Determination of impurities in heparin by capillary electrophoresis using high molarity phosphate buffers

Oversulfated chondroitin sulfate (OSCS), an impurity found in some porcine intestinal heparin samples was separated from intact heparin by capillary electrophoresis (CE) using a 600 mM phosphate buffer, pH 3.5 as the background electrolyte in a 56 cm × 25 μm i.d. capillary. This method was confirmed in two separate labs, was shown to be linear, reproducible, robust, easy to use and provided the highest resolution and superior limits of detection compared to other available CE methods. Glycosoaminoglycans such as dermatan sulfate and heparan sulfate were separated and quantified as well during a single run. The heparin peak area response correlated well to values obtained using the official assay for biological activity. A high speed, high resolution version of the method was developed using 600 mM lithium phosphate, pH 2.8 in a 21.5 cm × 25 μm i.d. capillary which provided limits of detection for OSCS that were below 0.1%.