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An efficient and sensitive analytical method based on high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) was established for the simultaneous separation and determination of glucosamine (GlcN)₁ and chitooligosaccharides (COS) ranging from (GlcN)₂ to (GlcN)₆ without prior derivatization. Detection limits were 0.003 to 0.016 mg/L (corresponding to 0.4-0.6 pmol), and the linear range was 0.2 to 10 mg/L. The optimized analysis was carried out on a CarboPac-PA100 analytical column (4 × 250 mm) using isocratic elution with 0.2 M aqueous sodium hydroxide-water mixture (10:90, v / v ) as the mobile phase at a 0.4 mL/min flow rate. Regression equations revealed a good linear relationship ( R ² = 0.9979-0.9995, n = 7) within the test ranges. Quality parameters, including precision and accuracy, were fully validated and found to be satisfactory. The fully validated HPAEC-PAD method was readily applied for the quantification of (GlcN) 1-6 in a commercial COS technical concentrate. The established method was also used to monitor the acid hydrolysis of a COS technical concentrate to ensure optimization of reaction conditions and minimization of (GlcN)₁ degradation.

Validated HPAEC-PAD Method for the Determination of Fully Deacetylated Chitooligosaccharides