Determination of 5-fluorouracil in human plasma by a simple and sensitive reversed-phase HPLC method.

A simple and sensitive reversed-phase HPLC method with UV detection was developed and validated for the quantitation of 5-fluorouracil (5-FC) in human plasma. After acidification and salting out, 5-FU was extracted into ethyl acetate and back-extracted into a basic buffer. The extract was adjusted to neutral pH before being injected onto the HPLC column. 5-FU was separated from the matrix components on a YMC ODS-AQ column at 40 degrees C using an aqueous mobile phase of 10 mM potassium phosphate at pH 5.5. A linear gradient of 0-25% methanol wash eluted late peaks, maintained column performance, and increased column stability. The run time was 20 min. The linear range was 25-300 ng ml-1 (r2 > 0.999). The limit of quantitation was 25 ng ml-1, with a signal-to-noise ratio of 23:1. Interday precision and accuracy of quality control samples were 6.2-8.4% relative standard deviation and -0.1(-)+1.9% relative error.