High-performance capillary electrophoresis of sialylated oligosaccharides of human milk

Oligosaccharides in human milk inhibit enteric pathogens in vitro and in vivo. Neutral milk oligosaccharides vary among individuals and over the course of lactation. To study such variation in the acidic milk oligosaccharides, a sensitive, convenient, quantitative method is needed. High-performance capillary electrophoresis of underivatized acidic oligosaccharides with detection by UV absorbance at 205 nm proved to be sensitive to the femtomole level. Eleven standard oligosaccharides ranging from tri- to nonasaccharide (3'-sialyllactose, 6'-sialyllactose, 3'-sialyllactosamine, 6'-sialyllactosamine, disialyltetraose, 3'-sialyl-3-fucosyllactose, sialyllacto-N-tetraose-a, sialyllacto-N-tetraose-b, sialyllacto-N-neotetraose-c, disialyllacto-N-tetraose, and disialomonofucosyllacto-N-neohexaose) were resolved; baseline resolutions of 3'-sialyllactose, 6'-sialyllactose, and other structural isomers were achieved. Peak areas were linear from 30 to 2000 pg and were reproducible with a coefficient of variation between 4 and 9%. There was no evidence of quantitative interference of one oligosaccharide with another. In studies using pooled human milk, addition of increasing amounts of authentic standard oligosaccharides produced the expected positive increments in detected values, indicating quantitative recovery without interference by other milk components. The identities of the major sialylated acidic oligosaccharides of pooled human milk agreed with the results of previous studies employing other analytical methods. Comparison of oligosaccharide profiles of milk samples from different donors revealed extensive variation, especially in the structural isomers of sialyllacto-N-tetraose. This sensitive, highly reproducible method requires only simple sample workup and is useful in defining variations in human milk acidic oligosaccharides and investigating their possible relationship with diseases of infants.