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 M_r 197 and 432 species was confirmed, by collision induced dissociation MS, to be selenomethionine and Se-adenosylhomocysteine, respectively. The compounds with larger M_r (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.