Utilization of Experimental Design for the Establishment and Optimization of RP-HPLC Method for the Estimation of Two Selective Serotonin Reuptake Inhibitors/benzodiazepines Combinations in the Presence of their Related Compounds

Background: Design of experiment (DOE) is considered the most powerful tool to identify factors that affect variation and improve the response by tuning these factors. In the present work, DOE was applied to establish an innovative, sensitive, and precise HPLC method for the simultaneous determination of Escitalopram oxalate, Paroxetine hydrochloride hemihydrate and Clonazepam in the presence of their related compounds in drug substance and drug products. Methods: Buffer molarity, % organic modifier (acetonitrile content) at the beginning/end of the gradient, flow rate at the beginning/end of the gradient, pH of the mobile phase, and column temperature were screened using Plackett-Burman design (PBD) model. The main effect plot showed that % organic modifier at the beginning/end of gradient and flow rate at the beginning of gradient were statistically significant variables influencing peaks resolution (p<0.05). Box-Behnken design (BB-D) was then used as an optimization model in order to achieve the highest possible resolution with the least possible experimental trials through studying the interaction and quadratic effects of these three factors. Finally, the optimum condition for predicated peak resolution could be achieved by the desirability function. Results: After optimization, the chromatographic separation was attained on Agilent Zorbax SB C-18 (4.6x250mm, 5 mu m) column using gradient elution of mobile phase: (A) potassium dihydrogen phosphate (pH 2.7; 0.025M) and (B) acetonitrile at ambient column temperature with the last eluted compound in less than 17 min. The flow rate was maintained at 1/2.3 mLmin(-1) with UV detection at 245/210 nm using time programming. Conclusion: The optimized chromatographic method was used for stability, indicating assay of the cited drugs in the presence of their related compounds according to ICH Q2R1 guidelines.