Determination of chitosan with a modified acid hydrolysis and HPLC method

Acid hydrolysis and subsequent quantification of glucosamine (GlcN) are widely used for chitosan quantification. Degradation of GlcN during chitosan hydrolysis was the main reason for the decrease of recovery, which made the method improper for the quantification of chitosan. Ten milligram of chitosan hydrolyzed with 10 mL mixed acid solution of HCl–H3PO4 (75:25 in molar ratio) showed the highest recovery, significantly higher than HCl hydrolysis. Further study revealed that the optimum conditions involved the hydrolysis with HCl–H3PO4 (4.5:1.5 M) for 24 h at 110 °C. The hydrolysate was neutralized and derived with 9-fluorenylmethoxycarbonyl chloride (FMOC-Cl) before HPLC quantification. The optimum ratio of FMOC-Cl:GlcN was 53:1, with excess FMOC-Cl induced by the high ionic strength of the solution. This quantification procedure was then validated and proved to be specific, with good linearity, accuracy, and precision, making it well-suited for the determination of chitosan.

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► HCl–H3PO4 (75:25 in molar ratio) showed higher recovery of chitosan than HCl hydrolysis.
► Hydrolysis with HCl–H3PO4 (4.5:1.5 M) for 24 h at 110 °C showed highest recovery for chitosan hydrolysis.
► In high ionic strength solution, optimum FMOC-Cl:GlcN ratio increased to 53:1 for GlcN derivation.
► The modified method improved the recovery from about 90% to 97.7% for the determination of chitosan.