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

Abstract

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 (R2 = 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.

Chemoenzymatic Production and Engineering of Chitooligosaccharides and N-acetyl Glucosamine for Refining Biological Activities

“…HPAEC, by reducing cost and time of the derivatization of COS, offered results with high resolution and sensitivity (Li et al, 2016 ). Cao et al ( 2016 ) conducted a study on the separation of the underivatized mixture of deacetylated COS (GlcN) 1−6 using HPAEC-PAD. The researchers used the HPAEC-PAD system comprised of ICS-3000 system, CarboPac-PA100 guard column (4 × 50 mm), and CarboPac-PA100 analytical column (4 × 250 mm).…”

Enzymatic Modification of Native Chitin and Conversion to Specialty Chemical Products

“…HPLC is probably the most frequently applied analysis method to gain structural insights of COS. Due to the acetyl group, a UV-light detector can be used to quantify GlcNAc units through monitoring of the absorbance rate at around 210 nm with an amino column [168], a reversed-phase column [169], or a carbohydrate column [170,171]. Chitosan-derived COS with a high D D can only be analysed by this method if the analytes are derivatized with a chromophore in a strenuous process, since an absorbing element would be missing otherwise [172].…”

“…High performance anionic exchange chromatography with pulsed amperometric detection (HAPAEC PAD) was validated as a sensitive tool to distinguish de-N-acetylated glucosamines with DP 1-6 rapidly and with low sample concentrations [171]. Nevertheless, no reliable predictions can be made for acetylated glucosamines or COS above DP 6 with this technique.…”

Application of Gelatin Decorated with Allura Red as Resonance Rayleigh Scattering Sensor to Detect Chito-Oligosaccharides

“…The present methods for the detection of COS include spectrophotometry [13], high performance liquid chromatography (HPLC) [14], ion chromatography [15], enzyme-linked immunosorbent assay (ELISA) [16], and capillary electrophoresis [17]. However, most of these methods have some limitations.…”