Development and validation of an ion-exchange chromatography method for heparin and its impurities in heparin products

An an ion-exchange liquid chromatography method for the determination of heparin andits impurities (dermatan sulfate and oversulfated chondroitin sulfate) was developed usingchemometric-assisted optimization, including multivariate experimental design and responsesurfacemethodology. The separation of heparin, dermatan sulfate, and oversulfatedchondroitin sulfate (Rs above 2.0) was achieved on aDionex RF IC IonPac AS22 column witha gradient elution of 10-70% of 2.5 M sodium chloride and 20 mM Tris phosphate buffer(pH 2.1) at a flow rate of 0.6 mL/min and UV detection at 215 nm.Method validation showsgood linearity (r > 0.99), acceptable precision (%relative standard deviations trueness (%recovery of 92.3-103.9%) for all analytes. The limits of detection for dermatansulfate and oversulfated chondroitin sulfate are equivalent to 0.11% w/w (10.5 g/mL) and0.07% w/w (7.2 g/mL), while the limits of quantification are 0.32% w/w (31.5 g/mL)and 0.22% w/w (22.0 g/mL) relative to heparin, respectively. The method is specific forheparin, dermatan sulfate, and oversulfated chondroitin sulfate without interference frommobile phase and sample matrices and could be used for accurate quantitation the drugand its impurities in a single run. Applications of the method reveal contents of heparinbetween 90.3 and 97.8%. Dermatan sulfate and oversulfated chondroitin sulfate were notdetected in any of the real-life samples.

Jaar van publicatie:2014

Trefwoorden:Anion-exchange chromatography