| Popis: |
A definitive method to determine pseudouridine and 5-fluoropyrimidines was established by introducing a new fluorescent reagent, 4-bromomethyl-6,7-dimethoxycoumarin, into high-performance liquid chromatography. The reagent reacted with compounds in acetone in the presence of crystalline water-free potassium carbonate and 18-crown-6. The derivatives gave intense fluorescence having its excitation and emission maxima at 340 and 420 nm in 60% methanol (mobile phase), respectively. Under the optimum conditions, standard curves were generated from serum samples containing 0.1–10 μg/ml of 5-fluorodeoxyuridine, 0.06–10 μg/ml of 5-fluorouracil, 0.006–10 μg/ml of Ftorafur, and 1–20 μg/ml of pseudouridine, respectively (5 μl injected). This fluorometric HPLC technique was shown to be useful for the simultaneous analysis of pseudouridine estimated as a cancer marker and 5-fluoropyrimidines used as a chemotherapeutic in small volumes of serum. |
