A stability indicating analytical method for imiquimod and its related impurities was developed by ultra-pressure liquid chromatography (UPLC) using design of experiments. This method could quantify imiquimod and all its eight known related impurities in a single run. The optimum separation was achieved on reverse phase Acquity UPLC column (2.1 mm × 100 mm, 1.7 μm) using 0.1% trifluoroacetic acid and acetonitrile as mobile phase. Preventing the use of ion pair reagents assured the compatibility of this method to liquid chromatography in tandem with mass spectrometry. All components were separated within 9 minutes, maintaining good resolution. The stability indicating nature of the developed method was assessed by analyzing the samples of imiquimod which were exposed to various environments such as acid, alkali, peroxide, light and heat. This method was found to be sensitive, precise and accurate. The method achieved the lower detection limit of 0.04 μg/mL and the quantification limit of 0.08 μg/mL for all analytes.
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