Advanced structural profiling of arabinoxylans from cereal grains by high performance anion exchange chromatography with parallel pulsed amperometric and mass spectrometric detection.
Arabinoxylans (AX) are major hemicellulosic polysaccharides of cell walls of cereal grains. Potential health benefits and techno-functional properties of AX are determined, among others, by the extent and distribution of arabinofuranose (Araf) units as substituents along the β-(1 → 4)-linked d-xylopyranose (Xylp) backbone. Conventional carbohydrate analytical methods, such as methylation analysis, are time-consuming and often insufficient to capture structural diversity. Therefore, a semi-quantitative profiling method based on enzymatic AX degradation and on high-performance anion-exchange chromatography with pulsed amperometric and mass spectrometric detection (HPAEC-PAD/MS) was developed. The method relies on enzymatic depolymerization of AX in cereal dietary fiber using endo-β-1,4-xylanases from glycoside hydrolase families 10 and 11, releasing xylooligosaccharides (XOS) and arabinoxylooligosaccharides (AXOS), which were analyzed by HPAEC-PAD/MS. These enzyme-dependent (A)XOS profiles, which do not cover enzymatically resistant AX regions, provide indirect insights into AX structural characteristics. Newly isolated higher substituted AXOS standards were combined with commercially available compounds, resulting in 17 structurally distinct (A)XOS standards for a more detailed characterization of AX substitution patterns with Araf. Furthermore, incubation conditions and chromatographic parameters were optimized, and relative response factors (RRF) of the (A)XOS standards relative to an internal standard were determined, providing a basis for routine semi-quantitative analysis. The applicability of the method was demonstrated using well-characterized cereal soluble and insoluble dietary fiber samples (five different wheat (Triticum) species, rye, and barley), confirming its ability to provide additional structural information on AX. Among others, it was demonstrated that the analyzed wheat species exhibited comparable (A)XOS profiles, being consistent with their close phylogenetic relationship.