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...

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Bibliographic Details
Published in:Journal of Chromatography A 2008-09, Vol.1203 (2), p.137-145
Main Authors: Yodoshi, Masahiro, Tani, Akiko, Ohta, Yumiko, Suzuki, Shigeo
Format: Article
Language:English
Subjects:
7-Amino-4-methylcoumarin
Analytical, structural and metabolic biochemistry
Biological and medical sciences
Chromatography, High Pressure Liquid - methods
Coumarins - chemistry
Fluorescent probe
Fluorometry
Fundamental and applied biological sciences. Psychology
Glycoprotein
Glycoproteins
Monosaccharides - analysis
Negative-ion electrospray mass spectrometry
Oligosaccharides - analysis
Polysaccharides - analysis
Proteins
Sensitivity and Specificity
Spectrometry, Mass, Electrospray Ionization - methods
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Summary: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.
ISSN:0021-9673
DOI:10.1016/j.chroma.2008.07.053