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

An analytical approach allowing the detection of previously unreported glut athione 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 se lenospecies, ICP-MS for the monitoring of the eluting selenium, and electro spray MS-MS for the identification of the eluted species. The presence of s ix compounds with molecular masses of 197, 603, 562, 584, 372 and 432 (in t he elution order from reversed-phase HPLC) was identified on the basis of t he selenium isotopic pattern. The identity of the M-r 197 and 432 species w as confirmed, by collision induced dissociation MS, to be selenomethionine and Se-adenosylhomocysteine, respectively. The compounds with larger M-r (5 62, 584, 604) were demonstrated, by reaction with dithiothreitol followed b y HPLC-ICP-MS and ES-MS-MS, to contain a Se-S bridge between glutathione (g amma Glu-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 pres ence of a selenium-containing fragment that could be fragmented only at hig h fragmentation energies. A similar Se-containing moiety (m/z 227), resista nt to collision induced dissociation, was found in the 372 compound attache d to a glutamine (Gln) residue as demonstrated by MS-MS.