A thermostable pyrimidine nucleoside phosphorylase from Brevibacillus borstelensis LK01 for synthesizing halogenated nucleosides.

OBJECTIVE: To isolate a thermostable pyrimidine nucleoside phosphorylase (PyNP) from mesophilic bacteria by gene mining. RESULTS: BbPyNP from Brevibacillus borstelensis LK01 was isolated by gene mining. BbPyNP had a highest 60% identity with that of reported PyNPs. BbPyNP could catalyze the phosphorolysis of thymidine, 2'-deoxyuridine, uridine and 5-methyuridine. BbPyNP had good thermostability and retained 73% of its original activity after 2 h incubation at 50 °C. BbPyNP had the highest activity at an optimum alkaline pH of 8.5. BbPyNP was stable from pH 7 to 9.8. Under preliminary optimized conditions, the biosynthesis of various 5-halogenated pyrimidine nucleosides by BbPyNP reached the yield of 61-84%. CONCLUSION: An efficient approach was estimated in isolating thermostable PyNP from mesophilic bacteria.

An in vitro liver model on microfluidic device for analysis of capecitabine metabolite using mass spectrometer as detector.

In this work, an in vitro liver model in a microfluidic device to imitate and detect prodrug metabolism was developed. A widely used prodrug capecitabine (CAP), which needs to be metabolized into active intermediate in the liver and then transformed into final effective drug in tumor cells, was selected as a model compound. The microfluidic device we exploited consists of a cell co-culture section, in which HepG2 cells were cultured to represent liver while MCF-7 cells were used to represent the tumor tissue, and an on-line solid phase extraction (SPE) section connecting to the ionization source of the ESI-Q-TOF mass spectrometer. The prodrug metabolism was realized and confirmed within this in vitro liver model as the intermediate product of the prodrug 5'-deoxy-5-fluorouridine (DFUR) was successfully detected with MS after the conditioning of HepG2 cells, and the anti-tumor effect of the active metabolite was observed through cell vitality assays of MCF-7 cells. The limit of detection (LOD) using on-chip SPE was at 10nM and semi-quantitative analysis could be realized. This system has been proved useful and practical, showing a potential to replace conventional drug screening methods.

Determination of gemcitabine and its metabolite in extracellular fluid of rat brain tumor by ultra performance liquid chromatography-tandem mass spectrometry using microdialysis sampling after intralesional chemotherapy.

The cytotoxic agent Gemcitabine (2',2'-difluoro-2'-deoxycytidine) has been proved to be effective in the treatment of malignant gliomas. A rapid, sensitive and specific ultra performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS) assay using microdialysis sampling was developed and validated to quantify gemcitabine and its major metabolite 2',2'-difluoro-2'-deoxyuridine (dFdU) in Sprague-Dawley rat bearing 9L glioma. Microdialysis probes were surgically implanted into the area of rat brain tumor in the striatal hemisphere, and artificial cerebrospinal fluid was used as a perfusion medium. The samples were analyzed directly by UPLC-MS/MS after the addition of 5-bromouracil as an internal standard (IS). Separation was achieved on Agilent SB-C(18) (50 mm × 2.1mm I.D., 1.8 µm) column at 40 °C using an isocratic elution method with acetonitrile and 0.1% formic acid (4:96, v/v) at a flow rate of 0.2 mL/min. Detection was performed using electrospray ionization in positive ion selected reaction monitoring mode by monitoring the following ion transitions m/z 264.0→112.0 (gemcitabine), m/z 265.1→113.0 (dFdU) and m/z 190.9→173.8 (IS). The calibration curves of gemcitabine and dFdU were linear in the concentration range of 0.66-677.08 ng/mL and 0.31-312.00 ng/mL, respectively. The lower limit of quantification of gemcitabine and dFdU were 0.66 ng/mL and 0.31 ng/mL, respectively. The lower limit of detection of gemcitabine and dFdU were calculated to be 0.2 ng/mL and 0.1 ng/mL, respectively. All the validation data, such as intra- and inter-day precision, accuracy, selectivity and stability, were within the required limits. The validated method was simple, precise and accurate, which was successfully employed to determinate the concentrations of gemcitabine and dFdU in the extracellular fluid of rat brain tumor.

