Analysis of crude heparin by ¹H NMR, capillary electrophoresis, and strong-anion-exchange-HPLC for contamination by over sulfated chondroitin sulfate

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• 作者：David A. Keire ; Michael L. Trehy ; John C. Reepmeyer ; Richard E. Kolinski ; Wei Ye ; Jamie Dunn ; Benjamin J. Westenberger ; Lucinda F. Buhse
• 关键词：Heparin ; Over-sulfated or fully-sulfated chondroitin sulfate ; Dermatan sulfate ; Glycosaminoglycan ; SAX-HPLC ; NMR ; CE
• 刊名：Journal of Pharmaceutical and Biomedical Analysis
• 出版年：2010
• 出版时间：11 March 2010
• 年：2010
• 卷：51
• 期：4
• 页码：921-926
• 全文大小：496 K

文摘

We previously published a strong-anion-exchange-high performance liquid chromatography (SAX-HPLC) method for the detection of the contaminant over sulfated chondroitin sulfate (OSCS) in heparin sodium active pharmaceutical ingredient (API). While APIs have been processed to remove impurities, crude heparins contain insoluble material, chondroitin sulfates, heparan sulfate, and proteins that may interfere with the recovery and measurement of OSCS. We examined 500 MHz ¹H NMR, capillary electrophoresis (CE), and SAX-HPLC to quantify OSCS in crude heparin. Using our standard API protocol on OSCS spiked crude heparin samples; we observed a weight percent LOD and LOQ for the NMR approach of 0.1 % and 0.3 % , respectively, while the SAX-HPLC method gave values of 0.03 % and 0.09 % , respectively. CE data was not amenable to quantitative measurement of OSCS in crude heparin. We developed a modified HPLC sample preparation protocol using crude dissolved at the 100 mg/mL level with a 2.5 M NaCl solution. This SAX-HPLC approach gave a weight percent LOD of 0.02 % and a LOQ of 0.07 % and had better performance characteristics than that of the protocol used for APIs.