Application of experimental design and response surface technique for selecting the optimum RP-HPLC conditions for the determination of moxifloxacin HCl and ketorolac tromethamine in eye drops

A method has been developed for the separation of moxifloxacin HCl and ketorolac tromethamine using reverse phase high-performance liquid chromatography (RP-HPLC) on C18 column (250 × 4.6 mm, 5 μm) with UV detection at 308 nm. Experimental designs were applied for multivariate optimization of the experimental conditions of RP-HPLC method. Three independent factors: methanol content in the mobile phase composition, buffer pH and flow rate were used to design mathematical models. Here faced central composite (FCC) experimental design was used to study the response surface technique and to study in depth the effects of these independent factors. Derringer's desirability function was applied to simultaneously optimize the retention time of last eluting peak (ketorolac tromethamine) and tailing factor of moxifloxacin. The predicted optimum assay condition consisted of methanol and potassium dihydrogen phosphate buffer (pH 3.2; 25 mM, 0.5% Triethylamine) in a proportion of 60:40% v/v, respectively, as the mobile phase at a flow rate of 1.2 mL min−1. Using this optimum condition, baseline separation of both drugs with good resolution and a run time of less than 7 min were achieved. The optimized assay condition was validated according to ICH guidelines to confirm specificity, linearity, accuracy and precision.