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

A high-resolution normal-phase high-performance liquid chromatography–fluorescence detection–electrospray ionization–mass spectrometry separation and structural characterization of the main oligosaccharides along with lactose from human milk samples is described. A total of 22 commercially available oligosaccharides were fluorotagged with 2-aminoacridone and separated on an amide column and identified on the basis of their retention times and mass spectra. Derivatized species having mass lower than approximately 800 to 900 exhibited mainly [M − H]−1 anions, oligomers with mass up to approximately 1000 to 1100 were represented by both [M − H]−1 and [M − 2H]−2 anions, and oligomers greater than approximately 1200 to 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 prepurification and derivatization steps also in the presence of lactose in high content. This analytical approach enabled us to perform the determination of species not detected by traditional techniques, such as sialic acid, as well as of species present in low content easily mistaken with other peaks. Finally, labeled 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.