Separation and detection of Se-compounds by ion pairing liquid chromatography-microwave assisted hydride generation-atomic fluorescence spectrometry

Abstract

Liquid chromatography coupled to a hydride generation atomic fluorescence spectrometer has been applied for the speciation of Se in extracts of Saccharomyces cerevisiae. In order to develop a method which allows the separation of the compounds and detection of the element, seven Se standards were used: Se-methionine (Se-Met), Se-cystine (Se-(Cys)₂), Se-cystamine (Se-Cya), Se-methylselenocysteine (Se-MeSeCys), Se-ethionine (Se-Et), selenate (Se^VI), selenite (Se^IV). Optimal chromatographic results were obtained with reversed-phase chromatography on an XTerra C₁₈ column using a positively charged ion-pairing agent. It was observed that for these standards precise control of the pH was of utmost importance. Attention was devoted to the compatibility of the mobile phase with hydride generation. Efficient formation of the hydrides was obtained by optimisation of different parameters. The redox mixture which allowed optimum conversion of all different species was HBr–KBrO₃. To assist in the conversion of the compounds, on-line microwave digestion was applied. The detection limits obtained for the standards were: 0.8 µg Se l⁻¹ for selenite(IV); 1.3 µg Se l⁻¹ for selenate(VI); 1.2 µg Se l⁻¹ for Se-methionine; 1.2 µg Se l⁻¹ for Se-cystine; 1.3 µg Se l⁻¹ for Se-cystamine; and 1.1 µg Se l⁻¹ for Se-methylselenocysteine, respectively. Se-compounds in Saccharomyces cerevisiae were extracted by hot water (50 °C) or proteolytic digestion with protease XIV (37 °C). The method developed for separation and elemental detection was applied to these extracts in order to distinguish between the different species extracted from the yeast matrix. Total Se concentration in the extracts was measured with pneumatic nebulization-inductively coupled plasma-mass spectrometry (PN-ICP-MS). Species transformation was investigated by analysing extracts preserved at 2 different temperatures (−20 °C and 4 °C). Only those extracts kept at −20 °C proved to be unchanged.