Characterization of heparin impurities with HPLC-NMR using weak anion exchange chromatography.

A weak anion exchange (WAX) chromatography method coupled to online UV and NMR detection was developed for the separation and structural identification of heparin impurities. This work describes the separation of dermatan sulfate, chondroitin sulfate A, heparin, and the semisynthetic fully sulfated chondroitin sulfate, FSCS, through a displacement-based mechanism. The WAX separation requires significantly less salt than comparable strong anion exchange chromatography, facilitating NMR detection using a standard commercially produced flow probe. Retention and elution of both heparin and FSCS were found to be highly dependent on pH/pD and salt concentration. The elution time of each analyte could be controlled simply by adjusting a delay prior to introduction of the appropriate salt concentration, with little impact of the extent of the delay on chromatographic peak shape. Both on-flow and stop-flow proton ((1)H) NMR spectra were acquired to monitor and identify eluting components following separation. The approach described for the online separation of heparin and its potential impurities adds to the arsenal of techniques available for the rapid identification of new natural and/or intentional contaminants that may be introduced into the heparin supply chain.