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Liquid Chromatography-Tandem Mass Spectrometry Approach for Determining Glycosidic Linkages.

The structural analysis of carbohydrates remains challenging mainly due to the lack of rapid analytical methods able to determine and quantitate glycosidic linkages between the diverse monosaccharides found in natural oligosaccharides and polysaccharides. In this research, we present the first liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method for the rapid and simultaneous relative quantitation of glycosidic linkages for oligosaccharide and polysaccharide characterization. The method developed employs ultrahigh-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC/QqQ-MS) analysis performed in multiple reaction monitoring (MRM) mode. A library of 22 glycosidic linkages was built using commercial oligosaccharide standards. Permethylation and hydrolysis conditions along with LC-MS/MS parameters were optimized resulting in a workflow requiring only 50 μg of substrate for the analysis. Samples were homogenized, permethylated, hydrolyzed, and then derivatized with 1-phenyl-3-methyl-5-pyrazolone (PMP) prior to analysis by UHPLC/MRM-MS. Separation by C18 reversed-phase UHPLC along with the simultaneous monitoring of derivatized terminal, linear, bisecting, and trisecting monosaccharide linkages by mass spectrometry is achieved within a 15 min run time. Reproducibility, efficacy, and robustness of the method was demonstrated with galactan ( Lupin) and polysaccharides within food such as whole carrots. The speed and specificity of the method enables its application toward the rapid glycosidic linkage analysis of oligosaccharides and polysaccharides.