CAPILLARY ELECTROPHORESIS OF INTACT HEPARIN AND IMPURITIES IN SHORT CAPILLARIES WITH HIGH CONCENTRATION PHOSPHATE BUFFERS USING LOW MOBILITY COUNTERIONS

Abstract

Capillary electrophoresis (CE) of heparin, oversulfated chondroitin sulfate (OSCS), dermatan sulfate (DS), and related impurities were separated in 5 min using 1 M lithium phosphate buffer, pH 2.5 on an 8.5 cm × 25 μm i.d. capillary. The separation was optimized for overall speed, resolution between heparin, OSCS and DS, and in particular, the limit of detection (LOD) for OSCS. The LOD for OSCS was less than 0.03% of heparin. The migration time precision was always less than 0.5% for all solutes. The peak area precision was 1.9% and 2% for OSCS and DS at the 0.4% and 2% concentration levels, respectively. The response factors of OSCS and DS were found to be 3.69 and 6.40 times greater than heparin, respectively. Optimization experiments including buffer counterion and concentration, buffer pH, sample concentration, and injection size are reported. The buffer counterion impacted the speed and resolution of the separation beyond consideration of the effect of solution conductivity alone. A correlation between data generated by both CE and NMR for 29 separate lots of heparin known to be contaminated with OSCS was reported for the first time. The differences in OSCS measured by both techniques was not statistically significant.

Keywords:

• capillary electrophoresis
• dermatan sulfate
• heparin
• heparin impurities
• oversulfated chondroitin sulfate

ACKNOWLEDGMENTS

The authors wish to thank Mark Nordhaus of the Baxter Technology Resources Molecular Design and Synthetic Chemistry for preparing the Over Sulfated Chondroitin Sulfate.