Analysis of exogenous nandrolone metabolite in horse urine by gas chromatography/combustion/carbon isotope ratio mass spectrometry

Nandrolone (17β-hydroxy-4-estren-3-one, NAD) is an endogenous steroid hormone; thus, the detection of its metabolites is not conclusive of NAD doping in racehorses. NAD doping control in male horses is based on the threshold, namely, the concentration ratio of 5α-estran-3β,17α-diol (ETA) to 5(10)-estren-3β,17α-diol (ETE). The ETA/ETE ratio of 1/1 was determined based on statistical data of authentic horses in International Federation of Horseracing Authorities. To individuals with complex metabolic disorders, however, such a threshold might not be applicable. The aim of this study was to establish an analytical method that discriminates endogenous steroids from exogenous ones in horse urine after NAD administration using gas chromatography/combustion/carbon isotope ratio mass spectrometry (GC/C/IRMS). Urine was sampled from NAD-administered and authentic horses. Ten millilitres of urine was hydrolyzed and subjected to liquid-liquid extraction and solid phase extraction. The residue of the extracts purified by HPLC was derivatized by acetylation. As a result of measurement of the 13C/12C ratio (δ13C) by GC/C/IRMS, the δ13C values of ETA for NAD-administered and authentic horses were −32.20 ± 0.35‰ and −27.85 ± 0.75‰ (n = 60), respectively. The detection limit of ETA in this GC/C/IRMS analysis was approximately 25 ng/ml. This study indicates that the measurement of δ13C by GC/C/IRMS enables us to discriminate exogenous ETA derived from NAD administration from endogenous ETA, proving that GC/C/IRMS is a useful technique to complement the ETA/ETE ratio.