Analysis of anabolic steroids in the horse: development of a generic ELISA for the screening of 17alpha-alkyl anabolic steroid metabolites.

The research article focuses on developing a new screening test that can detect a variety of anabolic steroids in horse urine through the identification of a specific class of metabolites.

Research Methodology

To address the problem, the researchers developed and tested an enzyme-linked immunosorbent assay (ELISA) for the detection of 17alpha-alkyl anabolic steroid metabolites in horse urine.

• The process involved synthesizing 16beta-Hydroxymestanolone from a commercially available substance called epiandrosterone.
• They then produced polyclonal antibodies using specific antigens that were raised in sheep.
• To evaluate the cross-reactivity of the antibodies, the researchers tested their ability to bind with 54 representative steroids.
• With the generated antibodies, a new ELISA was defined to identify the 16beta,17beta-dihydroxy-17alpha-methyl steroid metabolites.

Applying the ELISA Test

The ELISA they developed was then used to analyze urine samples collected from horses treated with an anabolic steroid called stanozolol.

• The samples were analyzed in their raw form, post beta-glucuronidase hydrolysis, and after solid-phase extraction procedures.
• The resulting absorbances indicated the detection of a metabolite chemical known as 16beta-hydroxystanozolol.
• The positive screening results were further validated by comparing them with the standard LCMS analyses.

Further Application of Developed Antibodies

The antibodies that were generated against mestanolone were further used to develop a new ELISA to detect metabolites with parent D-ring structures.

• This secondary application was used to determine the presence of these metabolites after administration of methandriol.

Conclusion and Further Application

In conclusion, the ELISA methods that were developed provide valuable tools for screening both new and known anabolic steroid metabolites in horse urine. This has significant potential in enhancing the monitoring process in horse racing and tackling the misuse of anabolic steroids.