Speciation Analysis And Structure Identification Of Selenium-containing Compounds In Typical Selenium-enriched Plants

As a safe and effective natural source of organic Se,Se-enriched plants can provide essential high-quality organic Se for human beings,and play a role in resisting oxidative stress,improving immune system function and maintaining normal metabolism of human body.However,Se-containing compounds in Se-enriched plants are not well understood due to the complex matrix,the poor stability of Se Species in the extraction process,and the limitations of standard substances,methods and ideas of the identification of Se-containing compounds.In this paper,firstly,by optimizing the compound enzymatic hydrolysis pretreatment and RPIC-ICP MS conditions and comparing the stability of different Se species in compound enzymatic hydrolysis process,the common known Se speciations in typical Se-enriched plants were analyzed.Secondly,the difference of total Se and Se species in different Se-enriched plants were analyzed,and their storage stability of total Se and various Se species was investigated.Finally,unknown Se-containing compounds in different Se-enriched plants were identified by UPLC-ESI-Triple TOF MS.It provided experimental scheme and identification idea for the speciation analysis and identification of Se-containing compounds in Se-enriched plants,thus laying a foundation for dietary supplement of high-quality organic Se.The main conclusions are as follows:Firstly,an efficient and accurate enzymatic hydrolysis pretreatment-RPIC-ICP MS method was optimized to analyze Se species in typical Se-enriched plants quantitatively.The optimal enzymatic hydrolysis conditions were as follows:100 mg of the sample was mixed in 15 m L30 mmol/L Tris-HCl buffer（p H 8.5）with 5%（enzyme-to-sample mass ratio,w/w）alcalase,5%protease K and 5%trypsin,and magnetic stirred at 45°C for 4 h.A Tech Mate C18-ST reversed-phase column（4.6 mm×250 mm,5μm）was selected.The mobile phase consisted of 30 mmol/L diammonium hydrogen phosphate,0.5 mmol/L tetrabutylammonium hydroxide and 3.0%（v/v）methanol was used,and its p H was adjusted to 6.0.Isometric elution was carried out at a flow rate of 1.0 m L/min and the column temperature was 30°C.Six selenium species,including selenocystine[Se Cys₂],methylselenocysteine[Me Se Cys],selenite[Se（IV）],selenomethionine[Se Met],selenate[Se（VI）]and Selenoethionine[Se Et],were separated within 10 min by this method.Then the method was validated by methodology.The linear relationship of six Se species was good within the linear range,and the correlation coefficients（r²）were all more than0.999.The limits of detection（LOD）were 0.08μg/L～0.15μg/L,and the limits of quantitation（LOQ）were 0.30μg/L～0.51μg/L.The six Se species showed good repeatability and stability with RSD less than 5%.The recoveries of the Se species ranged from 83.0%to 105.8%with RSD less than 6.4%.Secondly,the differences of the Se species in six different Se-enriched plants were compared.Se Cys₂ was the main Se specie in C.violifolia and Brassica oleracea L.var.italica Plenck.The main Se specie in Se-enriched Glycine max（Linn.）Merr.was Se Met.The main Se specie in Se-enriched Brassica oleracea var.capitata L.was Se（VI）.Three organic Se species:Se Met,Me Se Cys and Se Cys₂,were the main Se species in Se-enriched Asparagus officinalis L.The Se specie in Se-enriched Allium sativum L.was mainly Me Se Cys.The storage stability of the contents of total Se and Se species in six different Se-enriched plants for four months were also evaluted.The storage stability of total Se and Se（VI）in all Se-enriched plants was good.But the content of Se（IV）of the Se-enriched plants decreased significantly.The contents of the three organic Se species in Se-enriched plants showed a decreasing trend,which indicated that some Se species had undergone transformation during storage.Finally,unknown Se-containing compounds in six different Se-enriched plants were identified by UPLC-ESI-Triple TOF MS.Based on the unique isotope pattern filter and mass defect filter of Se,a comprehensive identification strategy of targeted analysis and untargeted analysis was established.A total of 27 different unknown Se-containing compounds were identified,and 8 of them have been reported.Among the 19 Se-containing compounds that had not been reported,the structural formulas of 3 unknown Se-containing compounds（C₄H₇NO₂Se,C₅H₈O₂Se and C₇H₉NO₂Se）,as well as the molecular formulas of 16 unknown Se-containing compounds were identified.