Milk oligosaccharide profiles by reversed-phase HPLC of their perbenzoylated derivatives

Human milk is rich in oligosaccharides, some of which inhibit toxins and pathogens involved in diseases of infants. To investigate qualitative and quantitative individual variation of human milk oligosaccharides, a sensitive method for routine identification and quantification of intact milk oligosaccharides was developed and applied to milk samples from 50 donors. The isolated, reduced neutral oligosaccharide fractions were perbenzoylated, resolved by reversed-phase HPLC, and detected at 229 nm. This method resolves most structural isomers and does not require stringent removal of lactose. Peaks were detected at the low nanogram (pmol) level and peak areas were linear from 1 to 1000 micrograms for a standard oligosaccharide. Oligosaccharide samples equivalent to 1 microliter of human milk give optimum chromatographic separation and resolution. The method gives quantitative results comparable to those obtained with classic total sugar analyses, and has an average coefficient of variation of 13%. The 12 major peaks in human milk coeluted with authentic oligosaccharide standards ranging from tri- to octasaccharides, and their identities were confirmed by mass spectrometry. Significant individual variation exists in oligosaccharide profiles; almost 70% of samples contained 2'-fucosyllactose and lacto-N-fucopentaose I as the major oligosaccharides; for the remainder, the major oligosaccharides were 3-fucosytlactose and lacto-N-fucopentaose-II or lacto-N-fucopentaose-III. This method can be used to investigate the extent and biological significance of oligosaccharide variation in human milk.