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

Abstract

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 mul aliquot of distilled water containing L-cysteine (100 mg/ml) was added to a 100 mul aliquot of biological sample. L-CySteme 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 mul aliquot of the mobile phase containing the internal standard (10 mug/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 mul aliquot of the mobile phase. A 50 mul aliquot was injected onto a C 18 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 1 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 1 in rat plasma and urine were 0.1 and 0.5 mug/ml, respectively. (C) 2003 Elsevier B.V. All rights reserved.