How to identify target glycosidic linkages by UPLC-MS/MS? Case study of oligosaccharides and polysaccharides composed of glucose

Glycosidic linkage analysis is crucial for elucidating the structure-activity relationships of polysaccharides, but existing GC-MS methods have limitations in practical applications. This study developed a simple, efficient, and sensitive UPLC-QqQ-MS/MS method for glycosidic linkage analysis, validated using glucose-based oligosaccharides and polysaccharides. 16 glucose glycosidic linkage standards were synthesized using anion reconstruction, partial methylation, and PMP derivatization. Samples underwent permethylation and PMP derivatization before UPLC-QqQ-MS/MS analysis. The accuracy and feasibility of the method were validated by comparing the results with the synthesized glycosidic linkage standards and known glycosidic linkage information. Results showed that the LC-MS-MRM spectra of the 1-4-Glc glycosidic linkages in lactose, maltose, and cellulose exhibited identical retention times and characteristic fragment ions, consistent with the synthesized 1-4-Glc standard. This confirmed the accuracy of the synthesized standards and the feasibility of the LC-MS method for detecting glycosidic linkages. Similarly, the retention times and characteristic fragment ions of the T-Glc linkages in maltose and Aspergillus Niger disaccharide, the 1-3-Glc linkage in Aspergillus Niger disaccharide, and the 1-6-Glc linkages in glucan were consistent with their corresponding standards. These results demonstrate that the UPLC-QqQ-MS/MS method can be used for qualitative and quantitative analysis of both unknown and known glycosidic linkages in oligosaccharides and polysaccharides.