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

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.

Carbon isotope ratio determination and investigation of seized testosterone preparations

“…Hence, the difference in 13 C/ 12 C isotope ratio can be effectively used to detect testosterone misuse. [7][8][9][10][11][12][13][14][15] The diagnostic metabolites selected are in most cases androstandiols, androsterone and etiocholanolone. They are isolated from urine together with an endogenous reference steroid, commonly 11b-hydroxyandrosterone, 16-androstenol or pregnandiol.…”

“…[6] The IRMS analysis is based on differences in isotope ratio between endogenous and synthetic testosterone and provides the basis for confirmatory analysis. [7][8][9][10][11][12][13][14][15] The 13 C/ 12 C ratio in natural compounds, such as steroids, is determined by the pathway by which they were produced. [16,17] Consequently, synthetic testosterone is generally less enriched in 13 C and shows a different 13 C/ 12 C ratio than human endogenous testosterone.…”

Development of a sensitive GC‐C‐IRMS method for the analysis of androgens

“…All doping control laboratories accredited by WADA have been confronted with the task of developing analytical methods and establishing criteria that allow endogenous steroids to be distinguished from their synthetic copies. It has been known for some time that GC-C-IRMS is capable of meeting this challenge by comparison of the 13 C/ 12 C ratios of the target compounds with those of endogenous reference compounds (Aguilera et al, 1996(Aguilera et al, , 1999(Aguilera et al, , 2001Shackleton et al, 1997aShackleton et al, , 1997bUeki and Okano, 1999;Saudan et al, 2004;Cawley et al, 2005). T itself and/or its main metabolites androsterone (A; 5a-androstane-3a-ol-17-one), etiocholanolone (Et; 5bandrostane-3a-ol-17-one), 5b-androstane-3a,17b-diol (bab-diol) and 5a-androstane-3a,17b-diol (aab-diol) normally function as target compounds.…”

Two‐dimensional gas chromatography with heart‐cutting for isotope ratio mass spectrometry analysis of steroids in doping control

“…Much of the early work in this area focused on the analysis of T metabolites such as 5a-and 5b-androstane-3a,17b-diols (5aDiol and 5b-Diol), androsterone (A) and etiocholanolone (E), due to their relative abundance in urine compared to T. [10,[17][18][19][20][21] The time window for confirming T administration using these analytes rather than T itself is decreased as they do not solely derive from T, and so any perturbation in the isotope ratio caused by the administration of T is diluted. [22] While successful methods have been developed for T analysis, the desire to achieve baseline separation for the analytes of interest in GC-C-IRMS analysis means these methods typically rely on multiple extensive and time consuming sample preparation steps such as liquid chromatography (LC) fraction collection.…”

“…[23,24] A further consideration for IRMS is derivatization, which improves chromatography and has therefore proved popular for steroid analysis using GC. [10,[17][18][19][20][21] Derivatization though results in the addition of extra carbons to the analyte which alters the carbon isotope ratio of the target compound. While this effect can be accounted for, extra uncertainty will be added to the reported delta value.…”