Aminoquinolines as fluorescent labels for hydrophilic interaction liquid chromatography of oligosaccharides

Abstract

In this study, we investigated the potential of four different aminoquinoline (AQ) compounds as fluorescent labels for glycan analysis using hydrophilic interaction liquid chromatography (HILIC) and fluorescence detection (FLD). We confirmed the optimal excitation and emission wavelengths of 3-AQ and 6-AQ conjugated to glycan standards using three-dimensional fluorescent spectral scanning. The optimal excitation and emission wavelengths for 6-AQ were confirmed at λ(ex)=355 nm and λ(em)=440 nm. We concluded that the optimal wavelengths for 3-AQ were λ(ex)=355 nm and λ(em)=420 nm, which differed considerably from the wavelengths applied in previous reports. HILIC-FLD chromatograms using experimentally determined wavelengths were similar to 2-aminobenzamide controls, but the peak capacity and resolution differed significantly when published 3-AQ λ(ex/em) values were applied. Furthermore, we found that 5-AQ and 8-AQ labeled maltohexaose did not display any fluorescent properties when used as a carbohydrate tag for HPLC analysis. Finally, we applied experimentally determined wavelengths to 3-AQ labeled N-glycans released from human IgG to illustrate changes in retention time as well as to demonstrate that AQ labeling is applicable to complex sample analysis via exoglycosidase sequencing.