A thymidylate synthase ternary complex-specific antibody, FTS, permits functional monitoring of fluoropyrimidines dosing.

5-Fluorouracil (5FU) and similar fluoropyrimidines induce covalent modification of thymidylate synthase (TS) and inhibit its activity. They are often used to treat solid cancers, but drug resistance and toxicity are drawbacks. Therefore, there is an unmet need for a functional assay to quantify fluorouracil activity in tissues, so as to individually tailor dosing. It is cumbersome to separately quantify unmodified and 5FU-modified TS using currently available commercial anti-TS antibodies because they recognize both forms. We report here the first monoclonal antibody (FTS) specific to 5FU-modified TS. By immunoblot assay, the FTS antibody specifically recognizes modified TS in a dose-dependent manner in 5FU-treated cells, in cancer xenograft tissues of 5FU-treated mice, and in the murine tissues. In the same assay, the antibody is nonreactive with unmodified TS in untreated or treated cells and tissues. Speculatively, a high-throughput assay could be enabled by pairing anti-TS antibodies of two specificities, one recognizing only modified TS and another recognizing both forms, to structurally quantify the TS-inhibiting effect of fluorouracil at a cellular or tissue level without requiring prior protein separation. Such a development might aid preclinical analytic studies or make practical the individual tailoring of dosing.

The achievement of mass balance by simultaneous quantification of floxuridine prodrug, floxuridine, 5-fluorouracil, 5-dihydrouracil, α-fluoro-ß-ureidopropionate, α-fluoro-ß-alanine using LC-MS.

5-Fluoro-2'-deoxyuridine (floxuridine, 5-FdUrd) and 5-fluorouracil (5-FU) are widely used for the treatment of colorectal cancers. The mechanisms of action of 5-FdUrd and 5-FU, as well as the biochemical pathway responsible for their metabolism, are well understood. Identification of every metabolite and achieving mass balance by conventional UV absorption-based HPLC analysis are not feasible because the metabolites beyond 5-FU in the 5-FdUrd metabolic pathway are undetectable by UV light. We therefore established a mass spectrometry method, designed for fast and convenient analysis, for simultaneously measuring 5-FdUrd, 5-FU, and their metabolites. Linearity, precision and accuracy were validated in the concentration ranges studied for each compound. Hydrolysis studies of 5-FdUrd and amino acid mono ester prodrugs of 5-FdUrd in Capan-2 cell homogenates were carried out and the achievement of mass balance was established with this method (recovery of 5'-O-l-leucyl-FdUrd was 96.6-108.2% and that of 5-FdUrd was 79.4-117.4%). This simple LC-MS method achieves reliable quantitation and mass balance of 5-FdUrd, 5-FU, and their metabolites and can be effectively utilized for further kinetic studies.

Mass flows of X-ray contrast media and cytostatics in hospital wastewater.

Little is known about the significance of hospitals as point sources for emission of organic micropollutants into the aquatic environment. A mass flow analysis of pharmaceuticals and diagnostics used in hospitals was performed on the site of a representative Swiss cantonal hospital. Specifically, we analyzed the consumption of iodinated X-ray contrast media (ICM) and cytostatics in their corresponding medical applications of radiology and oncology, respectively, and their discharge into hospital wastewater and eventually into the wastewater of the municipal wastewater treatment plant. Emission levels within one day and over several days were found to correlate with the pharmacokinetic excretion pattern and the consumed amounts in the hospital during these days. ICM total emissions vary substantially from day to day from 255 to 1259 g/d, with a maximum on the day when the highest radiology treatment occurred. Parent cytostatic compounds reach maximal emissions of 8-10 mg/d. A total of 1.1%, 1.4%, and 3.7% of the excreted amounts of the cytostatics 5-fluorouracil, gemcitabine, and 2',2'-difluorodeoxyuridine (main metabolite of gemcitabine), respectively, were found in the hospital wastewater, whereas 49% of the total ICM was detected, showing a high variability among the compounds. These recoveries can essentially be explained by the high amount administered to out-patients (70% for cytostatics and 50% for ICM); therefore, only part of this dose is expected to be excreted on-site. In addition, this study emphasizes critical issues to consider when sampling in hospital sewer systems. Flow proportional sampling over a longer period is crucial to compute robust hospital mass flows.

