Development and Validation of HPLC method for simultaneous estimation of Rifampicin and Ofloxacin using experimental design

Abstract

A accurate, precise and robust isocratic HPLC method has been developed and validated for simultaneous determination of Rifampicin and Ofloxacin. The chromatographic separation was carried out on Kinetex C18, 100 A Phenomenex column with a mixture of 0.03M Potassium dihydrogen phosphate buffer pH 3.0: Acetonitrile (55:45) as mobile phase at 230 nm. The retention times were 2.91 and 4.87 min for Ofloxacin and Rifampicin, respectively. Calibration plots were linear over the concentration range 1–5 and 2–10 µg/ml for Rifampicin and Ofloxacin, respectively. The method was validated for linearity, sensitivity accuracy, precision, and robustness. Percent recoveries were found to be close to 100% with low variability. Fractional factorial design with four factors was chosen for robustness testing. The volume of acetonitrile and flow rate showed significant effect on retention factor of both the drugs and asymmetry factor of ofloxacin. The method may be adopted for routine analysis at industry.

Keywords

• rifampicin
• ofloxacin
• fractional factorial design
• high performance liquid chromatography
• validation