Evaluation of different operation modes of high performance liquid chromatography for the analysis of complex mixtures of neutral oligosaccharides.

Chromatographic methods based on different HPLC operation modes, reverse phase (RP), high performance anion exchange chromatography (HPAEC), graphitized carbon chromatography (GCC) and hydrophilic interaction liquid chromatography (HILIC), have been developed and compared for the analysis of complex mixtures of neutral oligosaccharides with functional properties. Whereas GCC gave the best chromatographic separation of isomeric oligosaccharides with the same molecular weight (R(s) values in the range 1.0-4.0 and 2.4-5.6 for tetra- and pentasaccharides, respectively), HILIC provided the best results for mixtures including oligosaccharides of different degrees of polymerization (R(s) values of maltooligosaccharides between 3.4 and 6.2). Validation of the HILIC LC-MS method proved its utility for the analysis of oligosaccharide mixtures with functional properties: relative standard deviations lower than 10%, LOD's and LOQ's in the range 12.7-130.2 ng mL(-1) and 39.3-402.2 ng mL(-1), respectively, and linearity up to 10-20 μg mL(-1). Quantitative data for fructooligosaccharides, gentiooligosaccharides and dextransucrase cellobiose acceptor oligosaccharides were obtained by using this method.