Investigation of monosaccharide colorimetric efficiency by phenol-sulfuric acid method and optimization of quantitative models for polysaccharides

Phenol-sulfuric acid method, a classical approach for total sugar quantification, suffers from substantial variation in the colorimetric efficiency among different monosaccharides, which may result in significant measurement bias. In this study, we investigated the colorimetric efficiency of 14 monosaccharides across three different systems and found that the colorimetric efficiency followed the pattern: neutral > acidic > amino monosaccharides. Notably, the colorimetric efficiency of mannose was over 800-fold higher than that of glucosamine. Principal coordinate analysis (PCoA) revealed that functional groups (carboxyl, amino) were the major determinants of colorimetric efficiency. Based on these findings, we assessed three quantitative models for five polysaccharides. The traditional model was suitable for homogeneous glucans (e.g., starch, recovery ∼101.0 %). The dominant monosaccharide model was effective for neutral polysaccharides with a single monosaccharide composition (e.g., mannan, recovery ∼100.5% in the optimized system). In contrast, the weighted model, which integrates molar ratios and individual efficiencies, failed to improve accuracy for complex heterogeneous acidic polysaccharides (e.g., fucoidans, recovery <40.0%). Furthermore, amino groups in polysaccharides (e.g., chitosan) strongly inhibited the reaction, precluding accurate quantification. This study provides a theoretical basis for the precise application of the phenol-sulfuric acid method and offers methodological guidance for the standardization of polysaccharide analysis.