An LC-FLR-MS method was optimized on the BioAccord System with ACQUITY Premier LC fitted with an ACQUITY Premier Glycan C18 AX Column. Compared to a traditional HILIC-based method, the new mixed-mode C18 AX Column offered more resolving power for complex N-glycans with higher levels of sialic acid. The optimized LC-FLR-MS method enabled distinct charge-based separation and compositional assignments based on the number of sialic acids and mass data from the BioAccord System. More glycan structures were further resolved based on their hydrophilicity within the retention time window for the same charge (based on number of sialic acid). In summary, the combination of MaxPeak HPS Technology and the SmartMS functionality of the BioAccord LC-MS System enables the generation of high-quality data with respect to peak shape, resolution, and MS sensitivity in the analysis of highly sialylated complex N-glycans.
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