Determination of 5-fluorouracil and 5-fluoro-2'-deoxyuridine-5'-monophosphate in pancreatic cancer cell line and other biological materials using capillary electrophoresis.

Capillary zone electrophoresis (CZE) was used for the rapid determination of 5-fluorouracil (5-FU) and 5-fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP) in pancreatic cancer cell line (PANC-1), culture medium, plasma and pancreatic tissue. The assay is based upon protein precipitation with acetonitrile followed by a 9-min CZE analysis of the supernatant in an uncoated fused-silica capillary employing a borate buffer and on-column absorbance detection at 265 nm. Using 50 microl of sample, 5-FU levels between 4.12 and 132 microg/ml (31.7-1000 microM) were found to provide linear calibration graphs. Intra-day and inter-day RSD values evaluated from peak height ratios (n=5) were <7.6 and <8.8%, respectively. Corresponding RSD values of detection times (n=7) were <1 and <1.5%, respectively. The limits of detection for 5-FU and FdUMP were 1.72 and 5.29 microg/ml, respectively. As application, the accumulation of 5-FU by PANC-1 cells over a 4-h time period was investigated. Having a culture medium concentration of 100 microg/ml, the 5-FU cell content was estimated to become equal to that of the surrounding medium (i.e., 100 microg/ml or 3.61 fmol per cell with a volume of 4.7 pl) within that time period. The sensitivity of the assay was sufficient for the determination of 5-FU in all cell samples. FdUMP, however, could not be detected in these samples. Furthermore, the data obtained in uncoated capillaries are compared to those measured in a fused-silica capillary whose inner surface was coated with linear polyacrylamide (about 10-fold reduction of electroosmosis). The latter capillary format was found to be useless for simultaneous analysis of 5-FU and FdUMP in pancreatic cells but could be potentially useful for analysis of these compounds in plasma.

Determination of 5-fluorouracil and its main metabolites in plasma by high-performance liquid chromatography.

A relatively simple and sensitive ion-pair high-performance liquid chromatographic method has been developed for measuring the anticancer drug fluorouracil and its main metabolites, fluorouridine, fluorodeoxyuridine and fluorodeoxyuridine monophosphate in human plasma. A reversed-phase C18 column (150 mm x 2 mm I.D.) and ultraviolet detection (280 nm) were used. The influence of the tetrabutylammonium phosphate and monopotassium phosphate concentrations and pH of the mobile phase on the various k' values was investigated. The optimal k' values obtained for the four compounds ranged between 0.7 and 5.9. The optimized eluent was (10(-4) M tetrabutylammonium phosphate plus 2.10(-2) M potassium dihydrogen phosphate, pH 5.9)-methanol (95.5:4.5, v/v). The flow-rate was 0.3 ml/min. The procedure for plasma preparation included solvent extraction using ethyl acetate-methanol (80:20) followed by elution on C18 Sep-Pak cartridges to separate the four compounds from constituents normally occurring in plasma. The detection limit of the assay was 2 ng/ml (5-fluorouracil), 10 ng/ml (5-fluorouridine), 10 ng/ml (5-fluoro-2'-deoxyuridine) and 50 ng/ml (5-fluoro-2'-deoxyuridine 5'-monophosphate).

HPLC analysis of 5-FU and FdUMP in tissue and serum.

Tissue and serum concentrations of 5-fluorouracil (5-FU) and 5-fluoro-2'-deoxyuridine 5' monophosphate (FdUMP) can be determined using a simple and sensitive reverse phase HPLC technique. Samples are extracted with ethyl acetate, evaporated to dryness, resuspended in the mobile phase (water/methanol [85:15]), and injected into a C18 muBondapack column (250 mm x 30 mm) with a flow rate of 1 ml/min. Both fluorescence (266 lambda Excitation and 350 lambda Emission) and UV (250 nm) are used for detection. Standard solutions of 5-FU and FdUMP produce a linear relationship (r2 = 0.985) with a detection sensitivity of 50 pg. Recovery following this extraction procedure is 78%. This analytical method is simple and convenient to use for the simultaneous detection of 5-FU and FdUMP.

Conversion of encapsulated 5-fluoro-2'-deoxyuridine 5'-monophosphate to the antineoplastic drug 5-fluoro-2'-deoxyuridine in human erythrocytes.

The fluoropyrimidine deoxyribonucleotide 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP) was encapsulated in human erythrocytes by a procedure based on hypotonic hemolysis and isotonic resealing. Encapsulated FdUMP (up to 9 mumol/ml of packed erythrocytes) did not affect erythrocyte metabolism or morphology. Hemolysates were found to catalyze efficient dephosphorylation of FdUMP to yield nearly stoichiometric amounts of the corresponding deoxyribonucleoside 5-fluoro-2'-deoxyuridine (FdUrd), an antineoplastic drug showing selective cytotoxicity toward liver metastases from colorectal carcinomas. The dephosphorylation reaction had an apparent Km of 7.7 +/- 1.2 mM FdUMP at pH 7.4 and was remarkably slower at pH 8.2. ATP, GTP, and UTP inhibited both the disappearance of FdUMP and the formation of FdUrd in hemolysates. The enzyme responsible for the FdUMP-to-FdUrd conversion was identified with the deoxyribonucleotide-specific isozyme of erythrocyte pyrimidine 5'-nucleotidase (EC 3.1.3.5). Intracellular formation and subsequent release of FdUrd were observed in intact erythrocytes loaded with FdUMP. Inhibition of FdUrd release from these erythrocytes was obtained by raising the pH intracellularly and, alternatively, by coencapsulation of ATP. Autologous FdUMP-loaded erythrocytes might be used as endogenous bioreactors designed for time-programmed and liver-targeted delivery of FdUrd.

Competitive binding radioassay for the determination of 5-fluorodeoxyuridine and 5-fluorodeoxyuridine-5'-monophosphate levels in plasma and tumor tissue.

A competitive binding radioassay was developed to measure 5-fluoro-2'-deoxyuridine (FUDR) as well as 5-fluoro-2'-deoxyuridine monophosphate (FdUMP), FdUMP has been measured by a competitive binding radioassay with thymidylate synthase as the binding enzyme (TS assay). FUDR was enzymatically converted to FdUMP by thymidine kinase, and then the converted FdUMP was measured by the competitive binding assay to determine the concentration of FUDR in plasma and tumor tissue. As little as 100 pg/ml of FUDR or 50 pg/ml of FdUMP can be detected quantitatively by this method. When TS assay and high-performance liquid chromatography were compared for the measurement of FUDR and FdUMP levels in plasma and tumor tissue of Ehrlich carcinoma (EC)-bearing mice following administration of FUDR, a close agreement was observed for FUDR levels, though low FdUMP levels were detectable only by the TS assay method. The examination of intracellular metabolism of FUDR in EC cells by this method showed that metabolic conversion of FUDR into FdUMP or 5-fluorouracil is rapid. Thus, we have established a highly sensitive method for measuring not only FdUMP but also FUDR with TS assay. This should be very useful for experimental and clinical studies on fluoropyrimidines.