Carbohydrates are one of the most important ingredients in foods because they provide not only important nutritional value and organoleptic quality, but also functional properties beneficial to human health, which are strongly related to their chemical structure. Carbohydrates usually appear as complex mixtures of several isomers with different monosaccharide units, glycosidic linkages and degrees of polymerization. This complex composition makes their characterization very challenging. High performance liquid chromatography is one of the most widespread techniques for the analysis of carbohydrates. However, in many cases, the separation provided by monodimensional LC (1D LC) is not enough and the use of multidimensional liquid chromatography (LC×LC), with a higher resolving power, could be a good alternative. However, to the best of our knowledge, the application of LC×LC to the analysis of carbohydrate mixtures has not yet been performed. Thus, the aim of this work was to develop for the first time an LC×LC method for the analysis of carbohydrate mixtures with different glycosidic linkages and monomeric units. This method was applied to the analysis of commercial prebiotic oligosaccharides. Different stationary phases and chromatographic conditions were evaluated in 1D LC for the separation of di- and trisaccharide standard mixtures. These carbohydrates were derivatized using the 4-aminobenzoic acid ethyl ester (ABEE) reagent, previous to their analysis. Based on the data collected by 1D LC, the best orthogonality was achieved using a HILIC XBridge Amide column (2.1 x 150 mm, 3.5 μm particle size) in the first dimension and a partially porous Ascentis Express C18 column (50 x 4.6 mm, 2.7 μm particle size) in the second dimension. Optimization of the method involved the column dimensions, gradients, flow rates, injection volumes and modulation times. Moreover, the use of active modulation using two C18 trapping columns in the interface was able to efficiently minimize the solvent strength mismatch problems related to this coupling. The developed method was applied to the analysis of different commercial prebiotic formulations of galactooligosaccharides and gentiooligosaccharides. To the best of our knowledge, this is the first time that bioactive carbohydrates mixtures have been analysed by HILIC×RP-LC. Based on our results, LC×LC could be considered as a powerful analytical technique for the characterization of other oligosaccharides complex mixtures.

This research has been funded by MINEICO of Spain (projects AGL2016-80475-R, AGL2017-89417-R and AGL2017-84614-C2-1-R) and by Comunidad Autónoma of Madrid and European funding from FEDER program (project S2013/ABI-3028, AVANSECAL).

Trabajo presentado a las III Jornadas Científicas CIAL Fórum, celebradas del 22 al 23 de noviembre de 2018 en el Instituto de Investigación en Ciencias de la Alimentación (CIAL).

Peer reviewed