Determination of Eniluracil and 5-Fluorouracil in Human Plasma by LC–MS/MS

Abstract

5-fluorouracil (5-FU) has been one of the most widely used chemotherapeutic agents to treat various tumors, and eniluracil (5-ethynyluracil or EU) is being developed as a novel modulator of 5-FU. A simple and sensitive LC-MS/MS method was developed for reliably quantifying both EU and 5-FU in human plasma. The method was validated for EU over a dynamic concentration range from 4.13 ng/ml (LOQ) to 1030 ng/ml and for 5-FU over a dynamic concentration range from 8.61 ng/ml (LOQ) to 1080 ng/ml. The analog, 5-bromouracil, was used as the internal standard for calibration curves and quantitation. Method validation has covered the scope of precision, accuracy, specificity, LOQ, linearity/range, freeze-thaw cycles, benchtop integrity/stability, storage stability, matrix effect, recoveries and so on, in accordance with US FDA bioanalytical method validation guidelines. The validated method has shown good applicability for clinical studies and may be used for other clinical trials that involve measuring the concentration of EU and 5-FU simultaneously in human plasma and potentially in other similar biological matrices.