Speciation Analysis Of Selenium In Se-enriched Biological Samples By High Performance Liquid Chromatography

At present,the most convenient and quick instrument for speciation analysis of selenium in Se-enriched samples is high performance liquid chromatography followed by inductively coupled plasma mass spectrometry?HPLC-ICP-MS?,however,the expensive price limits its use in laboratory.In this work,an effective program of speciation analysis of selenium was established,that is,the content of total selenium in the Se-enriched sample was measured by fluorescence?FS?,then speciations of selenium in sample were analysed by high performance liquid chromatography?HPLC?,which successfully applied to analysis of other series of Se-enriched samples of protein selenium.The method is highly accurate and can be achieved in general laboratory,which provided a scientific basis for analysis of Se-riched samples.First,the content of total selenium of samples such as Se-enriched protein,yeast and tea was determined by fluorescence method?FS?,Secondly,the pre-column derivatization was carried out using 4-chloro-3,5-dinitro-trifluorotoluene?CNBF?with the organic selenium speciations?selenoaminoacids?,The retention times of selenomethionine?SeMet?and selenocystine?SeCys₂?in the standard chromatogram were determined by high performance liquid chromatography and mass spectrometry?HPLC-MS?,then determinated the speciations and content of seleno-aminoacids in protein by standard curve method.Based on the comparison of the acid hydrolysis and enzymatic hydrolysis of the protein,the optimal chromatographic conditions and experimental conditions were determined.The conditions could be applied to the analysis of the subsequent selenium-enriched yeast and selenium-enriched tea protein.The peaks of SeMet and SeCys2 in the chromatogram can achieve baseline separation.The linear correlation coefficient of the two standards reached0.9991.SeMet in the selenium-enriched protein accounts for 30.14%of total selenium and68.28%of SeCys₂,while inorganic selenium selenate and selenate are almost undetectable.Se-riched yeast and tea protein can use this method directly,the ratio of SeMet,SeCys₂and inorganic selenium in yeast was 21.25%,51.0%and 0.72%,respectively.As to the tea,because of its particularity,firstly,have to use the alkali to extract the protein and then to analyze.The extraction time?t?,protein settling pH,extraction temperature?T?,and pH of alkali solution have obvious effect on the extraction efficiency,so the Extraction factors are optimized by orthogonal test,the optimum extraction conditions were as follows:pH 14.0,V/W is 60,T is 40?and t is 5h.The contents of inorganic selenium and organic selenium in Se-enriched tea were determined,the mass ratio of SeMet to SeCys₂ in tea protein was 1:2.