Hydrophilic interaction liquid chromatography coupled to mass spectrometry for the characterization of prebiotic galactooligosaccharides

Three different stationary phases (sulfoalkylbetaine zwitterionic, polyhydroxyethyl aspartamide and ethylene bridge hybrid (BEH) with trifunctionally bonded amide), operating at hydrophilic interaction liquid chromatographic (HILIC) mode, have been assayed and compared for the analysis of complex mixtures of galactooligosaccharides (GOS). Chromatographic methods have been optimized to obtain the best separation between two consecutive galactose containing standards and maltodextrins, measured on the basis of resolution. Influence of several factors such as chemical modifiers (formic acid, ammonium acetate and ammonium hydroxide), organic solvent and gradients of the mobile phases in the separation of oligosaccharides have been studied. The best results were achieved on the BEH amide stationary phase, using acetonitrile:water with 0.1% ammonium hydroxide as mobile phase, where the most of oligosaccharides were successfully resolved. Characteristic MS2 fragmentation profiles of disaccharides containing galactose, glucose and/or fructose units with different linkages were evaluated and used for the characterization of di-, tri- and tetrasaccharides of three commercial prebiotic GOS mixtures (GOS-1, GOS-2 and GOS-3) by HILIC–MSn. Similar qualitative and quantitative composition was observed for GOS-1 and GOS-3, whereas different linkages and abundances were detected for GOS-2. In general, (1 → 4) and (1 → 6) glycosidic linkages were the main structures found in GOS, although (1 → 2) and (1 → 3) linkages were also identified. Regarding molecular weight, up to pentasaccharides were detected in these samples, disaccharides being the most abundant carbohydrates.