Speciation of organic and inorganic selenium in a biological certified reference material based on microbore ion-exchange chromatography coupled to inductively coupled plasma atomic emission spectrometry via a direct injection nebulizer or coupled to electrothermal atomic absorption spectrometry

Abstract

A speciation method for selenomethionine, selenite, selenate, selenocystine and trimethylselenonium is described, based on anion-exchange chromatographic separation using two different metal-free microbore anion-exchange columns followed by ICP-AES for detection of selenium. The chromatographic eluate is nebulized into the plasma at a flow rate of 80 µl min^–1 using a direct injection nebulizer (DIN). It is possible to separate selenomethionine, selenite, selenate and selenocystine within 5 min. Selenocystine was found to be completely retained and was therefore eluted with a plug of dilute HNO₃. On-line enrichment of selenocystine was investigated and was found to be quantitative. Since selenomethionine and trimethylselenonium were found to co-elute in the solvent front, a second column was used on which it was possible to separate both compounds without changing the mobile phase composition. Detection limits were between 20 and 38 ng ml^–1 (0.30–0.57 ng absolute) for the five selenium species (3s criterion). The relative standard deviation was 2–6% for the peak area and 3–12% for the peak height. The method was then applied to sample extracts from CRM 402, a certified reference material of biological origin certified for a high total concentration of selenium. This material is a lyophilized powder of white clover grown on selenium-enriched soil. Four different extraction procedures based on either sonication or mechanical shaking were compared with respect to their ability to extract selenium from CRM 402. Extraction efficiencies and losses of total selenium during extraction and work-up were monitored using ETAAS. In some instances the results obtained by ion chromatography (IC)–DIN-ICP-AES were checked by microbore IC coupled to a fraction collector followed by detection of selenium using ETAAS. Selenate and possibly selenocystine were found in the sample extracts along with unknown species. The extracts were spiked in order to check the separation efficiency and to verify the identity of incipient species. Evidence of breakdown of added selenocystine during the extraction is also presented.