In vitro generation of 34S-labelled steroid sulfates as reference material

Code: 18A16AK 

In anti-doping control, the detection of steroid conjugates is of increasing relevance. The wide detection window of certain conjugates in combination with efficient and highly sensitive LC-MS technology enables a prolonged traceability of hormone misuse. The reliable quantitation of steroid conjugates requires isotope-labelled reference materials, which are only available for a minority of doping relevant conjugates. An approach to generate steroid sulfates is the in vitro incubation with human liver S9 fraction.

The objective of the present study is to produce isotope-labelled steroid sulfates in vitro. This will be accomplished by the incubation of relevant steroids or their metabolites with liver S9 fraction and 34S-sodium sulfate. The in vitro generated reference material can be directly used for dilute-and-shoot quantification of the respective steroid sulfates in human urine samples. Moreover, the established approach could be rapidly expanded to comparable steroids and methods or materials may be shared with other WADA accredited laboratories.

Main Findings: 

The project aimed at the generation of 34S-labelled epiandrosterone sulfate as internal standard for doping routine analysis. This in vitro-synthesis should be accomplished by incubation of epiandrosterone with liver S9 fraction and 34S-labelled sodium sulfate as co-factor. Results: We introduced a modification of a previously described protocol which replaces phosphoadenosin-5’-phosphosulfate by sodium sulfate and adenosine-5’-triphosphate in the S9 fraction incubation. By using isotope-labelled sodium sulfate (Na234SO4), we generated 34S-labelled epiandrosterone-sulfate with a purity of ≥99.9 %. With this purity, the in vitro generated steroid sulfate fulfills the requirement for an internal standard for quantitation purposes. The sulfonation rate was found to be very low (13 %) and attempts to increase the reaction yield were not successful. 
Conclusions: In conclusion, we successfully introduced a protocol to generate isotope-labelled epiandrosterone sulfate with purity. Due to the low percentage of the epiandrosterone sulfonated by the S9 fraction, we were not able to provide the intented amount of min. 5 mg epiandrosterone-34SO4. Future plans: As the modified protocol was proven to generate 34S-isotope-labelled epiandrosterone, it is possible to expand investigations on comparable steroids. Future investigations should focus on the optimization of the sulonation rate of the subsrate.