Efficient HPLC separation of N-p-nitrophenylglycosylamines derived from complex oligosaccharide mixtures. Human orosomucoid as a model.
With human orosomucoid as model compound, a new method was developed to separate neutral oligosaccharides as N-p-nitro-phenylglycosylamines. Asialo orosomucoid was prepared by treatment with neuraminidase and purified by size exclusion HPLC on TSK 2000 SW. Oligosaccharides were isolated by reversed phase HPLC on Hamilton PRP-1 after hydrazinolysis and re-N-acetylation. Glycosylamination was performed with p-nitroaniline in DMSO-formic acid-water, where the whole mixture of oligosaccharide derivatives was isolated by reversed phase HPLC on Hamilton PRP-1 and separated into single glycosylamines on Shandon Hypersil ODS. The purified glycosylamines could be methylated by a new rapid method with sodium hydroxide and methyl iodide in DMSO, isolation and purification were carried out on Hamilton PRP-1 and Spherisorb ODS 2, respectively, as described for the glycosylamines. Preparative scale HPLC separations were performed on analytical columns using repetitive collection mode and automatic sample loading by means of a peristaltic pump operated by the HPLC controller. The purified glycosylamines can be used for sugar analyses or, after permethylation, for methylation analyses or related procedures.
D000606:Amino Sugars / Q000032:analysis*; D055598:Chemical Phenomena; D002621:Chemistry; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D009844:Oligosaccharides / Q000032:analysis*; D009961:Orosomucoid / Q000032:analysis*
D000606:Amino Sugars; D009844:Oligosaccharides; D009961:Orosomucoid