Development of Selective Analysis for Individual Functional Groups by High-performance Capillary Electrophoresis (HPCE) with Laser-induced Fluorescence (LIF) Detection

高效毛细管电泳是一种快速、分离性能好的多组分分离手段。本研究旨在寻找羧基、胺基、巯基、羟基等官能团的HPCE-LIF标记试剂。具有荧光素（如FITC）和4-硝基-2，1，3-苯并恶二唑（如NBD-F）结构的荧光体在波长和灵敏度方面适合氩离子（Ar）激光（激发波长488 nm）检测。而4-（N，N-二甲氨基磺酰基）-2，1，3-苯并恶二唑结构如DBD-F被推荐用于氦-镉（He-Cd）激光（激发波长442 nm）检测。与紫外检测相比，采用激光诱导荧光检测的试剂的检测灵敏度提高了两个数量级以上。He-Cd激光在325 nm处的检测灵敏度低于442 nm，但高于紫外检测。选择花菁染料用于630 nm处的二极管（DIO）激光检测。然而，试剂在溶液中的低稳定性和低衍生化产率是重要的缺点。作为HPCE-LIF检测的应用，分别用DBD-PyNCS和ABD-F标记大鼠肝细胞中的DL-氨基酸和生物活性硫醇进行手性分离。DL-氨基酸的衍生物完全分离的HPCE和检测灵敏度（几个fmol水平）与Ar-激光检测器在488 nm。用ABD-F标记后的细胞内巯基（如谷胱甘肽、半胱氨酸、N-乙酰半胱氨酸）也被分离并通过HPCE-LIF与He-Cd激光在325 nm处检测。单细胞内谷胱甘肽浓度为fmol水平。所提出的方法似乎适用于细胞中生物物质的高灵敏度检测。

HPCE is an excellent means for separation of multi-components with high speed and separatability. This study deals with search of labeling reagents for various functional groups such as carboxyl, amine, thiol and hydroxyl, by HPCE-LIF detection. Trace analysis of biologically importances by recommended CE-LIF procedures is also carried out with the selected fluorescent labeling reagents.Fluorophors possessing fluoresceine (e.g. FITC) and 4-nitro-2, 1,3-benzoxadiazole (e.g. NBD-F) moieties were suitable for argon-ion (Ar) laser (excitation wavelength, 488 nm) detection, in terms of wavelength and sensitivity. Whereas 4-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole structures such as DBD-F were recommended for helium-cadmium (He-Cd) laser (excitation wavelength, 442 nm) detection. The detectability using the reagents with LIF detection increased more than two orders of magnitude, as comparing with UV detection. On the other hand, the detection sensitivity with He-Cd laser at 325 nm was lower than that at 442 nm, but higher than that of UV detection. Cyanine dyes were selected for diode (DIO) laser detection at 630 nm. However, the low stability of the reagents in solution and the low derivatization yields were important disadvantages. As the application with HPCE-LIF detection , chiral separation of DL-amino acids and bioactive thiols in rat hepatocytes were performed after labeling with DBD-PyNCS and ABD-F, respectively. The derivatives of DL-amino acids were completely separated by HPCE and detected sensitively (a few fmol level) with Ar-laser detection at 488 nm. Intracellar thiols (e.g. glutathione, cysteine, N-acetylcysteine) after labeling with ABD-F were also separated and detected by HPCE-LIF with He-Cd laser at 325 nm. The concentration of glutathione in the single cell was fmol level. The proposed procedure seems to be applicable to highly sensitive detection of biological substances in a cell.