53 / 2019-12-09 20:36:28

Comparison of labled N-glycans analysis based on glycosylamine derivatization using HPLC-FLD with different chromatographic separations

glycosylamine derivatization,HPLC-FLD separation

Glycosylamine derivatization is increasingly being used for the analysis of N-glycans due to its multiple advantages. Hydrophilic interaction liquid chromatography (HILIC) has become a powerful N-glycan analysis technique because it has a significant advantage in separating polar compounds. Known methods of reverse-phase chromatography (RPC) and normal-phase chromatographic analysis (NPC) are also used for N-glycans separation. To fully understand the chromatographic action of glycosylamine-derivatives of N-glycans, we compared the separations of N-glycan derivatives of AQC from three standard glycoproteins (RNase B, Transferrin and Fetuin) containing neutral and acidic structures on three chromatographic columns of Amide 80, C18 and 5-NH2 (Figure 1). Firstly, various separation related conditions of different columns were studied in detail. Further analysis of the N-glycan derivatives of AQC in human serum was subsequently carried out. Results showed that HILIC achieved the best in summary, 5-NH2 column performed good in acidic glycan derivatives but was insufficient in neutral ones, unfortunately, the separation of C18 column was poor as the glycan derivatives did almost no retainment of acidic structures and serious co-elution was observed for neutral ones. This study can provide a useful reference for chromatographic separation of N-glycan derivatives.