Optimization of Sample Preparation for the Analysis of Selenium Metabolites in Trout

Abstract

When speciation of ultratrace level compounds is performed, sample preparation often results to be a crucial step in the analysis. The use of high performance liquid chromatography (HPLC) also requires in most cases a sample whose matrix was purified in order to preserve the chromatographic support and thus ensure a proper separation. Analysis of selenium metabolites in blood is a challenge because of the very low concentrations found and the presence of proteins that deteriorate the chromatographic separation. The aim of this work was to optimize the sample preparation of trout whole blood and blood plasma in order to minimize loss of analytes usually observed during the deproteinization step. In this context, two deproteinization reagents were tested and compared. The losses of analytes were evaluated by monitoring two isotopically enriched Se tracers (77Se-selenite and 76Se-selenomethionine) during the precipitation of proteins. These tracers were quantified after deproteinization of the sample in both the supernatant and the pellet by inductively coupled plasma-mass spectrometry (ICP-MS) and reverse isotope dilution (RID). The results showed that the losses caused by the conventionally used deproteinization reagent were in the order of 20 % higher than those obtained with an alternative reagent in trout blood plasma sample. On the other hand, the losses in trout whole blood sample were slightly higher with the conventionally reagent than with the alternative one. Both samples, trout whole blood and blood plasma with the added Se-enriched tracers, were compared by size exclusion chromatography (SEC) with ultraviolet (UV) detection and SEC-ICP-MS. Moreover, qualitative analysis of selenometabolites by HPLC-ICP-MS in trout tissues (liver, kidney and spleen), using acetonitrile as reagent in the deproteinization step, was carried out.