target compound from urinary matrices is an essential prerequisite for the GC/C/IRMS confirmation procedure in doping control analysis. Boldenone (Bo) is a natural anabolic androgenic steroid (AAS) and a derivative of testosterone. The GC/C/IRMS confirmation procedure for Bo and its main metabolite 5β-androst-1-en-17β-ol-3-one (BoM) is extremely complicated due to the low concentrations and the enormously complex matrices in urine. The present study demonstrated a sample purification procedure for GC/C/IRMS by using online 2D-HPLC to purify Bo and BoM in urine samples. Bo and BoM with concentrations as low as 2 ng/mL were isolated and enriched with superior purity and selectivity. The validity of the method was verified with the technical document issued by the world anti-doping agency. The online 2D-HPLC purification procedure featured high selectivity for the analytes and no isotopic fractionation in the collection process. The present method can be used as a routine method allowing doping control laboratories to perform Boldenone confirmation.