Speciation Analysis of Arsenic and Selenium by HPLC and Mass Spectrometry

Abstract

New methodologies have been developed for the determination of arsenic and selenium species in a variety of environmentally important matrices. A simple liquid chromatographic separation technique based upon mini-column technology was developed to obtain a simultaneous, fast, efficient and reliable separation of relatively toxic from relatively non-toxic arsenic and selenium species. The relatively toxic arsenic and selenium species studied were inorganic Asv, AsIII, SeVI and SeIV. The relatively non-toxic species of arsenic and selenium studied were AsBet, DMA and Se Met. Optimum conditions were found to be the use of a Hamilton PRP X100 12-20 µm anion-exchange resin with column dimensions of 100 x 3 mm. The mobile phase utilized a 10 mM K2S04 solution at pH 10.2 with a flow rate of 1 ml minˉ¹ and a sample injection loop of 100 µ1. Total analysis time was under 7 minutes with limits of detection in the range of 2.0 - 10 µg kgˉ¹ for arsenic and selenium species, respectively. Work was undertaken, using HPLC-ICP-MS instrumentation, as part of a feasibility study, into the production and certification of six new reference materials; these being analyzed for the species of arsenic, in chicken, fish, rice and soil samples, and selenium, in wheat and yeast samples. Enzyme extraction techniques were used throughout, except for soil where a microwave H3P04 extraction was used. Efficiencies were in the range 90-100%. The results obtained provided speciation information as well as total elemental concentrations with no operationally defined limits. Speciation analysis requires that the endogenous species are extracted without modification of their chemical form or disturbance to the equilibrium existing between the various species present. Work was undertaken to identify and quantify the selenium species present in two samples of novel, previously unstudied, bio-natured nutrients, these nutrients being: i) a selenized yeast from a new process and: ii) a probiotic bacteria-based dried milk sample (Biogurt®). Specific interest was directed towards enzyme, MeOH and KOH and TMAH extraction efficiencies together with retention of species information. Selenium speciation was performed using ion-exchange HPLC-ICP-MS. It was found that the selenium content, in the form of SeMet, was adequately extracted from the yeast (Pharma Nord) that was used for method validation using protease, which yielding 90% of the total selenium. However, the determination of selenium and selenium species in the bionatured nutrients proved to be quite problematic. Methods that avoided species conversion with the highest extraction efficiencies were found to be: i) the use of protease for the yeast sample (19%) and; ii) the use of 0.01 M HCl for the Biogurt® (71%). Information obtained from speciation of these samples by anion and cation-exchange HPLC-ICP-MS was limited due to the low extraction efficiencies of any procedure undertaken for the samples, by the retention of the analyte on-column and by the lack of standards available for matching of retention times. HPLC-ICP-MS has proved an efficient tool for the identification and determination of arsenic and selenium species providing detection limits at µg kgˉ¹ levels. However, a major concern with this instrumentation is the unambiguous assignment of peaks which relies on the chromatographic purity of the signal and the availability of standards. Anion-exchange chromatography employing Hamilton PRP X100 resin with NH4HC03 (10 mM, pH 10.2 for arsenic and 10-50 mM, pH 5 for selenium species) with methanol (10 %, v/v) as the mobile phase allowed separation of the arsenic and selenium species investigated under conditions that were compatible for both HPLC-ICP-MS and HPLC-ESMS. Molecular ions and structural fragmentation patterns of these by tandem MS have facilitated the identification of chromatographic peaks obtained using HPLC-ICP-MS. In the analysis of marine algae, arsenosugars were the major species found, and in yeast the dominant species was found to be selenomethionine.