Methodological Research For Analysis Of Heparin Sodium And Rapid Potency Determination

Heparin sodium (HP) is the sodium salt of sulfated glycosaminoglycans. Heparin is best known as an intravenous anticoagulant and was introduced into medical practice in the1930s, so it is one of the oldest drugs currently in widespread clinical use. However, serious adverse events associated with heparin administration were reported during2007-2008. This adulteration made the news and put heparin into the limelight. As reported, over-sulfated chondroitin sulfate (OSCS) in contaminated heparin products is the source of patients receiving heparin sodium resulting in acute allergic reactions and even death. OSCS is very similar with heparin sodium in structure, charge density, molecular weight and anticoagulant activity. Hence, to set up a fast and accurate method to determine OSCS, dermatan sulfate (DS) and other impurities in heparin sodium is not only the key to revise the pharmacopeia monographs, but also to improve the quality level of products, thus guarantees its durg safety.Firstly, to establish a precise method to determine simultaneously the related substances of OSCS and DS in heparin products, VWD was used to determine OSCS and DS. Five methods were compared according to their resolution of OSCS and DS. Method E was the optimized method with the greatest resolution and theoretical plates, the limit of detection (LOD) and limit of quantitation (LOQ) for the OSCS in heparin sodium were determined to be0.025%and0.03%, respectively. The LOD and LOQ for dermatan sulfate were determined to be0.03%and0.05%, respectively. This method is more accurate, sensitive, robust and reproducible, and it could be applied to replace the current pharmacopeia specifications of heparin products.In order to set up an analytical method suitable for small and medium-scale manufacturers, a comparative study of the three main heparin related substances detection methods:capillary electrophoresis, nuclear magnetic resonance and weak anion exchange HPLC, was carried out. The reliability, simplicity and economic feasibility indicators between the methods were investigated. The results indicated that WAX-HPLC method was proved to be the most appropriate method.To determine the potency of heparin sodium in the preparation process rapidly, dual wavelength spectrophotometry with azure A was used. Microplate reader was used to substitute the spectrophotometer in the method. This method is rapid and accurate, also can reduce the amount of samples and reagents. In comparison with the sheep plasma method, the difference between the results is within±4U/mg.