Validated high-performance anion-exchange chromatography with pulsed amperometric detection method for the determination of residual keratan sulfate and other glucosamine impurities in sodium chondroitin sulfate

An efficient and sensitive analytical method based on high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) was devised for the determination of glucosamine (G1cN) in sodium chondroitin sulfate (CS). Glucosamine (GIcN) is intended as marker of residual keratan sulfate (KS) and other impurities generating glucosamine by acidic hydrolyzation. The latter brings CS and KS to their respective monomers. Since GIcN is present only in KS we developed a method that separates GIcN from Ga1N, the principal hydrolytic product of CS, and then we validated it in order to quantify G1cN. Method validation was performed by spiking CS raw material with known amounts of KS. Detection limit was 0.5% of KS in CS (corresponding to 0.1 mu g/ml), and the linear range was 0.5-5% of KS in CS (corresponding to 0.1-1 mu g/ml). The optimized analysis was carried out on an ICS-5000 system (Dionex, Sunnyvale, CA, USA) equipped with a Dionex Amino Trap guard column (3 mm x 30 mm), Dionex CarboPac-PA20 (3 mm x 30 mm) and a Dionex CarboPac-PA20 analytical column (3 mm x 150 mm) using gradient elution at a 0.5 ml/min flow rate. Regression equations revealed good linear relationship (R-2=0.99, n = 5) 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 residual KS in CS in several raw materials and USP/EP reference substance. Results confirmed that the HPAEC-PAD method is more specific than the electrophoretic method for related substance reported in EP and provides sensitive determination of KS in acid-hydrolyzed CS samples, enabling the quantitation of KS and other impurities (generating glucosamine) in CS. (C) 2017 Elsevier B.V. All rights reserved.