Development and Validation of a New HPLC Method For the Detection of 5- Fluorouracil in Mobile Phase and in Plasma

Objective: To establish a particular, sensitive and accurate High Performance Liquid Chromatography (HPLC) technique for the specific and effective analysis of 5-Fluorouracil (5-FU) in mobile phase and in plasma. Method: Analytical methods for 5-FU in mobile phase and plasma were developed. An assay consisted of isocratic elution of 5-FU in prepacked column discovery R-HS C-18, 5micrometer, Catalog # 568523-U, column dimensions : 25cm x 4.6mm using mobile phase composed of 5 mmol/L potassium dihydrogen phosphate (pH 6.0) and methanol at a ratio of 96:4 v/v respectively. 5-FU was detected at 254 nm with the flow rate of 1 ml/min in both mobile phase and plasma. Results: Retention time was found to be 3.457 minutes in mobile phase and 2.543 minutes in plasma. System suitability test showed no robustness among the inter-day, intra-day analysis, mobile phase and column. Accuracy of the developed method proved reliability of the purposed procedure. %RSD analysis was found to be less than 2% and coefficient of correlation was found to be 1. Conclusion: Valuable precision, recovery and accuracy were observed indicating method reliability. Method can also be applicable for bioequivalence study.