Comparative in vitro metabolism of the ‘designer’ steroid estra-4,9-diene-3,17-dione between the equine, canine and human: identification of target metabolites for use in sports doping control.

The research involves the examination of the metabolism of a designer steroid, estra-4,9-diene-3,17-dione, in different species—equine, canine, and human — to determine suitable target metabolites for detecting steroid abuse in sports. The study provides the first account of this anabolic steroid’s metabolism and compares how it’s processed in the three major species subjected to sports doping control.

Methodology and Tools Used

• The researchers used a high-resolution, accurate-mass Thermo LTQ-Orbitrap LC-MS instrument to identify the metabolites in samples. This tool enabled a detailed evaluation of the samples and a more precise determination of the key metabolites involved in the in vitro metabolism of the steroid.
• Complementary methods were utilized to obtain a more detailed structural information of the plentiful metabolites. These included HR-LC-MS/MS analysis of the underivatised metabolites, functional group selective chemical derivatisation, enzyme inhibition experiments, and full scan electron ionization GC-MS analysis of methoxyamine-trimethylsilyl derivatives.

Findings

• The principal metabolite detected in all species, which could be the best candidate for identifying estra-4,9-diene-3,17-dione abuse, was found to be an isomer of 17-hydroxy-estra-4,9-dien-3-one.
• Minor metabolic pathways common to all three species involved hydroxylation and reduction followed by secondary hydroxylation.
• The canine species demonstrated less significant reductive metabolism compared to the other two species.
• The equine species was found to be unique in generating a di-reduced metabolite, hypothesized to be an isomer of estra-4,9-diene-3,17-diol, and also created relatively large quantities of d-ring hydroxy and hydroxy-reduced metabolites.

Significance of the Study

• The study sheds light on the in vitro metabolism of the ‘designer’ steroid estra-4,9-diene-3,17-dione across different species, which is crucial for improving doping control in both human and animal sports.
• The findings provide essential knowledge on the metabolites to target when testing for possible abuse of this particular steroid.
• The results also highlight the potential differences in drug metabolism between species which could influence the approach taken in both the detection and management of substance abuse cases.