Identification and verification of the anabolic steroid boldenone in equine blood and urine by HPLC/ELISA.

The research studies the development and application of an ELISA test to detect the presence of the anabolic steroid boldenone in horse blood and urine.

ELISA Method Development

• The research revolves around creating an enzyme-linked immunosorbent assay (ELISA) to study the occurrence of the anabolic steroid boldenone in equine blood and urine.
• Scientists used boldenone combined with a bovine serum protein to stimulate an immune response in rabbits, which yielded antibodies specific to boldenone. These antibodies were used in the ELISA test.
• The sensitivity of the assay was calculated to be approximately 26.0 pg/well—meaning the test could detect boldenone down to this small amount.
• Scientists also tested whether other common steroids could interfere with the assay. Only progesterone and testosterone showed slight cross-reactivity; however, these interactions were deemed not significant.

Sampling Procedure

• Background signals in serum samples were reduced by performing the extraction procedure prior to boldenone detection.
• Urine samples, meanwhile, were analyzed immediately after dilution, without performing hydrolysis of boldenone conjugates.
• Any positive results from the initial screening were retested using high-performance liquid chromatography (HPLC) systems combined with offline detection via the developed ELISA.

Confirmation and Validation

• Methandienone, another anabolic steroid, was used as an internal standard to ascertain retention factors—meaning it was used as a control to ensure consistency in the analysis.
• The method was tested on horses treated with boldenone.
• It was concluded that the presence of boldenone in serum could be confirmed up to 28 days post-application, and in urine up to 56 days post-application, thereby validating the usefulness and accuracy of the developed ELISA method.