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When humans are supplied with selenium-containing nutritional preparations, one of the selenium-containing metabolites in urine increases relatively more than the other selenium metabolites. The purpose of this study was to identify this major selenium metabolite. Urine samples from six male volunteers were collected and analysed by ion-pair chromatography with ICP-MS detection for this major selenium metabolite. Samples containing the metabolite were pooled and solid phase extracted to remove ionic substances. The extracted pool was purified and preconcentrated twice by preparative reversed-phase chromatography. The fractions containing the selenium metabolite were collected and further purified by size exclusion chromatography. It was not possible to ionize the selenium metabolite by electrospray ionization mass spectrometry, ESI-MS. Instead, atmospheric pressure chemical ionization, APCI, was applied. The m/z of the selenium metabolite was 300 for the Se-80 isotope. MS/MS experiments indicated that the metabolite was a selenosugar, and it is proposed that the selenium metabolite is a Se-methyl-N-acetylselenohexosamine