Simultaneous immunochemical detection of stanozolol and the main human metabolite, 3′-hydroxy-stanozolol, in urine and serum samples

Two enzyme-linked immunosorbent assays (ELISAs) have been established for the analysis of stanozolol (St) and 3′-hydroxy-stanozolol (3′OH-St), the main metabolite found in humans. The immunizing hapten N2′-(5-valeric acid)-androst-2-eno[3,2-c]-pyrazol-17a-methyl-17b-ol (hapten 8) has been designed with the aid of molecular modeling and theoretical tools to allow immunochemical detection of both compounds. Using an ELISA based on a homologous antisera/coating antigen combination, St can be selectively quantified without significant interference of the St metabolites or other steroids potentially present in the biological samples. On the other hand, St immunoreactivity equivalents due to the additional presence of 3′OH-St can also be quantified using an ELISA based on a heterologous antisera/coating antigen combination, in which the metabolite can be detected with 51% cross-reactivity. Thus, As147/5BSA detects 3′OH-St and St in buffer with IC50 values of 1.46 and 0.68 μg L−1, respectively. In contrast, As147/8BSA is highly specific for St with an IC50 of 0.16 μg L−1 and a limit of dection of just 0.022 μg L−1. Performance of both assays in urine and serum samples has been evaluated and demonstrate that inappropriate use of stanozolol by athletes or young people can be detected in these matrices after simple cleanup methods, with IC50 values below the minimum performance required levels established by the World Antidoping Agency.