Abstract:In this study, a high performance liquid chromatography-inductively coupled plasma-mass spectrometry (HPLC-ICP-MS) method was established to determine selenium speciation in Brazil nuts. The sample was treated with 40mg protease XIV and 12mLTris-HCl buffer solution, extracted by ultrasonic for 3 hours, and then eluted by C18 reversed-phase chromatographic column with 10mmol/L citric acid and 5mmol/L sodium hexanesulfonate (pH = 4. 0, containing 1% methanol) as mobile phases. The effective separation and quantification of selenium forms could be achieved within 6min. The method verification shows that the linear correlation coefficients (r) of the five selenium speciation methods were all greater than 0. 999, and the detection limits of Se (VI), Se (IV), MeSeCys and SeMet were 0.5 ~ 1.3 μg/kg. The recoveries of four selenium speciation at different concentration levels were 93.5% ~ 117.0%, the relative standard deviation (RSD) was less than 5%, and the recovery rate of SeCys2 was about 50%. It was speculated that the stability of SeCys2 might be related to the high protein matrix of Brazil nuts. The accuracy of the method was verified by analyzing the SeMet in the European Union standard material wheat flour (ERM? BC210a). The method established in this study is sensitive, rapid and reliable, and is suitable for the analysis and determination of selenium forms in Brazil nuts. It is found that the main selenium forms of Brazil nuts from Brazil, Peru, Bolivia and other places are SeMet and a small amount of SeCys2, of which SeMet content accounted for 95% of the sum of selenium forms. It provides reference for further study on nutritional value of selenium-enriched nuts.