DEVELOPMENT AND OPTIMIZATION OF HPLC ANALYSIS OF METRONIDAZOLE

Abstract

Two different reversed-phase HPLC techniques for the analysis of metronidazole and its related compound have been developed and optimized in this study. In RP-HPLC, isocratic water-acetonitrile and aqueous binary gradient of water+acetonitrile-0.1M sulphuric acid-acetonitrile were used as the mobile phase. Separation was carried out in the isocratic system using a 5 μm nova pack C18 column with 50:50 V/V water:acetonitrile as the mobile phase with a flow rate of 1 ml/min at 25 °C. Detection was carried out at a wavelength of 313 nm. Injection volume was 20 µl and retention time of 6.9 minutes was achieved. The aqueous binary gradient system was performed using a 5 μm hypersil C8 column with 75:25 V/V 0.1M sulphuric acid:acetonitrile as the initial mobile phase which would change direction to 10:90 V/V 0.1M sulphuric acid:acetonitrile. The flow rate was 1 ml/min at 25 °C with a total run time of 30 minutes. Detection was carried out at a wavelength of 313 nm and injection volume was 20 µl. Retention time for the first peak was 19.7 minutes and 26.6 minutes for the second. After both methods were utilized, the isocratic system was found to be more efficient and less time consuming for the separation of metronidazole and its related compound.

As a confirmation, an optimized method was developed through a complete forced degradation study of metronidazole utilizing a similar nova pack C18 column. A similar mobile phase of 50:50 V/V water:acetonitrile was used to carry optimum separation using different degraded samples of metronidazole. Visual observation as well as peak purity comparison was made from the separation of metronidazole with its degraded samples on the C18 column, showing that no degradation compounds were present in the 3UV dimension.