Enzyme-linked immunosorbent assays for doping control of 5alpha-reductase inhibitors finasteride and dutasteride.
Finasteride and dutasteride are 5alpha-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 microg L(-1) and an IC(50) of 0.75 microg 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 microg L(-1), and IC(50) values 0.18 and 1.18 microg 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.
Brun
Eva M
EM
Departamento de Química, Instituto de Reconocimiento Molecular y Desarrollo Tecnológico, Universidad Politécnica de Valencia, Camino de Vera s/n, 46071 Valencia, Spain.
Torres
Ana
A
Ventura
Rosa
R
Puchades
Rosa
R
Maquieira
Angel
A
D000818:Animals; D000906:Antibodies / Q000276:immunology; D000918:Antibody Specificity; D001378:Azasteroids / Q000494:pharmacology / Q000652:urine; D042944:Cholestenone 5 alpha-Reductase / Q000037:antagonists & inhibitors; D004300:Doping in Sports; D000068538:Dutasteride; D004791:Enzyme Inhibitors / Q000493:pharmacokinetics / Q000652:urine; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005260:Female; D018120:Finasteride / Q000493:pharmacokinetics / Q000652:urine; D006801:Humans; D057230:Limit of Detection; D008657:Metabolic Clearance Rate; D011817:Rabbits; D015813:Substance Abuse Detection / Q000379:methods
D000906:Antibodies; D001378:Azasteroids; D004791:Enzyme Inhibitors; D018120:Finasteride; D042944:Cholestenone 5 alpha-Reductase; D000068538:Dutasteride