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Selective isolation protocols of selenium (Se) species integrated to Se specific atomic absorption spectroscopy (AAS) detection were developed and optimized for Se speciation in food supplements, including selenized yeasts. By ultrafiltration, 69.18% of Se in the extract was found as a low molecular weight soluble form, the remaining 30.82% was bound to high molecular weight components. After a cation-exchange chromatography of the ultrafiltrate, 3.77% of the Se in the extract was found in the aqueous washings of the column indicating the presence of free inorganic anions of Se; the 65.41% of Se retained on the column corresponded to the free organic Se cations. The limit of detection for the HPLC-THG-AAS system was 1.85 ng of Se. Se was shown to be widely distributed over all the proteins with one sharp peak corresponding to the free forms of Se. Four major peaks were found at MW $>$ 250 000 Da (15.97% of Se recovered), between 102 330 and 117 490 Da (7.06%), between 48 977 and 53 703 Da (12.71%) and close to the dye migration band (17.25%). / Selective isolation and HPLC-AAS protocols were also developed and optimized for the determination of free organic forms e.g. selenomethionine (SeMet), selenocystine (SeCystine) and inorganic forms of selenium in aqueous solutions, and in complex matrices such as nutritional supplements and mixtures of free amino acids. The selenoamino acid in alkaline solution was first derivatized with 1-fluoro-2,4-dinitrobenzene. After removal of excess of reagent by partitioning with diethyl ether, the N-dinitrophenyl (DNP)-derivatized selenoamino acid was acidified and extracted with diethyl ether. Inorganic Se(IV) was extracted from the acidic aqueous phases by complexation with 1,2-phenylenediamine, forming a piazselenol. Se derivatives were determined selectively by HPLC-THG-AAS. A selective chromatographic mechanism based on $ pi$-electron interactions was optimized using a silica stationary phase derivatized with p-nitrophenyl moieties. Co-injections of DNP-SeMet, DNP-SeCystine and piazselenol save retention times of 3.7, 4.0 and 4.9 min, respectively, using a methanolic mobile phase containing 1.5% triethylamine and 0.013M acetic acid. Primary analytical validation parameters including stability, linearity and limits of detection were obtained using purified DNP-SeMet, DNP-SeCystine and piazselenol standards which were characterized by $ sp1$H-, $ sp{13}$C- and $ sp{77}$Se-NMR analysis and/or fast atom bombardment MS techniques. The calibration graphs for sequential dilutions of these Se standards were linear and the limits of detection from the resultant calibration graphs were 17 ng, 0.21 ng and 18.53 ng of Se, respectively. The purified DNP-SeMet and DNP-SeCystine were found to be photosensitive. The recovery of SeMet, SeCystine and inorganic Se from the stock solutions and/or nutritional supplements was virtually quantitative. In the presence of a 500-fold excess of other amino acids, the recovery of SeMet and SeCystine (96.1 $ pm$ 3.9% and 98.08 $ pm$ 4.2%, respec

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Selenium has been shown to have multiple biochemical effects ranging from nutrient deficiency at low levels to toxicity at high levels. This duality of concern has led to a demand for increased numbers of highly accurate and precise determinations of selenium in biological materials. A convenient procedure was developed for determining selenoamino acids by HPLC-THG-AAS, based on the derivatization of these analytes with Sanger's reagent. Selenomethionine, selenocystine and selenocysteine (after blocking the free selenol group with phenylmercurio cation) were converted to their N-2,4-dinitrophenyl derivatives, and separated on a Nucleosil 5-NO$ sb2$ column with methanolic mobile phase containing acetic acid and triethylamine. Furthermore, an improved HPLC-AAS interface design was modified and optimized for the detection of selenium in HPLC column eluate. The new design was (i) compatible with aqueous mobile phases containing volatile buffers and (ii) provided equivalent molar response to analytes containing Se($-$II), Se(+IV) and Se(+VI). A method for simultaneously determination of selenate, selenite, selenocystine, selenomethionine and selenoethionine was developed by using the HPLC-AAS system with aqueous acetic acid containing ammonium acetate as eluate solution on the cyanopropyl column. The equivalent low ng limits of detection (1-2 ng as Se) for different oxidation states of selenium analytes were obtained using several different mobile phases and/or columns. A phenol extraction procedure for selenate, selenite, selenocystine, selenomethionine and selenoethionine was evaluated for the determination of these selenium analytes in natural waters and wheat samples. The current HPLC-AAS system provides an inexpensive alternative to conventional techniques for the determination of selenium analytes in environmental samples.

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