Chitosan analysis using acid hydrolysis and HPLC/UV

In this study, a method for the characterization and quantification of chitosan was designed using acid hydrolysis, glucosamine derivatization, and high performance liquid chromatography. Based on a kinetic study of acid hydrolysis, we have demonstrated that chitosan can be quantitatively hydrolyzed into glucosamine in 6 h with either 10 M hydrochloric acid (HCl) at 105 °C, or 12 M HCl at 90 °C. Following N-(9-fluorenylmethoxycarbonyloxy) succinimide (Fmoc-OSu) derivatization, the glucosamine content can be separated from the rest of the hydrolysates and quantified using reverse-phase HPLC with UV detection. This method was validated for linearity, precision (repeatability and reproducibility), and accuracy using both chitosan and a dietary supplement formulation containing chitosan.