Enzyme-linked immunosorbent assays for doping control of 5α-reductase inhibitors finasteride and dutasteride

Abstract

Finasteride and dutasteride are 5α-reductase inhibitors included in the World Anti-Doping Agency's list of banned substances. Two highly sensitive and selective ELISA assays were developed for these compounds. Polyclonal rabbit antibodies were raised using synthesized haptens and other commercial products. The best immunoassay obtained, based on an antibody-coated format, showed a limit of detection of 0.01 μg L −1 and an IC 50 of 0.75 μg L −1 for finasteride (cross-reactivity with dutasteride < 4%). The second assay allowed finasteride and dutasteride determination, with limits of detection of 0.013 and 0.021 μg L −1, and IC 50 values 0.18 and 1.18 μg L −1, respectively. Both assays were highly selective to a set of anabolic steroids, but they showed 37% and 30% cross-reactivity with the major urinary metabolite of finasteride, allowing its determination. The developed ELISA had better sensitivity than HPLC/MS/MS method and was applied as a screening technique to quantify dutasteride, finasteride, and its main metabolite in human urine without sample pre-treatment. Moreover, the analysis of dutasteride's excretion urines by ELISA was used to obtain its human excretion rate, essential to improve the analytical strategies about this type of drugs (permitted as medicines and prohibited in sport) and to establish an effective anti-doping policy.