Analysis of sulfate metabolites of the doping agents oxandrolone and danazol using high performance liquid chromatography coupled to tandem mass spectrometry

• Sulfate metabolites as powerful tool in doping analysis.
• A sulfate metabolite of a long term metabolite of oxandrolone was identified an in excretion.
• Sulfate conjugates of the danazol metabolites ethisterone and 2-hydroxymethylethisterone were synthesized.
• The synthesized sulfate conjugates of the danazol metabolites were identified successfully in an excretion urine.
• Suitability of the identified sulfate metabolites for doping analysis was demonstrated by validation.

The direct detection of sulfate conjugates of anabolic androgenic steroids (AAS) can be a powerful tool in doping control analysis. By skipping the solvolysis step analysis time can be reduced, and due to long term sulfate metabolites the detection time can be significantly extended as demonstrated for some AAS. This study presents the successful identification of sulfate metabolites of the doping agents oxandrolone and danazol in excretion urines by high performance liquid chromatography coupled to tandem mass spectrometry (HPLC–MS/MS). The sulfate conjugate of 17β-hydroxymethyl-17α-methyl-18-nor-2-oxa-5α-androsta-13-en-3-one could be identified as a new metabolite of oxandrolone. Sulfate conjugates of the danazol metabolites ethisterone and 2α-hydroxymethylethisterone were identified in an excretion urine for the first time. In addition, these sulfate conjugates were synthesized successfully. For a confirmation analysis, the number of analytes can be increased by additional sulfate conjugates of danazol metabolites (2-hydroxymethyl-1,2-dehydroethisterone and 6β-hydroxy-2-hydroxymethylethisterone), which were also identified for the first time. The presented validation data underline the suitability of the identified sulfate conjugates for doping analysis with regard to the criteria given by the technical documents of the World Anti-Doping Agency (WADA).