Analysis of 2-Aminobenzoic Acid-Labeled Monosaccharides and Glycoprotein-Derived Oligosaccharides by Online Cleanup Liquid Chromatography in the Reversed-Phase and Hydrophilic Interaction Liquid Chromatography Modes

Quantitative analysis of monosaccharides or glycoprotein glycans by high-performance liquid chromatography (HPLC) often involves labeling of the saccharide aldehyde groups with fluorescent tags to enhance sensitivity and selectivity. However, the methods required to remove the large excess of labeling reagents from the reaction mixture are time-consuming. Furthermore, these methods often hinder the quantitative analysis of the labeled samples. Here, we developed an online sample cleanup procedure for HPLC analysis of 2-aminobenzoic acid (2-AA)-labeled monosaccharides or oligosaccharides using a ten-port valve and mini columns. An online purification system using a combination of short HLB columns with the valve was proposed for the analysis of 2-AA-labeled monosaccharides utilizing reversed-phase modes. In the analysis of 2-AA-labeled glycans derived from glycoproteins, a short CN column with the valve was proposed utilizing hydrophilic interaction liquid chromatography (HILIC) modes. Optimized conditions enabled the direct injection of the diluted labeling reaction mixture into the chromatographic system without any prior removal of the excess labeling reagents. These methods were successfully applied to the analysis of various monosaccharides and N -linked glycans released from specific glycoproteins.