On-line high-performance liquid chromatography-fluorescence detection-electrospray ionization-mass spectrometry profiling of human milk oligosaccharides dervatized with 2-aminoacridone

A high resolution normal phase HLPC-fuorescence detection-ESI-MS separation and structural characterization of the main oligosaccharides along with lactose from human milk samples is described. Twenty-two commercialy available oligosaccharides were fluorotagged with 2-aminoacridone and separated on an amide column and identified on the basisi of their retention times and mass spectra. Derivatized species having mass lower than about 800-900 exhibited mainly [M-H](-1) aninons, oligomers with mass up to 1000-1100 were represented by both [M-H](-1) and [M-H](-2) anions, and oligomer greater than 1200-1300 were characterized by a charge state of -3. Furthermore, the retention times were directly related to the glycans molecular mass. Human milk samples from the four groups of donors (Se+/-, Le+/-) were analyzed for their composition and amount of free oligosaccharides after rapid and simple pre-purifucation and derivatization steps also in the presence of lactose in high content. This analytical approach enebled us to perform the determination of species not detected by traditional tecniques, such as sialic acid, as well as of species present in low content easily mistaken with other peaks. Finally, labelled human milk oligosaccharides were analyzed without any interference from excess fluorophore, or interference from proteins, peptides, salts and other impurities normally present in this complex biological fluid