柱前衍生高效液相色谱-荧光检测法测定水稻中7种巯基化合物  

Determination of seven biothiols in rice by high performance liquid chromatography-fluorescence detection with pre-column derivatization

作　　者：周蓉[1] 曹赵云[1] 牟仁祥[1] 李正翔[1] 陈铭学[1] 

机构地区：[1]中国水稻研究所,农业部稻米产品质量安全风险评估实验室(杭州),浙江杭州310006

出　　处：《色谱》2015年第1期35-39,共5页Chinese Journal of Chromatography

基　　金：国家粮油作物产品质量安全风险评估项目(GJFP2014006)

摘　　要：建立了水稻中半胱氨酸（Cys）、谷胱甘肽（GSH）和植物螯合肽（phytochelatin，PC：PC2、PC3、PC4、PC5、PC6）7种巯基化合物的柱前衍生高效液相色谱-荧光检测分析方法。样品经0.1％三氟乙酸（ TFA）（含6.3 mmol／L 二乙烯三胺五乙酸（DTPA））超声提取，然后以单溴二胺（mBrB）为衍生剂在 pH 8.0的4-羟乙基哌嗪丙磺酸（HEP-PS）缓冲溶液中衍生化。采用的色谱分离柱为 Agilent Eclipse plus Cl8柱，流动相为0.1％TFA（pH 2.5）和100％乙腈（ ACN），梯度洗脱，流速为0.8 mL／min。荧光检测的激发波长和发射波长分别为380 nm 和470 nm。结果表明，7种巯基化合物在0.7-100.0 mg／L范围内，峰面积与质量浓度之间的线性关系良好（ r2≥0.9991）；检出限为0.03-0.20 mg／L；加标回收率为89.26％-99.42％，相对标准偏差为2.05％-5.87％。该方法准确、灵敏度高、重现性好，为水稻中巯基化合物的研究提供了检测手段。A high performance liquid chromatographic method with fluorescence detection and pre-column derivatization（ HPLC-FLD）has been developed for the determination of seven bio-thiols including Cys,GSH,and phytochelatins（ PCs：PC2,PC3,PC4,PC5 and PC6）in rice. The samples were ultrasonically extracted with 0. 1% trifluoroacetic acid（ TFA）containing 6. 3 mmol/L diethylenetriaminepentaacetic acid（ DTPA）,and then the seven biothiols were derivat-ized with monobromobimane（ mBrB ） as derivatization agent in 4-（ 2-hydroxyethyl ）-1-pipera-zine propanesulfonic acid（ HEPPS）buffer solution（ pH 8. 0）. The separation was performed on an Agilent Eclipse Plus C18 column（50 mm＆#215;4. 6 mm,5 μm）with gradient elution of 0. 1%TFA solution（the pH value was adjusted to 2. 5 with hydrochloric acid）and acetonitrile as mobile phases at a flow rate of 0. 8 mL/min. The detection was performed at 380 nm for excitation and 470 nm for emission. The calibration curves of the seven biothiols showed good linearity in the concentration range of 0. 7-100. 0 mg/L with the correlation coefficients（ r2）≥0. 999 1. The limits of detection were 0. 03-0. 20 mg/L. The recoveries of standard addition were in the range of 89. 26%-99. 42% with the relative standard deviations（ RSDs,n=6）of 2. 05%-5. 87%. The method is sensitive,accurate,reproducible and suitable for the simultaneous determination of Cys,GSH,PC2,PC3,PC4,PC5 and PC6 in rice.

关 键 词：柱前衍生 高效液相色谱法 巯基化合物 水稻 

分 类 号：O658[理学—分析化学]