Determination of 5-Fluorouracil and 5-Fluoro-2′-deoxyuridine in Human Serum by High Performance Liquid Chromatography with Fluorescence Detection

Abstract

Described is a high performance liquid chromatographic method using fluorescence detection for the simultaneous determination of 5-fluorouracil and 5-fluoro-2′-deoxyuridine in human serum. After extraction with ethyl acetate from serum, these compounds and 5-chlorouracil (internal standard) are converted into the corresponding fluorescent derivatives by reaction with 3-bromomethyl-6, 7-dimethoxy-1-methyl-2(1H)-quinoxalinone in the presence of potassium hydrogen carbonate and 18-crown-6 in acetone. The derivatives are separated by reversed phase chromatography on a Radial Pak C₁₈ cartridge, with stepwise elution using aqueous 35 and 50% (v/v) methanol. The detection limits for 5-fluorouracil and 5-fluoro-2′-deoxyuridine added to serum are 12.5ng (95pmol) and 25ng (63pmol) per ml of serum, respectively, at a signal-to-noise ratio of 5. This method permits the determination of 5-fluorouracil in serum (0.1ml) from a healthy volunteer and a stomach cancer patient administered 5-fluorouracil intravenously.