Quantification of chitosan in aqueous solutions by enzymatic hydrolysis and oligomer analysis via HPLC-ELSD

A new method for quantitative analysis of chitosan in aqueous solution is introduced, comprising an enzyme-driven cleavage to water-soluble chitooligosaccharides (COS), N-acetylation, separation via UHPLC and detection by use of an evaporative light scattering detector (ELSD). Chitosans with different fractions of acetylation (F_A) and molecular weights (M_w) were hydrolyzed using a chitosanase/chitinase mixture. By subsequent N-acetylation with isotopically labelled acetic anhydride, COS mixtures with F_A = 1 were obtained allowing for chromatographic separation solely based on their degree of polymerization (DP). ELSD data conversion into molar concentrations was realized using COS-specific external calibration curves, and mass spectrometry (MS) data informed about the chitosan's F_A. The overall chitosan concentration was determined by simple addition of the COS concentrations multiplied by their DP. Validity of the method is shown for chitosan in presence of various co-solutes such as the protein BSA, the polysaccharide dextran and the monosaccharide glucosamine.