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A separation method based on ion-pairing liquid chromatography was combined with both elemental (inductively coupled plasma mass spectrometry (ICP-MS)) and molecular (electrospray tandem mass spectrometry (ES-MS-MS)) mass spectrometry in order to unravel the identity of the Se-species present in the complex matrix of Brazil nuts rich in Se. Via enzymatic digestion, Se-species were released from the matrix. Subsequently the species were separated and the Se was monitored on-line by ICP-MS. By HPLC–ES-MS-MS, the species were identified based on their molecular mass and their specific product ions. The main compound was identified as Se-Methionine. Another compound was identified as Se-Cystine, partly on the basis of the isotopic pattern of Se. This research was further extended to the analyses of in vitro gastrointestinal digests of the Brazil nuts. These digests were analyzed for their Se-content and screened for the presence of the different Se-species by HPLC–ICP-MS. In both the gastric and the intestinal digests, we were able to identify the Se-species as Se-Methionine and Se-Cystine by HPLC–ES-MS-MS. By coupling HPLC to both elemental and molecular mass spectrometry, the species present in Brazil nuts and supposedly extractable by our body were fully characterized.