An analytical procedure based on ultra‐performance liquid chromatography‐mass spectrometry was developed to screen and to confirm dutasteride and its metabolites in human urine. Sample preparation included an enzymatic hydrolysis followed by solid‐phase extraction using the strong cation exchange cartridges OASIS® MCX. The chromatographic separation was carried out on C18 column, employing as mobile phases ultra purified water and acetonitrile, both containing 0.1% formic acid. Detection was achieved using a triple quadrupole as a mass spectrometric analyzer, with positive ion electrospray ionization and multiple reaction monitoring as acquisition mode. The analytical procedure developed was validated according to ISO 17025 and World Anti‐Doping Agency guidelines. The extraction efficiency was estimated to be greater than 75% for both dutasteride and its hydroxylated metabolites. Detection capability was determined in the range of 0.1–0.4 ng/mL. Specificity and repeatability of the relative retention times (CV% < 0.5) and of the relative abundances of the characteristic ion transitions selected (CV% < 10) were confirmed to be fit for purpose to ensure the unambiguous identification of dutasteride and its metabolites in human urine. The developed method was used to characterize the urinary excretion profile of dutasteride after both chronic and acute administration of therapeutic doses. After chronic administration, dutasteride and its hydroxylated metabolites were easily detected and confirmed. After acute administration, instead, only the two hydroxylated metabolites were detected for 3–4 days.

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