Application of Box-Behnken experimental design and response surface methodology for selecting the optimum RP-HPLC conditions for the simultaneous determination of methocarbamol, indomethacin and betamethasone in their pharmaceutical dosage form

An isocratic RP-HPLC method has been developed for the separation and determination of methocarbamol (MTL), indomethacin (IND), and betamethasone (BET) in combined dosage form using an Inertsil ODS-3v C18 (250 × 4.6 mm, 5 μm) column with UV- detection at 235 nm. Experimental design using Box-Behnken design (BBD) was applied to study the response surface during method optimization and to achieve a good separation with a minimum number of experimental runs. The three independent parameters were pH of buffer, % of acetonitrile and flow rate of the mobile phase while the peak resolution of IND from MTL and the peak resolution of BET from IND (R2) were taken as responses to obtain mathematical models. The composite desirability was employed to optimize a set of responses overall (peak resolutions). The predicted optimum assay conditions include a mobile phase composition of acetonitrile and phosphate buffer (pH 5.95) in a ratio of 79:21, v/v, pumped at a flow rate of 1.4 mL min-1. With this ideal condition, the optimized method was able to achieve baseline separation of the three drugs with good resolution and a total run time of less than 7 min. The linearity of MTL, IND, and BET was determined in the concentration ranges of 5-600 µg mL- 1, 5-300 µg mL- 1, and 5-300 µg mL- 1 and the regression coefficients were 0.9994, 0.9998, and 0.9998, respectively. The average percent recoveries for the accuracy were determined to be 100.41 ± 0.60%, 100.86 ± 0.86%, and 100.99 ± 0.65% for MTL, IND, and BET, respectively. The R.S.D.% of the intra-day precision was found to be less than 1%, while the R.S.D.% of the inter-day precision was found to be less than 2%. The RP-HPLC method was fully validated with regard to linearity, accuracy, precision, specificity, and robustness as per ICH recommendations. The proposed method has various applications in quality control and routine analysis of the investigated drugs in their pharmaceutical dosage forms and laboratory-prepared mixtures with the goal of reducing laboratory waste, analysis time, and effort.