Method development for identification of streptokinase in pharmaceutical formulation by SDS-PAGE/Western Blot as an alternative to pharmacopoeia method

Abstract

       Streptokinase is currently used for the treatment of thromboembolic blockages such as stroke. Due to its high price, illegal streptokinase has been found in pharmaceutical market. According to Drug Act of B.E. 2510 (1967), the punishment can be done if the object belonging to the culprit is proved as real medicine. Thus, the identification test of the drug is especially important for the investigation process. The identification method for streptokinase in pharmacopoeia requires the use of animal plasma; however, the reduction of animal usage in drug test by using alternative method is widely accepted in every corner of the world. Therefore, our study aimed to develop the identification test for streptokinase using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot as an alternative for pharmacopoeia method. Various factors including concentrations of primary and secondary antibodies, incubation period, and limit of detection (LOD) for streptokinase standard and streptokinase pharmaceutical formulation were validated. The optimal concentrations of primary and secondary antibodies were at 7.5 × 10^-3 and 2 × 10^-3 mg/ml, respectively. The incubation periods for both primary and secondary antibodies at 1 h provided the strongest signal. LOD values of streptokinase standard and streptokinase pharmaceutical formulation were at the concentrations of 0.5 and 20 IU/ml, respectively. The human albumin, which is used as stabilizer in the streptokinase pharmaceutical formulation, was not interfered with the test. The results of the study confirmed that the method is reliable and applicable for the identification of streptokinase in pharmaceutical formulation.