高效液相色谱-电感耦合等离子体质谱法测定富硒碎米荠中的硒形态

Determination of Selenium Speciations in Selenium-enriched Cardamine Violifolia by High Performance Liquid Chromatography-Inductively Coupled Plasma Mass Spectrometry

建立了富硒碎米荠中Se(Ⅵ)、Se(Ⅳ)、硒代胱氨酸(SeCys2)、甲基硒代半胱氨酸(MeSeCys)、硒代蛋氨酸(SeMet)5种硒形态的高效液相色谱-电感耦合等离子体质谱(HPLC-ICP-MS)分析方法。通过风味蛋白酶和蛋白酶E两种酶先后添加的顺序提取不同形态硒化合物,经两次酶解提取后,稀释到合适浓度。选取Thermo Scientific Hypersil GOLD C8色谱柱(250 mm×4.6 mm×5μm),以含0.05%七氟丁酸与3%甲醇的20 mmol/L KH2PO4作为流动相进行等度洗脱,可在6 min内将5种硒形态完全分离。5种硒形态在线性范围内相关系数(r2)均大于0.999;加标回收率均在85.8%~106.3%之间;变异数小于5%;方法的检出限为0.08~0.25μgSe/L;定量限为0.25~0.54μgSe/L。应用该方法测定实际样品,发现不同碎米荠中有机硒形态占总硒的71.2%~87.9%,其中以SeCys2和SeMet两种硒形态为主,同时还含有少量无机硒和未知形态硒。

A high performance liquid chromatography coupled inductively plasma mass spectrometry(HPLC-ICP-MS)method was established for the determination of selenium species in selenium-enriched Cardamine violifolia,including selenate(Se(Ⅵ)),selenite(Se(Ⅳ)),selenocysteine(SeCys2),methylselenocysteine(MeSeCys)and selenomethionine(SeMet).Different speciations of selenium compounds were extracted by adding two enzymes i.e.,flavor protease and protease E successively.After twice enzymolysis,it was diluted to the appropriate concentration.The samples were separated with a Thermo Scientific hypersil gold C8 column(250 mm×4.6 mm,5μm)by isocratic elution using 20 mmol/L potassium dihydrogen phosphate containing 0.05%heptafluorobutyric acid and 3%methanol as mobile phase.The five speciations of selenium could be completely separated within 6 min.There were linear relationships for five selenium speciations with correlation coefficients(r2)higher than 0.999.Recoveries for five speciations of selenium were in the range of 85.8%-106.3%with relative standard deviations less than 5%.The limits of detection and limits of quantitation were 0.08-0.25μgSe/L and 0.25-0.54μgSe/L,respectively.Using this method,it was found that the organic selenium forms accounted for 71.2%-87.9%of the total selenium in Cardamine violifolia of which SeCys2 and SeMet were the main selenium speciations,and the sample also contained a small amount of inorganic selenium and unknown forms of selenium.