Characterization of structures of galacto-oligosaccharides by high-performance liquid chromatography coupled with mass spectrometry

As a supplement for infant formulae, Galacto-oligosaccharides (GOS) are considered as prebiotic compounds to maintain the proliferation of colonic microflora. GOS are producted by transgalactosylation from lactose with β-galactosidase in food industry, and result in several linkage isomers. In this study, we developed a fast and effective analytical method to solve the complicated isomers of GOS products. First, six commercial GOS products were subjected to degree of polymerization (DP) analysis using High-performance size exclusion chromatography. Then, native (without derivatization) sample and p-aminobenzoic acid ethyl ester (ABEE)-derived sample were analyzed with two HPLC-tandem MS methods: GCC-ESI-MSn and Polyamine II-PAD-ESI-MSn respectively. Experimental results showed the DP of all structures was less then six, while DP2 and DP3 were the major components. In total, 9 different kinds of DP2, 18 of DP3 and 21 of DP4 isomers of GOS have been identified, and the composition between each product were varied. For linkage isomers determination, isomers contained β-1→4 and β-1→6 linkages were the major, ones followed by β-1→3 and then β-1→2 linkages in quantity; β-1→1 was also detected in some of the samples. These results demonstrated the tendency to linkage formation of β-galactosidase in transgalactosylation. Overall, the methods developed in this study are capable of analyzing the isomer components and structure characterization simultaneously. The proposed method of determining GOS products can be used for other relevant samples or for a qulity control measurement in related industries.