Analysis of 2-Aminobenzamide-Labeled Oligosaccharides by High-pH Anion-Exchange Chromatography with Fluorometric Detection

Abstract

To establish high-pH anion-exchange chromatography with fluorometric detection (HPAEC-FD) for the sensitive oligosaccharide analysis, the system for HPAEC with pulsed amperometric detection (PAD) was modified by placing the anion micromembrane suppressor between PAD and fluoromonitor to neutralize the alkaline eluate and connecting the flow path. N-Linked oligosaccharides were released from various glycoproteins by hydrazinolysis followed by labeling with 2-aminobenzamide, and their asialooligosaccharides were analyzed by HPAEC-FD using an isocratic elution with 0.3 M NaOH solution. The results indicated that the new system using FD offers fine separation of oligosaccharides at the subpicomole level. Coinjection of reduced dextran oligomers as internal standards which are detected by PAD permits us to express elution positions of the fluorescent oligosaccharides as glucose units, resulting in a much more reliable analysis. The system was also effectively used for structural analysis of oligosaccharides by sequential glycosidase digestion. Thus, HPAEC-FD promises to be an alternative tool for the sensitive analysis of oligosaccharides.