Quantification of galacturonic acid in pectin-containing samples using a stable isotope dilution approach and LC-MS

Reliable quantification of galacturonic acids (GalA) is essential for understanding the structural and functional properties of pectin-containing materials. However, conventional photometric methods often suffer from low reproducibility, limited sensitivity, and poorly understood reactions involved during analysis. Here, an LC-MS-based method for the sensitive and precise determination of total GalA contents in soluble and insoluble dietary fiber fractions of pectin containing samples is presented. The method is based on the degradation of GalA to the characteristic product 5-formyl-2-furancarboxylic acid (5FFA) in concentrated sulfuric acid under optimized conditions. The degradation product is extracted and quantified by UHPLC-ESI-MS. To compensate for degradation and extraction variability, the internal standard ¹³C₆-galacturonic acid is used. Quantification is achieved by single ion monitoring (SIM) of 5FFA and the equivalent ¹³C-labeled degradation product. The validated method was successfully applied to various sample materials, including isolated galacturonic acid oligosaccharide standards with defined degrees of polymerization and plant-derived dietary fiber samples such as carrot, apple, and citrus pulp. Comparison to a widely used colorimetric assay demonstrated that the results of the two methods differ if applied to soluble fiber samples. Thus, the LC-MS approach represents a robust alternative to photometric assays, offering enhanced sensitivity, precision, and applicability for pectin analysis.