Determination of 1-(3-fluoro-4-hydroxy-5-mercaptomethyltetrahydrofuran-2-yl)-5-methyl-1H-pyrimidine-2,4-dione in rat plasma and urine by high-performance liquid chromatography

A high-performance liquid chromatographic method using liquid–liquid extraction was developed for the determination of 1-(3-fluoro-4-hydroxy-5-mercaptomethyl-tetrahydrofuran-2-yl)-5-methyl-1H-pyrimidine-2,4-dione (l-FMAUS; I) in rat plasma and urine. A 100 μl aliquot of distilled water containing l-cysteine (100 mg/ml) was added to a 100 μl aliquot of biological sample. l-Cysteine was employed to protect binding between the 5′-thiol of I and protein in the biological sample. After vortex-mixing for 30 s and adding a 50 μl aliquot of the mobile phase containing the internal standard (10 μg/ml of 3-aminophenyl sulfone), 1 ml of ethyl acetate was used for extraction. After vortex-mixing, centrifugation, and evaporating the ethyl acetate, the residue was reconstituted with a 100 μl aliquot of the mobile phase. A 50 μl aliquot was injected onto a C18 reversed-phase column. The mobile phases, 50 mM KH2PO4 (pH=2.5):acetonitrile (85:15, v/v) for rat plasma and 50 mM KH2PO4 (pH 2.5):acetonitrile:methanol (85:10:5, v/v/v) for urine samples, were run at a flow-rate of 1.2 ml/min. The column effluent was monitored by an ultraviolet detector set at 265 nm. The retention times for I and the internal standard were approximately 9.7 and 12.5 min, respectively, in plasma samples and the corresponding values in urine samples were 16.8 and 14.9 min. The quantitation limits of I in rat plasma and urine were 0.1 and 0.5 μg/ml, respectively.