Development and Validation of Stability Indicating Assay Method For Simultaneous Estimation of Azithromycin, Fluconazole and Ornidazole In Bulk and Its Dosage Form by RP-HPLC

The present study was designed to develop simple accurate, precise, reproducible and validating of a stability indicating reverse phase high performance liquid chromatographic (RP-HPLC) method for the simultaneous estimation of azithromycin, ornidazole and fluconazole in bulk and its pharmaceutical dosage forms. Chromatographic separation of the three drugs was performed on a Phenomenex C18 column (250X4.6mm 5μm) as stationary phase with a mobile phase comprising of 20mM potassium dihydrogen phosphate : Acetonitrile (pH 4.8) in the ratio 30:70% v/v at a flow rate of 1ml/min and peak monitored at 254nm using PDA detector. The retention time of azithromycin, fluconazole, ornidazole and procaine hydrochloride (internal standard) was Rt1-2.8, Rt2-5.0, Rt3-6.3 and Rt-3.8minutes respectively. The linearity of azithromycin, fluconazole and ornidazole were in the range of 20-100μg/ml, 3-15μg/ml and 15-75μg/ml with an internal standard, procaine hydrochloride 5µg/ml respectively. The accuracy of the method was found to be 98-102% and %RSD was found to be less than 2% indicating high degree of accuracy and precision of the proposed HPLC method. The limit of detection for azithromycin, fluconazole and ornidazole was found to be 0.34, 2.80 and 0.76μg/ml respectively whereas, the limit of quantification was found to be 1.05, 8.6 and 2.31μg/ml respectively. Forced degradation studies were conducted to know the stability of the drug samples under various stress conditions like acid, base, peroxide and photolytic degradation according to ICH guidelines. Results are validated statistically as per ICH guidelines.