Quantification of Sugar Epimers in Polygalactomannans by ESI-MS/MS

Polygalactomannans (PGMs) represent an important family of polysaccharides. They are obtained from the endosperm of leguminous plant seeds and are employed in a growing number of industrial applications as thickening agents and rheology modifiers. Each PGM has a typical mannose/galactose (M/G) ratio (from ~1 to ~5), which determines its solubility, viscosity and other physico-chemical properties. Complex polysaccharides are commonly characterized by proton nuclear magnetic resonance (1H-NMR), whereas mass spectrometry (MS) has not yet seen wide application. In this work, a new quantification method for sugar epimers in PGMs is presented, based on the characteristic fragmentation patterns of mannose and galactose in tandem MS (MS/MS) analyses. Standard galactose and mannose have been analyzed and fragmented in negative-ion mode on a hybrid quadrupole/time-of-flight (qTOF) mass spectrometer equipped with a nano electrospray ionization (ESI) source. The MS/MS spectra indicate accumulation of the same fragments for the two monosaccharides, but significant and reproducible differences in the relative intensities of the product ions. Known mixtures of mannose and galactose have been analyzed by the same procedure to test the applicability of the method for quantification purposes. The resulting peak intensities over the entire MS/MS spectrum can be deconvoluted as a linear combination of the signals from pure mannose and galactose standards, obtaining reliable and reproducible quantification of the epimers. The method has been applied to the characterization of hydrolysis products of PGMs from different species of leguminous plants, such as guar (Cyamopsis tetragonolobus), sesbania (Sesbania bispinosa) and tara (Caesalpinia spinosa), in order to assess specific susceptibility to hydrolysis conditions.