Analysis of anabolic steroids in the horse: Development of a generic ELISA for the screening of 17α-alkyl anabolic steroid metabolites

Abstract

Due to the potential for misuse of a wide range of anabolic steroids in horse racing, a screening test to detect multiple compounds, via a common class of metabolites, would be a valuable forensic tool. An enzyme-linked immunosorbent assay (ELISA) has been developed to detect 17alpha-alkyl anabolic steroid metabolites in equine urine. 16beta-Hydroxymestanolone (16beta,17beta-dihydroxy-17alpha-methyl-5alpha-androstan-3-one) was synthesised in six steps from commercially available epiandrosterone (3beta-hydroxy-5alpha-androstan-17-one). Polyclonal antibodies were raised in sheep, employing mestanolone (17beta-hydroxy-17alpha-methyl-5alpha-androstan-3-one) or 16beta-hydroxymestanolone conjugated to human serum albumin, via a 3-carboxymethyloxime linker, as antigens. Antibody cross-reactivities were determined by assessing the ability of a library of 54 representative steroids to competitively bind the antibodies. Antibodies raised against 16beta-hydroxymestanolone showed excellent cross-reactivities for all of the 16beta,17beta-dihydroxy-17alpha-methyl steroids analysed and an ELISA has been developed to detect these steroid metabolites. Using this 16beta-hydroxymestanolone assay, urine samples from horses administered with stanozolol (17alpha-methyl-pyrazolo[4',3':2,3]-5alpha-androstan-17beta-ol), were analysed raw, following beta-glucuronidase hydrolysis, and following solid-phase extraction (SPE) procedures. The suppressed absorbances observed were consistent with detection of the metabolite 16beta-hydroxystanozolol. Positive screening results were confirmed by comparison with standard LCMS analyses. Antibodies raised against mestanolone were also used to develop an ELISA and this was used to detect metabolites retaining the parent D-ring structure following methandriol (17alpha-methylandrost-5-ene-3beta,17beta-diol) administration. The ELISA methods developed have application as primary screening tools for detection of new and known anabolic steroid metabolites.

Metabolism of Anabolic Steroids and Their Relevance to Drug Detection in Horseracing

“…The use of both MS/ MS [107][108][109] and high-resolution accurate mass MS have been proposed for this application [91,92]. A generic approach to the use of ELISA has also been proposed and demonstrated [110]. In this case, rather than raising an antibody to a specific steroid the antibodies were targeted against specific substructures common to several steroids.…”

Selective manipulation of steroid hydroxyl groups with boronate esters: efficient access to antigenic C-3 linked steroid–protein conjugates and steroid sulfate standards for drug detection

“…2 The development of antibodies and their application to the development of ELISA is in progress using previously reported methods and will be reported elsewhere. 2 Treatment of the ketones with carboxymethoxylamine hemihydrochloride, gave the corresponding carboxymethyloximes 33-36.…”

“…Our first goal was the development of assays targeting other commonly occurring D-ring substitution patterns associated with World Anti-Doping Agency (WADA) and International Federation of Horseracing Authorities (IFHA) prohibited anabolic steroids such as 17a-ethyl and 17a-ethynyl steroids. 2 In addition to the development of antibodies already reported, the conjugate 5 was required as a reagent for the ELISA and this bottleneck to the large-scale synthesis potentially limited the future application of the successful assay. It was anticipated that 17a-ethyl antigens 9 and 10 would afford Scheme 2 antibodies suitable for the detection of prohibited steroids such as ethylestrenol 11, norethandrolone 12 ( Fig.…”

A high-throughput screening (HTS) immunochemical method for the analysis of stanozolol metabolites in cattle urine samples

“…Barret et al [11] designed a hapten for the specific recognition of St introducing the spacer arm for the OH 17 of norstanozolol (NorSt), however the paper does not provide information on the antibody features and the immunoassay specificity. Hungerford et al [12] described a generic immunoassay for 17␣ alkyl anabolic steroids in equine urine by using 16␤-hydroxymestanolone as immunizing hapten. The polyclonal antibodies raised recognized very well several 16␤,17␤-dihydroxy-17␣-methyl steroids, including 16␤OH-St, while St was barely recognized.…”