Effective Separation of Human Milk Glycosides using Carbon Dioxide Supercritical Fluid Chromatography

Carbohydrate purification remains problematic due to the intrinsic diversity of structural isomers present in nature. Although liquid chromatography-based techniques are suitable for analyzing or preparing most glycan structures acquired either from natural sources or through chemical or enzymatic synthesis, the separation of regioisomers or linkage isomers with a clear resolution remains challenging. Herein, a carbon dioxide supercritical fluid chromatography (SFC) method was devised to resolve 18 human milk glycosides: oligomers (disaccharides to hexasaccharides), fucosylated regioisomers (lacto-N-fucopentaose I, III, and V; lacto-N-neofucopentaose V; lacto-N-difucohexaose III; blood group H₁ antigen; and TF-LNnT), and connectivity isomers (lacto-N-tetraose/lacto-N-neotetraose and para-lacto-N-hexaose/para-lacto-N-neohexaose/type-1 hexasaccharide). The analysis of these glycosides represents a major limitation associated with conventional carbohydrate analysis. The unprecedented resolution achieved by the SFC method indicates the suitability of this key technology for revealing complex human milk glycomes.