Performance Characteristics of a Carbon Isotope Ratio Method for Detecting Doping with Testosterone Based on Urine Diols: Controls and Athletes with Elevated Testosterone/Epitestosterone Ratios

Abstract

Abstract Background: Carbon isotope ratio methods are used in doping control to determine whether urinary steroids are endogenous or pharmaceutical. Methods: Gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) was used to determine the δ13C values for 5β-androstane-3α,17β-diyl diacetate (5βA), 5α-androstane-3α,17β-diyl diacetate (5αA), and 5β-pregnane-3α,20α-diyl diacetate (5βP) in a control group of 73 healthy males and 6 athletes with testosterone/epitestosterone ratios (T/E) >6. Results: The within-assay precision SDs for 5βA, 5αA, and 5βP were ± 0.27‰, ± 0.38‰, and ± 0.28‰, respectively. The between-assay precision SDs ranged from ± 0.40‰ to ± 0.52‰. The system suitability and batch acceptance scheme is based on SDs. For the control group, the mean δ13C (SD) values were −25.69‰ (± 0.92‰), −26.35‰ (± 0.68‰), and −24.26‰ (± 0.70‰), for 5βA, 5αA, and 5βP, respectively. 5βP was greater than 5βA and 5αA (P <0.01), and 5βA was greater than 5αA (P <0.01). The means − 3 SD were −28.46‰, −28.39‰, and −26.37‰ for 5βA, 5αA, and 5βP, respectively. The maximum difference between 5βP and 5βA was 3.2‰, and the maximum 5βA/5βP was 1.13. Three athletes with chronically elevated T/Es had δ13C values consistent with testosterone administration and three did not. Conclusions: This GC-C-IRMS assay of urine diols has low within- and between-assay SDs; therefore, analysis of one urine sample suffices for doping control. The means, SDs, ±3 SDs, and ranges of δ13C values in a control group are established. In comparison, testosterone users have low 5βA and 5αA, large differences between 5βA or 5αA and 5βP, and high 5βA/5βP and 5αA/5βP ratios.