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Comparison of established and novel purity tests for the quality control of heparin by means of a set of 177 heparin samples

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An Erratum to this article was published on 30 October 2010

Abstract

The widespread occurrence of heparin contaminated with oversulfated chrondroitin sulfate (OSCS) in 2008 initiated a comprehensive revision process of the Pharmacopoeial heparin monographs and stimulated research in analytical techniques for the quality control of heparin. Here, a set of 177 heparin samples from the market in 2008 as well as pure heparin sodium spiked with defined amounts of OSCS and DS were used to evaluate established and novel methods for the quality control of heparin. Besides 1H nuclear magnetic resonance spectroscopy (NMR), the assessment included two further spectroscopic methods, i.e., attenuated total reflection-infrared spectroscopy (ATR-IR) and Raman spectroscopy, three coagulation assays, i.e., activated partial thromboplastin time (aPTT) performed with both sheep and human plasma and the prothrombin time (PT), and finally two novel purity assays, each consisting of an incubation step with heparinase I followed by either a fluorescence measurement (Inc-PolyH-assay) or by a chromogenic aXa-assay (Inc-aXa-assay). NMR was shown to allow not only sensitive detection, but also quantification of OSCS by using the peak-height method and a response factor determined by calibration. Chemometric evaluation of the NMR, ATR-IR, and Raman spectra by statistical classification techniques turned out to be best with NMR spectra concerning the detection of OSCS. The validity of the aPTT, the current EP assay, could be considerably improved by replacing the sheep plasma by human plasma. In this way, most of the contaminated heparin samples did not meet the novel potency limit of 180 IU/mg. However, also more than 50% of the uncontaminated samples had <180 IU/MG. In contrast to the aPTT, the PT specifically detects OSCS and other heparin mimetics (LOD 3%). About ten times more sensitive are both the Inc-PolyH-assay and the Inc-aXa-assay, two rapid and simple quantification assays for heparin mimetics. The determined OSCS contents of the heparin samples excellently correlated with those calculated from the NMR spectra. In conclusion, NMR proved to be the current spectroscopic method of choice. The two two-step-assays represent options to supplement NMR, especially as tests for the initial screening, since they detect any heparin mimetic without requiring special expertise for interpretation of the results.

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Acknowledgment

Thanks are due to the Federal Institute for Drugs and Medical Devices for providing financial support to UH.

Author information

Authors and Affiliations

  1. Pharmaceutical Institute, Christian-Albrechts-University, Gutenbergstr. 76, 24118, Kiel, Germany

    Susanne Alban, Susanne Lühn & Simone Schiemann

  2. Institute of Pharmacy and Food Chemistry, University of Wuerzburg, Am Hubland, 97074, Wuerzburg, Germany

    Tanja Beyer & Ulrike Holzgrabe

  3. Federal Institute for Drugs and Medical Device, Kurt-Georg-Kiesinger-Allee 3, 53175, Bonn, Germany

    Jochen Norwig

  4. Ratiopharm Group, Graf-Arco-Str. 3, 89079, Ulm, Germany

    Claudia Schilling, Oliver Rädler & Bernhard Wolf

  5. Institute of Pharmaceutical Chemistry, University of Technology Braunschweig, Beethovenstrasse 55, 38106, Braunschweig, Germany

    Magnus Matz & Knut Baumann

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  1. Susanne Alban
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  2. Susanne Lühn
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  11. Ulrike Holzgrabe
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Correspondence to Ulrike Holzgrabe.

Additional information

Published in the special issue Heparin Characterization with Guest Editor Cynthia K. Larive.

An erratum to this article can be found at http://dx.doi.org/10.1007/s00216-010-4317-0

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Alban, S., Lühn, S., Schiemann, S. et al. Comparison of established and novel purity tests for the quality control of heparin by means of a set of 177 heparin samples. Anal Bioanal Chem 399, 605–620 (2011). https://doi.org/10.1007/s00216-010-4169-7

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  • DOI: https://doi.org/10.1007/s00216-010-4169-7

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