Analysis for selenium speciation in selenized yeast extracts by two-dimensional liquid chromatography with ICP-MS and electrospray MS-MS detection

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Abstract
An analytical approach allowing the detection of previously unreported glutathione S-conjugates with selenocompounds in water extracts of nutritional yeast supplements was developed. The procedure was based on the use of two-dimensional size-exclusion and reversed-phase HPLC for the separation of selenospecies, ICP-MS for the monitoring of the eluting selenium, and electrospray MS-MS for the identification of the eluted species. The presence of six compounds with molecular masses of 197, 603, 562, 584, 372 and 432 (in the elution order from reversed-phase HPLC) was identified on the basis of the selenium isotopic pattern. The identity of the Mr 197 and 432 species was confirmed, by collision induced dissociation MS, to be selenomethionine and Se-adenosylhomocysteine, respectively. The compounds with larger Mr (562, 584, 604) were demonstrated, by reaction with dithiothreitol followed by HPLC-ICP-MS and ES-MS-MS, to contain a Se–S bridge between glutathione (γGlu–Cys–Gly) and a selenocompound that did not respond in the positive ES-MS mode. The MS-MS analysis of the original compound confirmed the presence of a selenium-containing fragment that could be fragmented only at high fragmentation energies. A similar Se-containing moiety (m/z 227), resistant to collision induced dissociation, was found in the 372 compound attached to a glutamine (Gln) residue as demonstrated by MS-MS.

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