Analysis of Pharmaceutical Heparins and Potential Contaminants Using 1 H-NMR and PAGE ZHENQING ZHANG, 1,2 BOYANGZI LI, 1,2 JIRAPORN SUWAN, 2,3 FUMING ZHANG, 2,4 ZHENYU WANG, 2,5 HAIYING LIU, 1,2 BARBARA MULLOY, 6 ROBERT J. LINHARDT 1,2,4,5 1 Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy, New York 12180 2 Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York 12180 3 Faculty of Medicine, Department of Biochemistry, Thailand Excellence Center for Tissue Engineering, Chiang Mai University, Chiang Mai 50200, Thailand 4 Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, New York 12180 5 Department of Biology, Rensselaer Polytechnic Institute, Troy, New York 12180 6 National Institute for Biological Standards and Control, South Mimms, Potters Bar, Herts EN6 3QG, UK Received 11 December 2008; revised 20 January 2009; accepted 26 January 2009 Published online 29 July 2009 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.21729 ABSTRACT: In 2008, heparin (active pharmaceutical ingredient, API) lots were associated with anaphylactoid-type reactions. Oversulfated chondroitin sulfate (OSCS), a semi-synthetic glycosaminoglycan (GAG), was identified as a contaminant and der- matan sulfate (DS) as an impurity. While DS has no known toxicity, OSCS was toxic leading to patient deaths. Heparins, prepared before these adverse reactions, needed to be screened for impurities and contaminants. Heparins were analyzed using high-field 1 H-NMR spectroscopy. Heparinoids were mixed with a pure heparin and analyzed by 1 H-NMR to assess the utility of 1 H-NMR for screening heparin adulterants. Sensitivity of heparinoids to deaminative cleavage, a method widely used to depolymerize heparin, was evaluated with polyacrylamide gel electrophoresis to detect impurities and con- taminants, giving limits of detection (LOD) ranging from 0.1% to 5%. Most pharma- ceutical heparins prepared between 1941 and 2008 showed no impurities or contaminants. Some contained DS, CS, and sodium acetate impurities. Heparin pre- pared in 2008 contained OSCS contaminant. Heparin adulterated with heparinoids showed additional peaks in their high-field 1 H-NMR spectra, clearly supporting NMR for monitoring of heparin API with an LOD of 0.5–10%. Most of these heparinoids were stable to nitrous acid treatment suggesting its utility for evaluating impurities and contaminants in heparin API. ß 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:4017–4026, 2009 Keywords: heparin; contamination; 1 H-NMR; heparinoids; nitrous acid INTRODUCTION Heparin is a polydisperse mixture of linear acidic polysaccharides and has been in clinical use as an anticoagulant over 75 years. 1–3 Heparin is unique as one of the oldest drugs currently still in widespread clinical use, one of the first bio- polymeric drugs, and one of the few carbohydrate Additional Supporting Information may be found in the online version of this article. Correspondence to: Robert J. Linhardt (Telephone: 518-276- 3404; Fax: 518-276-3405; E-mail: linhar@rpi.edu) Journal of Pharmaceutical Sciences, Vol. 98, 4017–4026 (2009) ß 2009 Wiley-Liss, Inc. and the American Pharmacists Association JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 11, NOVEMBER 2009 4017