Galactooligosaccharides (GOS) are important prebiotics with function closely related to their structure. However, a comprehensive overview of the structure-function relationship is still limited due to the challenge in characterizing multiple isomers in GOS. This study presents a strategy of combining both hydrophilic interaction liquid chromatography (HILIC) retention time and tandem mass spectrometry (MS/MS) fragmentation pattern to distinguish α/β-linkages and linkage positions of disaccharide isomers in GOS through HILIC-MS/MS analysis. The results indicated that the ratio of m/z 203.0524 to m/z 365.1054 could distinguish α/β-linkages, while the ratios of m/z 347.0947 to m/z 365.1054, m/z 245.0642 to m/z 365.1054 and HILIC retention time could distinguish (1 → 2), (1 → 3), (1 → 4) and (1 → 6) linkages. The above rules enabled effective characterization of disaccharides in GOS-containing food samples, including milk powder, rice flour, drink, yogurt. This method can be used in the quality control of GOS and future research on the structure-specific health effects of GOS.
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