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 0.2 fmol to 1 pmol, 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.
Yodoshi
Masahiro
M
School of Pharmacy, Kinki University, 3-4-1, Kowakae, Higashi-osaka 577-8502, Japan.
Tani
Akiko
A
Ohta
Yumiko
Y
Suzuki
Shigeo
S
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003374:Coumarins / Q000737:chemistry; D005470:Fluorometry; D009005:Monosaccharides / Q000032:analysis; D009844:Oligosaccharides / Q000032:analysis*; D011134:Polysaccharides / Q000032:analysis; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods
D003374:Coumarins; D009005:Monosaccharides; D009844:Oligosaccharides; D011134:Polysaccharides; C028743:7-amino-4-methylcoumarin