Development of an off-line heart cutting 2D-HPLC method for monosaccharide analysis: focus on the resolution of the three epimers glucose, mannose, and galactose in grape-pomace fiber

Background Monosaccharide analysis presents several challenges due to their structural complexity as well as for their occurrence as enantiomers, diastereomers, and epimers. Their hydrophilicity and lack of chromophores further complicate direct analysis and easy detection. Nevertheless, as key components of food, in free or polysaccharide form, sugar analysis is essential, highlighting the need for simple and accessible methods for quality control and compositional studies. Results A two-dimensional (2D) achiral-chiral chromatography method was developed for the direct analysis of monosaccharides, focusing on the separation of the three epimers glucose, mannose, and galactose, largely overlooked despite their nutritional relevance. HILIC elution mode was applied in both dimensions, employing an ELS detector coupled to a HPLC system. The achiral amino column used in the first dimension proved effective in achieving the resolution of five out of eight investigated saccharides. The limited selectivity of the first dimension was coped with a teicoplanin-based column in the second dimension, which enabled the separation of the three epimers, through an off-line heart cutting protocol. The method was successfully applied to grape pomace extract, enabling the characterization of free sugars and fiber-derived monosaccharides, with sugar identity confirmed by GC-FID. Significance The developed 2D-HPLC approach represents an unprecedented strategy for monosaccharide analysis in complex matrices. The study further demonstrates that high levels of chemoselectivity can be achieved using stationary phases originally designed for enantioselective analysis. Another major strength of the study lies in the development of HILIC methods that are readily transferable to mass spectrometry-based analyses, enabling accurate quantification in complex matrices.