RP-HPLC Method Development and Validation for the Estimation of Ofloxacin in Eye Drop

The present study deals with development of reversed phase HPLC method for the quantitation of Ofloxacin in ophthalmic solutions utilizing as an internal standard (Ornidazole). The approach is easy, sensitive, accurate, reliable, and precise. The stationary phase used was a Qualisil-5 BDS C8 column (250cm x 4.6mm, 5μm size of particle), chromatographic separation was carried out. Mobile phase consisted of phosphate buffer (pH 3) and acetonitrile (78:22 %v/v). For the chromatographic analysis the volume of injection was 20μL and flow rate was set at 1.0mL/min with UV detection at 280nm. It was found that developed method was linear for Ofloxacin (3- 15μg/mL, r²=0.999). The method validation was carried out as per ICH Q2 (R1) guidelines. In the accuracy assessment, recovery rates at 80%, 100%, and 120% levels ranged from 98.14% to 100.13%. The values of detection limit (LOD) and quantification limit (LOQ) were 2.02μg/mL and 6.14μg/mL, respectively. All validation criteria met the required acceptance limits, confirming that the method is fit for analysis of Ofloxacin in ophthalmic preparations.