色谱

色谱 ›› 2015, Vol. 33 ›› Issue (1): 35-39.DOI: 10.3724/SP.J.1123.2014.09024

• 研究论文 • 上一篇    下一篇

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

周蓉, 曹赵云, 牟仁祥, 李正翔, 陈铭学   

  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
  • 收稿日期:2014-09-16 修回日期:2014-10-24 出版日期:2015-01-08 发布日期:2014-12-26
  • 通讯作者: 陈铭学
  • 基金资助:

    国家粮油作物产品质量安全风险评估项目(GJFP2014006).

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

ZHOU Rong, CAO Zhaoyun, MOU Renxiang, LI Zhengxiang, CHEN Mingxue   

  1. Laboratory of Quality & Safety Risk Assessment for Rice (Hangzhou), Ministry of Agriculture, China National Rice Research Institute, Hangzhou 310006, China
  • Received:2014-09-16 Revised:2014-10-24 Online:2015-01-08 Published:2014-12-26

PDF

210

被引次数

4

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(HEPPS)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.

关键词: 高效液相色谱法, 巯基化合物, 水稻, 柱前衍生

Abstract:

A high performance liquid chromatographic method with fluorescence detection and pre-column derivatization (HPLC-FLD) has been developed for the determination of seven biothiols 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 derivatized with monobromobimane (mBrB) as derivatization agent in 4-(2-hydroxyethyl)-1-piperazine propanesulfonic acid (HEPPS) buffer solution (pH 8.0). The separation was performed on an Agilent Eclipse Plus C18 column (50 mm×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)≥0.9991. 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.

Key words: biothiols, high performance liquid chromatography (HPLC), pre-column derivatization, rice

中图分类号: