Optimized conditions for high-performance liquid chromatography analysis of oligosaccharides using 7-amino-4-methylcoumarin as a reductive amination reagent

Reductive amination reaction using 7-amino-4-methylcoumarin (AMC) as a fluorescent probe enabled analyses of glycoproteins' monosaccharides and N-linked oligosaccharides. Reductive amination of N-acetylhexosamines and AMC using sodium cyanoborohydride or dimethylamine-borane complex indicated slight recovery of derivatives, but pyridine-borane achieved better recoveries. Reversed-phase high-performance liquid chromatography (HPLC) analyses of monosaccharides constituting glycoprotein glycans using fluorimetric detection revealed linearity for 0.2fmol to 1pmol, with less than 5% RSD quantitation reproducibility. Reversed-phase HPLC analyses of glycoprotein glycans, combined with negative-ion electrospray ionization mass spectrometry (LC-ESI-MS), enabled their structural determination. Using this highly hydrophobic reagent, AMC-labeled oligosaccharides displayed one-order to two-order higher ESI-MS intensity than derivatives labeled using other reagents.