Detection of sulfo-conjugated anabolic steroids metabolites in antidoping initital and confirmatory analysis

Code: 11A13CG

Anabolic androgenic steroids (AAS) are the most frequent abused substances in sports and are included in the World Antidoping Agency (WADA) List of prohibited substances. AAS are extensively metabolized in the liver and their target tissues. The metabolic pathways are divided into phase-I and phase-II reactions. Phase-I reactions involve oxidation, hydrolysis and reduction, which introduce new functional groups for the subsequent phase-II reactions (i.e. conjugation).

For the endogenous androgens and for exogenous AAS the main phase-II reactions are conjugation with glucuronic acid (glucuronidation) or with a sulfo-moiety (sulphatation). The screening methods used by the Antidoping Laboratories usually focus on those metabolites, that are excreted unconjugated or as glucuronides into the urine. Extraction of the gluco- deconjugated steroids from the matrix and concentration of the analytes is performed by liquid-liquid extraction (with diethylether or tertbutylmethylether) or solid phase extraction followed by mass spectrometric detection either by liquid chromatography mass spectrometry or gas chromatography mass spectrometry. By using this initial screening extraction protocol the Antidoping Laboratories substantially ignore the sulfo-conjugated part of AAS. Nevertheless there are AAS for which the longer detected metabolite is a sulfo-conjugate. Sulfo-conjugated AAS are relatively easy to be detected directly since the development of instrumentation providing interfacing of liquid chromatographic (LC) separation to mass spectrometric (MS) detection, especially via electrospray ionisation (ESI), has opened up broad possibilities for the direct analysis of sulfo-conjugated substances. Moreover, sulfo-conjugated substances can be extracted from urine using ethyl acetate, instead of diethylether, as extraction solvent. The objectives of this project will be:

a) To develop a screening method for the already known sulfo-conjugated metabolites of AAS, and

b) To investigate the existence of not yet reported sulfo-conjugated metabolites of AAS, that can improve detectability and identification in either initial screening protocol or confirmation methods.

Main Findings

Anabolic steroids such as oxandrolone, madol, formebolone, methenolone, 17- methylnandrolone and mesterolone were tested for the existence of sulfo-conjugated metabolites. Metabolic samples from long-term excretion studies were tested for any sulphate metabolite and where any sulphate metabolite was found, an evaluation of its retrospectivity was performed in comparison with their free and gluco-conugated metabolites used for their monitoring in GC/HRMS analysis. In most cases where new metabolites were found, a detailed characterization of their structures based on mass spectrometry techniques was also performed. Additionally, spotted metabolic samples for oxymetholone, drostanolone, norethandrolone, danazol, clostebol, methandriol, calusterone, furazabol, fluoxymesterone, oxymesterone, boldenone, mesterolone, methandienone, methyltestosterone, oral turinabol, methenolone, and tibolone that include the known, up to that time, anabolic steroids with sulphates metabolites, as well as other AASs with unknown sulphate metabolism were tested in order to develop a new screening method for sulphate metabolites. Samples from the above listed steroids were extracted and analyzed using a screening method based on alkaline extraction with ethylacetate and LC/QTOF analysis in a negative mode. Potential sulfate metabolites of these steroids were drawn and the molecular ions were calculated and extracted using the instrument software.

Conclusions

The investigation of sulfo-conjugated metabolites of methenolone and mesterolone led to the discovery of new metabolites of at least equal or better retrospectivity compared to the already known gluco-conjugated metabolites detected by GCMS. Furthermore, a sulfo-conjugated long-term metabolite of 17-methylnandrolone was discovered (unpublished results). The analysis of madol, formebolone and oxandrolone didn’t lead to any new (sulfo-) metabolite, at least to a concentration level that would be detectable by the technology used in this study. A screening method for sulfo-conjugated metabolites was developed for several anabolic steroids based on literature data, using accurate mass measurements for the extracted ion chromatograms and leading to a number of new sulfo-conjugated metabolites. Their structures, as well as their retrospectivity for the monitoring of their parent compounds abuse for doping control purposes were not evaluated in the framework of this project.