Abstract

Background: A reversed-phase high-performance liquid chromatographic method for determining thrombin activity is described. The improved method can provide a high resolution between substrate Nα-P-toluenesulfonyl-L-arginine methyl ester (TAME) and the product of enzyme reaction, Nα-P-toluenesulfonyl-L-Arginine (TA).

Methods: When the substrate concentration was 5 mg/mL, the Michaelis equation approached the zero-order reaction, and the thrombin enzyme activity must have a good linear relationship with the consumption of TAME or the yield of TA.

Results: The method is suitable for quantitatively analyzing thrombin activity in formulation or serum.

Conclusion: Three batches of thrombin lyophilized preparations were determined for activity.

Keywords: Thrombin, reversed-phase high-performance liquid chromatography, pharmaceutical formulations, Nα-Ptoluenesulfonyl- L-arginine methyl ester (TAME), Nα-P-toluenesulfonyl-L-Arginine (TA), HPLC analysis.