Korean Journal of Pharmacognosy (생약학회지)

The Korean Society of Pharmacognosy (한국생약학회)
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Analysis of Contaminated Heparins by the Combination of Glycosaminoglycan Degrading Enzymes and Nuclear Magnetic Resonance Spectroscopy

글리코사미노글칸 분해 효소와 핵자기공명을 이용한 오염된 헤파린 시료의 분석

  • 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)
  • 임아랑 (서울대학교 약학대학/천연물과학연구소) ;
  • 박유미 (서울대학교 약학대학/천연물과학연구소) ;
  • 홍정희 (식품의약품안전청) ;
  • 이수정 (식품의약품안전청) ;
  • 이선희 (식품의약품안전청) ;
  • 김영식 (서울대학교 약학대학/천연물과학연구소)
  • Published : 2009.06.30
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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

References

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