Inserting chloramphenicol (CL) and hydrocortisone acetate (HCA) in cream preparation is intended to have activity against skin infection and dermatitis and such a product is available in the Indonesian market. Due to its capability as 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 an experimental design for an effective analysis of CL and HCA in a cream formulation. In this study, the experimental BoxBehnken design (BBD) was used. BBD is one of the useful experimental designs for the optimization of chromatographic separation and analysis and for getting a better understanding of the interaction of studied factors on HPLC separation quality. Separation and HPLC analysis of CL and HCA were performed using a Shimadzu LC-20AD chromatograph, a Waters X-Bridge C-18 column (250 × 4.6 mm ID, 5 μm), and a UV-Vis detector at 261 nm. HPLC method was validated according to the International Conference on Harmonization by determining several analytical performances intended for the methods purpose. Based on BBD, the optimal condition of HPLC was obtained using a mobile phase of acetonitrile 47% 53%, with a flow rate of 0.9 ml/minutes and a column temperature of 38°C. The validation of HPLC resulted in the selectivity of a method with a resolution value of ≥1.5, linearity with a correlation coefficient of >0.999, intraday and inter-day precisions with relative standard deviation values of ≤1.9%, and recovery values in the range of 98%−102%. The validated method is successfully used for the analysis of CL and HCA in cream formulations. BBD could be an effective design to get the optimum reversed HPLC condition for the separation of CL and HCA in a cream formulation.
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