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Background: Retapamulin is the first pleuromutilin antibacterial approved for the treatment of impetigo. The objective of the current research was to utilize the Design of Experiments approach for the development and optimization of robust RP-HPLC method for the quantitation of Retapamulin in marketed cream and in-house developed microemulsion based formulations with an oily matrix. Methods: The impact of various chromatographic conditions (independent variables) was assessed using Plackett–Burman design on critical analytical attributes (response) to screen initial experimental conditions. The Box-Behnken design was employed to optimize the selected chromatographic factors on the responses. Further, validation of optimized RP-HPLC was carried out as per the ICHQ2(R1) guideline. Results: Pareto ranking analysis showed that % organic phase, flow rate, and volume of injection were found statistically significant (p < 0.05) variables influencing the retention time, number of plates, and tailing of the Retapamulin peak. The optimized RP-HPLC method with the stationary phase, C18 (250 mm × 4.6 mm, 5 µm) column, and mobile phase as a mixture of methanol and potassium dihydrogen orthophosphate buffer (50 mM, pH 7.0, 90:10 % v/v,isocratic), the flow rate of 1.0 mL/min, 10 µL injection volume, 25°C column oven temperature, 247 nm as detection wavelength, was successfully validated based on ICHQ2(R1) guideline. Conclusion: RP-HPLC method was successfully used to separate (retention time 4.34 ± 0.2 min) and assay Retapamulin in microemulsion and marketed cream. The outcomes of the investigation exhibited the effective application of a multivariant approach in the optimization of the RP-HPLC for routine analysis of Retapamulin in quality control laboratories.

̒ulūm-i dārūyī

<b>Design of Experiment Centered Multivariate Optimization Strategy for RP-HPLC Method to Quantitate Retapamulin in Topical Cream and Microemulsion</b>