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Development and validation of stability indicating assay method for simultaneous estimation of metronidazole and ofloxacin in pharmaceutical dosage form by using RP-HPLC

Ravi Challa, B.Ramachandra, N.V.S.Naidu

A simple, accurate, precise sensitive, repeatable and stability indicating RP-HPLC method was developed for simultaneous determination ofMetronidazole (MET) andOfloxacin (OFL) in combine dosage form. Themethod was developed by using reverse phase Hypersil BDS C18, 150x4.6, 5µ as stationary phase with phosphate buffer and acetonitrile : (90:10 %v/v) as a mobile phase, pH was adjusted to 5.0 ± 0.1 by ortho-phosphoric acid at a flowrate of 1.0ml/min and column temperaturemaintained at 30ºC. Quantification of eluted compound was achieved with UV-Vis detector, Thermostat column compartment connected withWaters (alliance) Empower software at 289 nm. The combination drug product was exposed to water, acid, base hydrolytic, and oxidative stress conditions and the stressed samples were analyzed by proposed method.Metronidazole and Ofloxacin followed linearity in the concentration range of 25-150 µgml-1and 12-75 µgml-1with r2=0.9993 (n=6) and r2=0.9997 respectively. Limit of detection (LOD) and limit of quantification (LOQ) values forMetronidazole was 0.3795 and 1.15 µgml-1and for Ofloxacinwas 0.818 and 2.477 µgml-1respectively.Chromatographic peak purities are 9999 and 1000 for MET and OFL respectively. This demonstrated the specificity of assay method for their estimation in presence of degradation products. The validation study is carried out as per International Conference on Harmonization (ICH) guidelines. This method was also successfully applied for estimation ofMetronidazole and Ofloxacin in pharmaceutical formulation.