High Performance Liquid Chromatographic Determination of Pyrimidine Nucleobases and 5-Fluorouracil Labeled by Fluorescent Reagent

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Abstract
A rapid fluorescent labeling method of imides with 4-bromomethyl-7-methoxycoumarin (Br-Mnc) is described. Mmc-derivatives of imides are prepared by adding 10 mg or Br-Mmc to a solution containing 1 mg of imides (pyrimidine nucleobases or barbitals) and 10 mg K2CO3 in 5 ml dimethylsulfoxide (DMSO). The reaction is completed for 5 min at room temperature when Br-Mmc concentration in the mixture is 2 mg/ml. The excess Br-Mmc is treated with p-nitrobenzoic acid, which gives the Mmc-derivative with no fluorescence. High performance liquid chromatographic separation of Mmc-derivatives of imides are obtained on a C18 column. With this method the reaction time for labeling is dramatically reduced without catalyst such as crown ether. The reaction system is discussed according to donor-acceptor theory.