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(W/EH017) Optimization of clean-up procedure for determination of selenium compounds by HPLC-MAD-HG-AFS from biota samples. Gómez-Ariza, José Luis1, Caro de la Torre, Marco 1, Sánchez-Rodas, Daniel1, Velasco, Alfredo1, Giráldez, Inmaculada1, 1 ABSTRACT- Selenium is an essential trace element for most living organisms. It exists in the environmental and biological samples both as inorganic (selenite and selenate) and organic species (selenomino acids, selenoproteins). The nutritional bioavailability, toxicity and cancer chemoprotective activity of selenium depend on the concentration and the chemical form in wich it is present. Simultaneous determination for inorganic selenium and selenoamino acids, in biological samples, is a more complicated task, due to the inherent differences in physicochemical characteristics of these species. The chromatographic separation of selenocysteine, selenomethionine, selenoethionine, selenite and selenate was carried out using HPLC, with anion exchange and a reversed-phase column, both connected through a six-port switching valve. On-line microwave-asssisted digestion and hydride generation steps were performed prior to the atomic fluorescence detection. The elution of selenoamino acids was using water as mobile phase in the reversed-phase. The inorganic selenium were separated in the anion exchange column, using gradient elution with an acetate buffer. The extraction from the sample and clean-up procedure constitute a crucial step when biota samples are considered, due to possible analyte losses, changes of the species or incomplete extraction of selenium species. In this study the extraction procedure of selenium species based on enzimatic treatments from clams was performed. The procedure involved mixing 1 gr of freeze-dried clams with 30 mg of protease and 6 ml water in a teflon centrifuge tube followed by mechanical shaking at 300 rpm for 24 hours at ambient temperature. The suspended material was centrifuged for 10 min at 10.000 rpm and the supernatant was collected. Due to the complexity of the extracts, clean-up procedures are required for removing interferences and prolonging the lifetime of the chromatographic columns. This was been carried out using the solvent partitioning (hexane and dichlorometane) and solid-phase extraction (C-18, DIOL, CN, SCX and SAX). The efficiency of these clean-up methods by means of spiking and recovery experiments from real samples were studied Key words: Selenium speciation, Clean-up, Atomic fluorescence detection, LC |
