Box–Behnken design-based HPLC optimization for quantitative analysis of chloramphenicol and hydrocortisone acetate in cream

ABSTRACT

Inserting chloramphenicol (CL) and hydrocortisone acetate (HCA) in cream preparation is intended to have activityagainst skin infection and dermatitis and such a product is available in the Indonesian market. Due to its capabilityas a separation technique, chromatography is widely used for the analysis of mixture in pharmaceutical products.The objective of this study was to develop high-performance liquid chromatography (HPLC) combined with anexperimental design for an effective analysis of CL and HCA in a cream formulation. In this study, the experimentalBox–Behnken design (BBD) was used. BBD is one of the useful experimental designs for the optimization ofchromatographic separation and analysis and for getting a better understanding of the interaction of studied factors onHPLC separation quality. Separation and HPLC analysis of CL and HCA were performed using a Shimadzu LC-20ADchromatograph, a Waters X-Bridge C-18 column (250 × 4.6 mm ID, 5 µm), and a UV-Vis detector at 261 nm. HPLCmethod was validated according to the International Conference on Harmonization by determining several analyticalperformances intended for the method’s purpose. Based on BBD, the optimal condition of HPLC was obtained usinga mobile phase of acetonitrile 47%– 53%, with a flow rate of 0.9 ml/minutes and a column temperature of 38°C. Thevalidation of HPLC resulted in the selectivity of a method with a resolution value of ≥1.5, linearity with a correlationcoefficient of >0.999, intraday and inter-day precisions with relative standard deviation values of ≤1.9%, and recoveryvalues in the range of 98%−102%. The validated method is successfully used for the analysis of CL and HCA in creamformulations. BBD could be an effective design to get the optimum reversed HPLC condition for the separation of CLand HCA in a cream formulation