Issue 8, 2004

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

Abstract

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, Se-methylseleno-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.

Article information

Article type
Paper
Submitted
04 May 2004
Accepted
09 Jun 2004
First published
19 Jul 2004

J. Anal. At. Spectrom., 2004,19, 950-957

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

L. Bendahl and B. Gammelgaard, J. Anal. At. Spectrom., 2004, 19, 950 DOI: 10.1039/B406589A

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