An improved method for galactosyl oligosaccharide characterization.

Due to beneficial effects of galactosyl oligosaccharides (GOS) on digestive and immune health, their characterization has become increasingly important. This is especially so as GOS are synthesized enzymatically and contain oligosaccharides of different sizes and linkages. High performance anion exchange chromatography with pulsed amperometric detection (HPAE-PAD) is widely used for GOS characterization. With its high resolving power, it can separate structural isomers. Here we present a significant improvement to currently used methods. Our approach combines high resolution HPAE separation on a CarboPac PA300 column with 4 µm particle size with PAD and Orbitrap mass spectrometry (MS) detections to provide in-depth information on GOS composition. Oligosaccharide resolution, especially in the disaccharide region, is significantly improved and can be routinely achieved. Improvement in technology to remove sodium before MS results in minimal peak dispersion, allowing GOS degrees of polymerization 2 to 6 to be identified based on mass spectra obtained from intact oligosaccharides and confirmed using fragmentation patterns observed in MS/MS data. Combining HPAE with MS led to identification of 28 oligosaccharides in a commercial GOS sample. We attempted to correlate oligosaccharide structure with observed elution behavior. To our knowledge this is first such attempt and can form a basis for a comprehensive structure vs HPAE elution behavior database.