Advancing total uronic acid quantification using a stable isotope dilution approach: validation and application to plant- and algal-derived polysaccharides

Uronic acids (UAs) are key components of plant and algal polysaccharides and serve as crucial markers for their structural and functional characterization. The widely applied conventional colorimetric Blumenkrantz and Asboe-Hansen assay suffers from matrix-dependent interferences and poor reproducibility. In this study, an LC-MS-based stable isotope dilution approach was optimized for reliable quantification of total UA content in complex biopolymer matrices. Calibration and degradation parameters were systematically adapted to various UA compositions, including galacturonic, glucuronic (GlcA), mannuronic (ManA), and guluronic acids (GulA). Structural factors such as degree of polymerization, O4-methyl substitution of GlcA, and the stability of aldobiuronic acid linkages in glucuronoxylan were also evaluated. Comparison with spectrophotometric results revealed that the LC-MS method provides higher robustness and selectivity, particularly in samples prone to over- or underestimation by traditional assays. The method further allows selective determination of alginate-derived UAs (ManA + GulA) in mixed matrices, exemplified by food materials containing xanthan. The optimized LC-MS method thus represents a versatile and accurate approach for the quantitative assessment of total UA contents in diverse biological and industrially relevant polysaccharides.