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A sensitive HPLC method has been developed and validated for the determination of 5-Fluorouracil (5-FU) and related substances in a novel topical formulation. The highly polar molecule requires aqueous mobile phase for the elution and separation of 5-Fluorouracil and its impurities (Impurity A, B and C official in EP). The developed method is found to be specific, reproducible, and stability indicating. The Phenomenex Synergi Polar RP 250×4.6mm 4µ column was used and mobile phase consisted of 0.1M potassium di hydrogen phosphate (KH2PO4) buffer to achieve good resolution and retention of the analyte and its impurities. The detector linearity was established from concentrations ranging from 0.01 µg/ml to 500 µg/ml for 5-Flurouracil and from 0.01 µg/ml to 0.08 µg/ml for related substances with a correlation co-efficient of 0.999. The relative response factor (RRF) values of impurity A, impurity B and impurity C determined from linearity plots were 1.9, 0.9 and 1.4 respectively. The limit of detection (LOD) and limit of quantification (LOQ) found to be in a range of 0.004µg/ml and 0.014µg/ml for 5-Fluorouracil and related substances respectively. The molecule was stable in all the stress conditions such as acid, base, oxidation, heat and photolysis as per the recommendations of ICH guidelines. The method was proved to be robust with respect to changes in flow rate, pH and column temperature. The proposed method is found to be sensitive, precise, rapid, reproducible, and offers good column life.
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