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 LC-MS/MS-based method for the rapid and simultaneous relative quantitation of glycosidic linkages for oligosaccharide and polysaccharide characterization. The method developed employs ultra-high 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 reverse-phase UHPLC along with the simultaneous monitoring of derivatized terminal, linear, bisecting, and trisecting monosaccharide linkages by mass spectrometry is achieved within a 15-minute 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.

The goal of food sustainability is possible if greater utilization of plants is achieved. In corn, only the kernels are currently used for human consumption; however, edible carbohydrates that may function as dietary fiber are present throughout the plant. A glycomic map of the maize plant was obtained providing a broad structural view of the carbohydrate distribution revealing that non-cellulosic material was present throughout. Newly developed rapid throughput liquid chromatography tandem mass spectrometry-based methods for analyzing monosaccharide and linkage compositions show unique structural features in the respective segments and parts of the plants from the roots to the tassel. The most abundant monosaccharides of the 14 that were monitored included glucose, xylose, and arabinose. Additionally, galactose, fructose, rhamnose, mannose, galacturonic acid, and glucuronic acid were found in lower abundances. The relative abundances of each monosaccharide varied with the parts of the plants. Linkage compositions also varied and provided further structural information that included the presence of polysaccharides such as xylans, starch, pectins, xyloglucans, arabinans, galactans, and β-glucans. The nonstructural carbohydrate components including the free mono-and disaccharides were also measured to provide a unique geographical map of their abundances. The glycomic map of corn would guide traditional plant breeding methods and new genome editing tools toward tissue-specific enhancements of carbohydrate polymers that have unique and specific functional utility.