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.