Method development and validation of fluorouracil in bulk and pharmaceutical dosage form by RP-HPLC

Background: Fluorouracil (5-FU) is a widely used chemotherapeutic agent with a narrow therapeutic index, making accurate and reliable quantification essential for quality control and stability assessment.Aim and Objective: The present study aimed to develop and validate a simple, rapid, precise, and stability-indicating reverse-phase high-performance liquid chromatography (RP-HPLC) method for the estimation of Fluorouracil in bulk drug and pharmaceutical dosage forms.Materials and Methods: Chromatographic separation was achieved on a Waters X-Bridge Phenyl column (150 × 4.6 mm, 3.5 µm) using an isocratic mobile phase of acetonitrile and 0.1% formic acid in the ratio of 25:75 (v/v) at a flow rate of 1.0 mL/min. Detection was performed at 245 nm with an injection volume of 10 µL, and the retention time of Fluorouracil was 2.423 minutes. The method was validated in accordance with ICH Q2(R1) guidelines for specificity, linearity, accuracy, precision, robustness, sensitivity, and system suitability. Forced degradation studies were performed under acidic, alkaline, oxidative, reductive, hydrolytic, thermal, and photolytic stress conditions.Results: The method demonstrated excellent linearity in the concentration range of 12.5–75 µg/mL with a correlation coefficient (r²) of 0.9997. Accuracy studies showed mean recoveries between 99.3% and 100.2%. Precision studies gave %RSD values below 2.0% for system, method, and intermediate precision. The LOD and LOQ were 0.60 µg/mL and 2.00 µg/mL, respectively. Robustness testing confirmed that minor variations in flow rate and mobile phase composition did not significantly affect performance. Forced degradation studies revealed significant degradation under acidic, alkaline, reductive, and thermal conditions, while oxidative, photolytic, and hydrolytic conditions showed minimal degradation. Peak purity confirmed the stability-indicating capability of the method.Conclusion: The developed RP-HPLC method is simple, sensitive, accurate, precise, and stability-indicating. With a short run time and robust validation, it is suitable for routine quality control, assay, and stability testing of Fluorouracil in bulk and pharmaceutical dosage forms.