Separation and identification of Se-methylselenogalactosamine -: a new metabolite in basal human urine -: by HPLC-ICP-MS and CE-nano-ESI-(MS)2

Three minor metabolites were isolated from human urine. Two of these were identified by nano electrospray ionisation mass spectrometry (nESI-MS) as Se-methylseleno-N-acetylglucosamine and Se-methylselenogalactosamine, respectively. A human urine pool was lyophilised and reconstituted in methanol prior to fractionation by preparative reversed phase HPLC. In addition to the major urinary metabolite, Semethylseleno-N-acetylgalactosamine, more than seven minor metabolites were separated by this system and detected by ICP-MS. Three of the metabolite fractions were isolated, re-chromatographed in the reversed phase system and further purified in different separation systems before analysis by nESI-MS. By CE-nESI-MS analysis of one of the fractions, the characteristic selenium pattern was recognized around m/z 285 and ( MS) 2 fragmentation resulted in a fragments at m/z 267, 173 and 155, respectively. It was not possible to identify this selenium compound on basis of the available data. The selenium compound in the second fraction showed co-elution with a Se-methylseleno-N-acetylglucosamine standard. The identity of this compound was verified by nESI-MS after further purification by size exclusion chromatography. The third fraction was further purified by ion-pair and anion exchange chromatography, reconstituted and subjected to CE-nESI-MS. The m/z of the compound was 258 and ( MS) 2 resulted in a fragment at m/z 162, corresponding to loss of methylselenium. This indicated that the structure of the compound was Se-methylselenogalactosamine. To verify the identity of the compound, the Se-methylselenogalactosamine and the Se-methylselenoglucosamine were prepared by hydrolysis of the corresponding N-acetylhexosamines. The mass spectra of these standards were identical and also identical to the mass spectra of the purified urine compound. The urine selenium compound co-eluted with Se-methylselenogalactosamine in a reversed phase chromatographic system able to separate Se-methylselenogalactosamine and Se-methylselenoglucosamine. Analysis of basal urine samples from volunteers who had not been supplemented with selenium showed the presence of Se-methylselenogalactosamine when only traces of the metabolite Se-methylseleno-N-acetylgalactosamine, which is the major metabolite in urine after selenium supplementation was present. Hence, this new metabolite may be the main metabolite in basal urine.