Determination of ¹³C/¹²C ratios of urinary epitestosterone and its main metabolites 5α‐ and 5β‐androstane‐3α, 17α‐diol

Abstract

Epitestosterone (17alpha-hydroxy-androst-4-en-3-one, EpiT) belongs to the list of prohibited substances of the World Anti-Doping Agency (WADA). Although it possesses no anabolic effect, it is presumed to be misused by athletes in order to mask administration of testosterone (T) by lowering the urinary T/EpiT ratio.To improve detection, an excretion study with 40 mg of orally administered EpiT was conducted focusing on the metabolites of EpiT: 5alpha- and 5beta-androstane-3alpha,17 alpha-diol (5aEpiD and 5bEpiD). A reference population of n = 74 volunteers was investigated to elucidate the urinary concentrations of these steroids.In order to prove whether an unusual finding in urinary concentrations or ratios is due to an illicit intake of steroids or due to physiological elevation, determination of carbon isotope ratios is advisable. A method for isotope ratio determination was developed to enable (13)C/(12)C ratios of EpiT, 5bEpiD, 5aEpiD, pregnanediol and androsterone and etiocholanolone to be measured from a single urine specimen. The method's validity was tested by applying linear mixing models and specificity was ensured by means of gas chromatography/mass spectrometry analysis. delta(13)C values at natural levels were determined with a reference population and both Delta values and corresponding reference limits were calculated.Considering the implemented EpiT-metabolites, a more than twofold extension of the detection time of EpiT administration was achieved with both the urinary concentration thresholds and the (13)C/(12)C ratios.

Recent developments in the use of isotope ratio mass spectrometry in sports drug testing

“…This finding was supported by data from routine doping control samples and another RefPop investigated [19,79]. PD showed the same pattern, whereas EpiT, investigated in the latter population [79], was not significantly different for males and females, but its metabolites 5aEpiD and 5bEpiD were. Steroids excreted as sulfates demonstrated the same trend towards more depleted values for females, but only DHEA and 5EN17a were found to be significant [83].…”

“…more negative, δ 13 C values in their urinary steroids OHA and A. This finding was supported by data from routine doping control samples and another RefPop investigated [19,79]. PD showed the same pattern, whereas EpiT, investigated in the latter population [79], was not significantly different for males and females, but its metabolites 5aEpiD and 5bEpiD were.…”

“…Investigations on the misuse of 4-androstene-3,11,17-trione, a nutritional supplement [77], and a comprehensive method to detect cortisone misuse, again using oxidation of target analytes, were published [78]. Another research focus was on EpiT and its metabolites 5aEpiD and 5bEpiD, which provided data for another RefPop (n=74), again showing differences between the genders in their CIR [79], and the first extensive results on the 2 H/ 1 H ratio of endogenous urinary steroids were reported [80]. As expected, HIR showed a larger variation in their δ 2 H values, but the associated Δ values were found to be stable; this might become a promising approach for doping control.…”

“…A closer look at the data revealed that the difference could not be attributed to gender but rather could be attributed to the use of oral contraceptives [71,79]. Only those women using contraceptives showed significantly different CIR in several endogenous steroids (Fig.…”

“…Usually a decrease in the amount of some steroids excreted can be found. But the concentration of steroids excreted is usually not correlated to the corresponding δ 13 C values [72,79]. One possible explanation may be the influence of administered contraceptives on the different enzymatic reactions taking place inside the body, transforming steroids.…”