Simultaneous Separation of Five Fluoroquinolones - Norfloxacin, Moxifloxacin, Enrofloxacin, Sparfloxacin and Prulifloxacin Using an Isocratic HPLC: Application to Determination of Norfloxacin in Pharmaceutical Formulations

Abstract

A rapid, simple, precise, specific, highly sensitive, accurate and reproducible isocratic reversed phase HPLC method was developed for the separation of five fluoroquinolones and validated for determination of norfloxacin for routine pharmaceutical quality control analysis in bulk and formulations. One of the salient aims for the method development is to accomplish constant, reproducible separation. The selection of good method is essential if this goal is to be achieved. RPHPLC method was developed by using WELCHROM C18 Column (4.6 X 250mm, 5µm), SHIMADZU LC-20AT prominence liquid chromatograph. The mobile phase consisting of phosphate buffer (pH-6.8)and acetonitrile in the proportion of 50:50 v/v under isocratic elution at a flow rate of 1mL/min was employed. The responses were measured at 281nm using SHIMADZU SPD-20A prominence UV-Vis detector. The retention time of norfloxacin, moxifloxacin, enrofloxacin, sparfloxacin and prulifloxacin were found to be 2.940 min, 3.363 min, 3.790 min, 4.013 min and 7.520 min, respectively. Chromatographic runtime was 10 min with elution window of 6 min and with a resolution of greater than 2.0 for all compounds. The developed method was validated according to ICH guidelines.The average recoveries ranged from 99.03 - 100.36 %, with %RSD less than 2%. The concentration of calibration solutions was in the range of 2-10 μg/mL and LOD and LOQ were 0.1491 and 0.4519 μg/mL respectively. The method was successfully applied to analysis of norfloxacin in pharmaceutical formulation. It can also be extended for the determination of other four fluoroquinolones or their combinations.