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

Philipp Schröder; Jasper Wattjes; Monika Schönhoff; Bruno M Moerschbacher; Cornelia Cramer; Stefan Cord-Landwehr

doi:10.1016/j.carbpol.2022.119141

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

Carbohydr Polym. 2022 May 1:283:119141. doi: 10.1016/j.carbpol.2022.119141. Epub 2022 Jan 14.

Authors

Philipp Schröder¹, Jasper Wattjes², Monika Schönhoff³, Bruno M Moerschbacher⁴, Cornelia Cramer⁵, Stefan Cord-Landwehr⁶

Affiliations

¹ Institute of Physical Chemistry and Center for Soft Nanoscience, University of Muenster, 48149 Münster, Germany. Electronic address: schroedp@uni-muenster.de.
² Institute for Biology and Biotechnology of Plants, University of Muenster, 48143 Münster, Germany. Electronic address: jasper.wattjes@wiwi.uni-muenster.de.
³ Institute of Physical Chemistry and Center for Soft Nanoscience, University of Muenster, 48149 Münster, Germany. Electronic address: schoenho@uni-muenster.de.
⁴ Institute for Biology and Biotechnology of Plants, University of Muenster, 48143 Münster, Germany. Electronic address: moersch@uni-muenster.de.
⁵ Institute of Physical Chemistry and Center for Soft Nanoscience, University of Muenster, 48149 Münster, Germany. Electronic address: dr.cornelia.cramer@uni-muenster.de.
⁶ Institute for Biology and Biotechnology of Plants, University of Muenster, 48143 Münster, Germany. Electronic address: stefan.cord-landwehr@uni-muenster.de.

PMID: 35153014
DOI: 10.1016/j.carbpol.2022.119141

Abstract

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.

Keywords: Chitin/chitosan oligosaccharides; Chitosan degradation; Chitosan determination; Chitosanase/chitinase; HILIC.