A detailed analysis of neutral and acidic carbohydrates in human milk

Reverse- and normal-phase chromatography have been used to separate a number of standard human milk oligosaccharides derivatized via a reductive amination reaction with 2-aminoacridone (2-AMAC). Analytes were detected by spectrofluorimetry and injected simultaneously with a hydrolyzed dextran ladder derivatized with methyl-4-aminobenzoate. The latter probe does not fluoresce at the wavelength of emission by the 2-AMAC derivatives, and the derivatized, hydrolyzed dextran components were visualized by their ultraviolet absorbance. This procedure gave precise measurements of the "size" of 2-AMAC oligosaccharides in terms of their glucose equivalent values. Analytical amounts of 2-AMAC oligosaccharide standards were also isolated for further characterization by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) and electrospray ionization (ESI) mass spectrometry. MS-MS was also used to provide information on oligosaccharide sequences. This methodology was used successfully to characterize mixtures of neutral and acidic oligosaccharides from samples of human milk. This approach could be usefully applied to the study of glycoforms from a variety of samples such as those released from glycoproteins/glycolipids; these have been reported to be altered in a number of diseases, for example, cancer, cystic fibrosis, and autoimmune disease such as rheumatoid arthritis.