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Analysis of Contaminated Heparins by the Combination of Glycosaminoglycan Degrading Enzymes and Nuclear Magnetic Resonance Spectroscopy  

Im, A-Rang (Natural Products Research Institute, College of Pharmacy, Seoul National University)
Park, You-Mie (Natural Products Research Institute, College of Pharmacy, Seoul National University)
Hong, Chong-Hui (Korea Food Drug Administration)
Lee, Su-Jung (Korea Food Drug Administration)
Lee, Sun-Hee (Korea Food Drug Administration)
Kim, Yeong-Shik (Natural Products Research Institute, College of Pharmacy, Seoul National University)
Publication Information
Korean Journal of Pharmacognosy / v.40, no.2, 2009 , pp. 109-117 More about this Journal
Abstract
We examined the purity of six heparin samples by using heparinase, chondroitinase, $^{1}H-NMR$, and polyacrylamide gel electrophoresis. To obtain high molecular weight contaminants from heparin samples, heparinase I - digested samples were subjected to the exhaustive microcon filtration. The filtration process removed heparin-derived di- and oligosaccharides effectively. By combining chondroitinase ABC treatment and strong anion exchange - high performance liquid chromatography, the result showed all six samples contained chondroitin sulfate as a contaminant ranging from 1.3 to 14.9%. Among them, sample S3 showed the highest content of 14.9%, which was further analyzed by chondroitinase AC treatment to confirm chondroitin sulfate B (dermatan sulfate). $^{1}H-NMR$ chemical shifts of N-acetyl groups clearly suggested the existence of chondroitin sulfate B (sample S3) and oversulfated chondroitin sulfate (samples S2 and S4) as contaminants. In addition, polyacrylamide gel electrophoresis was useful for qualitative detection on the sample's purity. These results suggest that the tools of heparinase I and chondroitinase ABC in combination with NMR spectroscopy would give very useful information for investigation of heparin contaminants such as oversulfated chondroitin sulfate and dermatan sulfate in heparin samples.
Keywords
Heparin; Oversulfated chondroitin sulfate; Heparinase; Chondroitinase; Analysis; Dermatan sulfate;
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