Touching the High Complexity of Prebiotic Vivinal Galacto-oligosaccharides Using Porous Graphitic Carbon Ultra-High-Performance Liquid Chromatography Coupled to Mass Spectrometry

[Image: see text] Galacto-oligosaccharides (GOS) are used in infant formula to replace the health effects of human milk oligosaccharides, which appear to be dependent upon the structure of the individual oligosaccharides present. However, a comprehensive overview of the structure-specific effects is still limited as a result of the high structural complexity of GOS. In this study, porous graphitic carbon (PGC) was used as the stationary phase during ultra-high-performance liquid chromatography–mass spectrometry (UHPLC–MS). This approach resulted in the recognition of more than 100 different GOS structures in one single run, including reducing and non-reducing GOS isomers. Using nuclear magnetic resonance-validated structures of GOS trisaccharides, we discovered MS fragmentation rules to distinguish reducing isomers with a mono- and disubstituted terminal glucose by UHPLC–PGC–MS. UHPLC–PGC–MS enabled effective recognition of structural features of individual GOS components in complex GOS preparations and during, e.g., biological conversion reactions. Hence, this study lays the groundwork for future research into structure-specific health effects of GOS.