Identification and fractionation of human milk oligosaccharides by proton-nuclear magnetic resonance spectroscopy and reverse-phase high-performance liquid chromatography.

It has been shown that the retention times of oligosaccharides containing N-acetyl amino sugars in reverse-phase high-performance liquid chromatography are sensitive both to chain length and to stereochemical differences. Data showing that oligosaccharides of human milk can be fractionated according to stereochemistry on C-18 columns using water as eluant, is presented. Detection at the nanomolar level is possible using ultraviolet absorption at 190 nm as a result of the acetamido group. The separation of linkage isomers by fractionation of two tetrasaccharides of identical carbohydrate composition, lacto-N-tetraose and lacto-N-neotetraose, and by the separation of two isomeric pentasaccharides, lacto-N-fucopentaose I and II, is shown. In some cases it was possible to preparatively fractionate pure oligosaccharides from complex mixtures. The identity and purity of the oligosaccharides was determined by proton-nuclear magnetic resonances spectroscopy at 300 MHz using the method of Vliegenthart and co-workers (B. Fournet, G. Montreuil, L. Strecker, J. Dorland, J. Haverkamp, J. F. G. Vliegenthart, J. P. Binette, and K. Schmid (1978) Biochemistry 17(24), 5206; J. F. G. Vliegenthart, H. V. Halbeek, and L. Dorland (1981) Pure Appl. Chem. 53, 45) in which "structural reporter" protons are identified for the different isomeric oligosaccharides. In addition to assignment of the anomeric proton resonances, it was possible to assign H4 of galactose linked at O3 as well as fucose H5 and H6 resonances.