Development of a Novel Separation Technique by High Performance Liquid Chromatography for the Determination of Bivalirudin

Abstract

The separation of a long peptide chain without derivatization, without amide columns and by using routine reversed-phase HPLC columns is itself a big task. Increase in the basic nature of the mobile phase as well as in column temperature and low flow rate of the mobile phase were demonstrated to permit the separation of polar compounds containing long peptide chains. The aim of this study was to develop a procedure for the determination of an antithrombing agent Bivalirudin containing a long peptide chain. This method was developed on a Princeton SPHER-C₁₈ 100 Å (4.6 × 250 mm, 5 µm) column and a mobile phase consisting of water-acetonitrile (pH 7, adjusted with triethylamine) in the ratio of 95 : 5. The flow rate was 0.7 mL/min at the thermostat temperature of 45°C. Validation of the method was performed as per the International Conference on Harmonization. The method was applied to the analysis of a pharmaceutical formulation. The developed method was accurate, fast, easy for implementation and economy as it did not require sophisticated equipment or tedious derivatization process.