Direct Identification of Disaccharide Structural Isomers Using Ambient Ionization Tandem Mass Spectrometry with In Situ Methylation

Carbohydrates play critically important roles in energy supply and biological functions in living systems. However, it has been a great challenge to identify saccharides and distinguish their isomers because they have highly similar structures and many possible positions for glycosidic linkages. In this work, an ambient ionization tandem mass spectrometry method was developed to characterize disaccharide structural isomers with in situ methyl-ation. The direct analysis in real time ion source can be used to facilitate the methylation reaction of disaccharides with tetrame-thylammonium hydroxide. The hydroxyl groups of disaccharides can be methylated instantaneously, and the products can be ionized at the same time. The methylated product ions from full scan mass spectrometry (MS) and tandem MS can be used to distinguish a variety of disaccharide structural isomers with different glycosidic linkages, compositions, and configurations. Characteristic marker ions were discovered, and they can be used for the assignment of linkage type and identification of specific isomeric forms. The method was used for the direct identification of disaccharide isomers from real commercial products such as honey, wine, and milk without complex sample pretreatment or chromatographic separation.

Automated summary

Carbohydrates are essential for energy supply and biological functions, but identifying and distinguishing their isomers has been challenging due to their similar structures and possible glycosidic linkages. This study developed an ionization tandem mass spectrometry method to characterize disaccharide isomers by in situ methylation. The method allowed for immediate methylation of hydroxyl groups on disaccharides, enabling their ionization and distinction through mass spectrometry. This approach was used to directly identify disaccharide isomers in real commercial products without complex pretreatment or chromatographic separation.