Compositional analysis and structural characterization of raffinose family oligosaccharides from Eupatorium

Raffinose family oligosaccharides (RFOs) are commonly applied as functional food ingredients. RFOs mixture derived from Eupatorium were analyzed by hydrophilic interaction liquid chromatography-evaporative light scattering detector (HILIC-ELSD) method with a Click Xion column, and then separated by graphitized carbon black (GCB) chromatography. RFOs with a specific degree of polymerization (DP) from 3 to 6 were obtained. The specific ions from glycosidic bond cleavage and cross-ring cleavage of RFOs were produced in Electrospray-collision-induced dissociation-mass spectroscopy/mass spectroscopy (ESI-CID-MS/MS), which are in accordance with the linkage of (1→6) between galactans. The anomeric configuration and glycosidic linkage of RFOs were confirmed to be [α-Gal-(1→6)]_n-α-Glc-(1→2)- β-Fru (n = 1–4) by 2D-NMR. In this study, we developed a HILIC-ELSD method for the compositional analysis of RFOs and obtained RFOs with a specific DP and high purity, and their structures were completely elucidated. Our work provides a basis for further research on the structure–activity relationship of RFOs.