Selenium speciation and isotope composition in 77Se-enriched yeast using gradient elution HPLC separation and ICP-dynamic reaction cell-MS

A batch of ⁷⁷Se-labelled and enriched yeast was characterised with regard to isotopic composition and content of selenium species for later use in a human absorption study based on the method of enriched stable isotopes. The abundance of the six stable selenium isotopes was determined by ICP-MS equipped with a dynamic reaction cell (DRC). The results showed that the ⁷⁷Se isotope was enriched to 98.5 atom-%, whereas the remaining selenium was present as the other five isotopes at low abundance. The low-molecular ⁷⁷Se containing species, which were biosynthesised by the yeast during fermentation using the enriched ⁷⁷Se-selenite as substrate, were released by enzymatic hydrolysis using (I), a β-glucosidase followed by a protease mixture, and (II), a commercial protease preparation. For selenium speciation the chromatographic selectivity of the cation exchange HPLC system was adjusted to the separation of over 30 selenium species occurring in the hydrolysates by applying gradient elution using pyridinium formate as mobile phase. The quantitative results obtained by detection with ICP-DRC-MS of ⁷⁷Se and ⁸⁰Se showed that both enzymatic sample preparation systems released 90–95% of the yeast's selenium content. The total area of the cation exchange chromatograms, however, amounted to 64% of the total selenium content in the yeast, which was 1390 µg g⁻¹. In the enzymatic extracts selenomethionine (SeMet) constituted 82% of all separated and quantified selenium species, which was equivalent to 53% of the total selenium content in the yeast. Oxidation of SeMet to selenomethionine-Se-oxide (SeOMet) occurred during sample preparation. The degree of formation of SeOMet was large and variable when using enzyme system I, but low when using enzyme II.