Abstract

Robustness tests were performed on a gradient RP-HPLC method that had been developed for rapid simultaneous separation and determination of clarithromycin, norfloxacin, doxycycline, tinidazole and omeprazole in their pharmaceutical dosage forms. Separation was carried out on a 250 x 4.6 mm (i.d.), 5µm ODS column (Inertsil, Tokyo, Japan) and multiwavelength overlay chromatograms at max of 210 and 310 nm were used for quantitative analysis. The standard curve was linear in the concentration range of 100 – 900, 5 – 150, 20 – 220, 10 - 150 and 0.5 - 110 µg ml^-1 for clarithromycin, norfloxacin, doxycycline, tinidazole and omeprazole, respectively. Robustness tests were performed using twolevel fractional factorial designs with resolution III, so only main effects can be estimated. Factors chosen for this study were the column temperature (^˚C), pH, ion pairing agent concentration (g/l), phosphate buffer concentration (g/l), the fraction of mobile phase B (B% start) at 4 min of the gradient and fraction of mobile B (B% end) at 7 min of the gradient run. The significance of the factor effects was determined statistically, using algorithm of Dong in the calculation of critical effects, and graphically, by means of half-normal plots. Non-significant intervals for significant factors and system suitability limits were calculated based on robustness tests' results. The method was validated according to ICH guidelines. The proposed method was found to be accurate, reproducible, and consistent which is useful for the routine determination of clarithromycin, norfloxacin, doxycycline, tinidazole and omeprazole in combined mixtures and pharmaceutical dosage forms.