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Anal Biochem. 2014 Sep 15;461:46-8. doi: 10.1016/j.ab.2014.05.028. Epub 2014 Jun 6.

Heparin stability by determining unsubstituted amino groups using hydrophilic interaction chromatography mass spectrometry.

Author information

1
Department of Chemistry and Chemical, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180, USA; Department of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180, USA.
2
Department of Chemistry and Chemical, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180, USA.
3
Department of Chemistry and Chemical, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180, USA; College of Food Science and Technology, Ocean University of China, Qingdao, Shandong 266003, China.
4
Department of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180, USA.
5
Department of Chemistry and Chemical, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180, USA; Department of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180, USA; Department of Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180, USA; Department of Biomedical Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180, USA. Electronic address: linhar@rpi.edu.

Abstract

The thermal instability of the anticoagulant heparin is associated, in part, with the solvolytic loss of N-sulfo groups. This study describes a new method to assess the increased content of unsubstituted amino groups present in thermally stressed and autoclave-sterilized heparin formulations. N-Acetylation of heparin samples with acetic anhydride-d6 is followed by exhaustive heparinase treatment and disaccharide analysis by hydrophilic interaction chromatography mass spectrometry (HILIC-MS). The introduction of a stable isotopic label provides a sensitive probe for the detection and localization of the lost N-sulfo groups, potentially providing valuable insights into the degradation mechanism and the reasons for anticoagulant potency loss.

KEYWORDS:

Amino group; Heparin; Mass spectrometry; Stability assay; Sulfate

PMID:
24909446
PMCID:
PMC4119833
DOI:
10.1016/j.ab.2014.05.028
[Indexed for MEDLINE]
Free PMC Article
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