Biosynthesis of isotopically enriched selenomethionine: application to its accurate determination in selenium-enriched yeast by isotope dilution analysis-HPLC-ICP-MS

Abstract

Isotope dilution analysis is proposed for the determination of selenomethionine (SeMet) in Se-enriched yeast material by high performance liquid chromatography inductively coupled plasma mass spectrometry (HPLC-ICP-MS) using a ⁷⁷Se-enriched selenomethionine spike obtained by yeast growth on a ⁷⁷Se-rich culture medium. Different methods of yeast enrichment with selenium were evaluated to determine the most effective one. The use of two different culture media, amount of selenium added, amount of carbon source (glucose) and harvest time were evaluated in order to characterize the total selenium incorporated by the yeast cells. The yeast proteins were hydrolysed with protease XIV, and the isotopically enriched SeMet isolated by anion exchange chromatography. The isotopic composition of the SeMet in the spike solution was determined by on-line HPLC-ICP-MS and reverse isotope dilution was used for the characterization of the spike by means of a natural SeMet standard. In the analysis of the Se-enriched yeast material, the sample was spiked with ⁷⁷Se-enriched SeMet, and hydrolyzed with a non-specific enzyme (protease XIV) at 37 °C for 20 hours or using an ultrasonic probe for 1 min. Both ⁷⁸Se/⁷⁷Se and ⁸⁰Se/⁷⁷Se isotope ratios were measured by ICP-MS with collision and reaction cell after the separation of selenomethionine by HPLC. The method was applied to the determination of selenomethionine in a candidate yeast reference material (SEAS project), and the results obtained were in agreement with those reported by other laboratories.