Separation and identification of zaleplon metabolites in human urine using capillary electrophoresis with laser-induced fluorescence detection and liquid chromatography-mass spectrometry

A capillary electrophoresis (CE) method using laser-induced fluorescence (LIF) detection for the determination of the hypnotic drug zaleplon and its metabolites in human urine could be developed using carboxymethyl-p-cyclodextrin as a charged carrier. By the help of a complementary HPLC method coupled to mass spectrometry, three metabolites present in human urine could be identified as 5-oxozaleplon, 5-oxo-N-deethylzaleplon and 5-oxozaleplon glucuronide. N-Deethylzaleplon, a previously described zaleplon metabolite, as well as zaleplon itself could not be detected in human urine by the CE-LIF assay. The results were confirmed by spiking with reference compounds of the phase I metabolites. The metabolites differed very much concerning their fluorescence intensities, thus the 5-oxo metabolites present as lactam tautomer fluoresced tenfold lower than the unchanged drug zaleplon and its N-deethylated metabolite. The glucuronide of the 5-oxozaleplon, however, showed high fluorescence due to its lactim structure. Limits of quantification yielded by the CE-LIF assay including a ten-fold preconcentration step by solid-phase extraction were 10 ng/ml for zaleplon and N-deethylzaleplon and 100 ng/ml for 5-oxozaleplon and 5-oxo-N-deethylzaleplon. (C) 2003 Elsevier B.V. All rights reserved.