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Size exclusion and anion-exchange chromatographies coupled with inductively coupled plasma-mass spectrometry (ICP-MS) were used for the speciation of selenium (Se) in a dietary supplement. A sequential extraction method resulted in 85% recovery of Se and 78% of the Se extracted could be identified. The results obtained show that selenomethionine and its oxide are the predominant compounds, while selenite and selenomethylcysteine are present at low concentrations. Methane seleninic acid, probably arising from the oxidation of selenomethylcysteine, accounted for 22% of total Se. High-molecular-weight compounds, probably proteins, were detected in sodium dodecyl sulfate (SDS) and driselase extracts by size exclusion chromatography.