新型荧光衍生试剂TMDEPAB-Ⅰ的设计合成及其在HPLC分离荧光检测巯基化合物中的应用  

The Design and Synthesis of Novel Fluorescence Reagent TMDEPAB-Ⅰ and Its Application on HPLC-Fluorescence Detection for Thiol Compounds

作　　者：郭小峰[1] 马丽娜 王红[1] 张华山[1] 

机构地区：[1]生物医学分析化学教育部重点实验室,武汉大学化学与分子科学学院,湖北武汉430072

出　　处：《分析科学学报》2014年第5期631-636,共6页Journal of Analytical Science

摘　　要：本研究以二氟化硼二吡咯甲烷（BODIPY）为荧光团,碘乙酰胺为衍生基团,设计并合成了新型巯基化合物荧光衍生试剂1,3,5,7-四甲基-2,6-二乙基-8-苯基-（4-碘乙酰胺）-二氟化硼二吡咯甲烷（TMDEPAB-I）。以TMDEPAB-I作为柱前衍生试剂,建立了高效液相色谱-荧光检测法（HPLC-FD）测定谷胱甘肽、L-半胱氨酸、N-乙酰半胱氨酸、高半胱氨酸、谷氨酰半胱氨酸、半胱氨酰甘氨酸和6-巯基嘌呤7种巯基化合物的新方法。TMDEPAB-I和巯基化合物在pH=11.0的NaOH-H3BO3缓冲溶液中,温度40℃下25min即可衍生完全。利用反相C18色谱柱,以pH=6.0的柠檬酸-NaOH缓冲溶液为流动相,梯度洗脱,各巯基化合物的衍生产物在22min内可实现基线分离,检测限（S/N=3）为1.0∽3.0nmol/L,回收率在94%∽102%之间。In this study,a novel fluorescence reagent based on difluoroboraindacene as fluorophore and iodoacetamido as labeling moiety,1,3,5,7-tetramethyl-2,6-diethyl-8-phenyl-（4-iodoacetamido）-difluorobo radiaza-s-indacene（TMDEPAB-I）,has been designed and synthesized for determination of thiol compounds.Taking TMDEPAB-I as pre-column derivatization reagent,a new HPLC-fluorescence detection method was developed for the determination of glutathione,L-cysteine,N-acetylcysteine,homocysteine,glutamylcysteine,cysteinylglycine and 6-mercaptopurine.After optimization of derivatization and separation conditions,the derivatization could be finished within 25 min under 40℃,and baseline separation of the derivatives could be achieved in 22 min on a reversed-phase C18 column using gradient elution.With the detection wavelength ofλex/λem=522/532 nm,the detection limits were in the range of 1.0-3.0nmol/L（S/N=3）.The developed method was successfully applied to determinate thiols in mouse brain with recoveries in the range of 94%-102%.The proposed method is suitable for the quantification of thiol compounds in clinical and biological samples with advantages of simpleness,good reproducibility,high sensitivity and good selectivity.

关 键 词：巯基化合物 荧光衍生试剂 高效液相色谱 

分 类 号：O657.72[理学—分析化学]