Simultaneous quantification of 5-FU, 5-FUrd, 5-FdUrd, 5-FdUMP, dUMP and TMP in cultured cell models by LC-MS/MS.

To specifically quantify several metabolites of 5-fluorouracil (5-FU) and two endogenous monophosphate nucleotides, we developed an original method based on a liquid chromatography-tandem mass spectrometry (LC-MS/MS). This assay allowed the determination of: (i) the intracellular production of 5-fluoro-2'-deoxyuridine-5'-monophosphate (5-FdUMP) from 5-FU or 5-fluoro-2'-deoxyuridine (5-FdUrd), (ii) the impact of 5-FdUMP concentration on the intracellular 2'-deoxyuridine-5'-monophosphate (dUMP)/thymidine-5'-monophosphate (TMP) ratio, and (iii) the secretion extent of 5-FdUMP and 5-FU from human cultured cells by ABC transporters. Under our experimental conditions, cells were incubated with 5-FU or 5-FUrd. Then, cellular proteins were precipitated by methanol. This procedure provided high extraction recovery. In addition, to measure 5-FU and 5-FdUMP secretion from cells, we carried out quantification of these molecules in culture medium. Media were either directly injected (5-FU) or underwent a solid phase extraction using Oasis Wax extraction cartridge (5-FdUMP). Separation of analytes was performed on a dC18 Atlantis 3.5microm, (100mmx2.1mm i.d) column with isocratic mode using ammonium formate buffer/methanol/water (5/5/90, v/v) as mobile phase. The run time did not exceed 6.2min. The analytes were ionized in an electrospray interface under negative ion mode. We validated the method over a range of 2.5-150ngmL(-1) according to the compounds. Intra- and inter-assay variability was lower than 10% over seven days. All compounds were stable in cells or in culture medium when samples were stored at -20 degrees C for at least two weeks, and after three freeze-thaw cycles. No matrix effect was observed in both media.

Retention studies of 2'-2'-difluorodeoxycytidine and 2'-2'-difluorodeoxyuridine nucleosides and nucleotides on porous graphitic carbon: development of a liquid chromatography-tandem mass spectrometry method.

The development of a method for the separation of 2'-2'-difluorodeoxycytidine (gemcitabine, dFdC), 2'-2'-difluorodeoxyuridine (dFdU) and their mono-, di- and triphosphates using a porous graphitic carbon column (Hypercarb), without ion-pairing agent, is described. The retention of dFdC and dFdU could be controlled with an organic modifier (acetonitrile, CH(3)CN) and the retention of the anionic nucleotides with an eluting ion (bicarbonate). Separation of all analytes was achieved using a 0-25 mM ammonium bicarbonate gradient in CH(3)CN-H(2)O (15:85, v/v). Under these conditions, however, very long re-equilibration times were required. Injection of an acidic solution (100 microL 10% formic acid in H(2)O, v/v; 2.65 M) after running a gradient directly restored the separation capabilities of the column. Still, separation between the analytes slowly deteriorated over a period of months. These problems were solved by preconditioning the column with a pH buffered hydrogen peroxide (H(2)O(2)) solution (0.05% H(2)O(2) in CH(3)CN-H(2)O (15:85, v/v), pH 4) before starting an analytical run. The oxidation of the stationary phase with H(2)O(2) prevented its slow reduction, which most likely caused the decreasing retention times. The analytes were detected using tandem mass spectrometry.

Challenge of high polarity and low concentrations in analysis of cytostatics and metabolites in wastewater by hydrophilic interaction chromatography/tandem mass spectrometry.

A method for solid phase extraction and HPLC-MS/MS of the cytostatics 5-fluorouracil, cytarabine, and gemcitabine and human metabolites uracil 1-beta-d-arabinofuranoside and 2',2'-difluorodeoxyuridine in wastewater was established. Wastewater samples from a Swiss hospital were analyzed for 5-fluorouracil, gemcitabine and 2',2'-difluorodeoxyuridine. The limits of quantification were 5.0, 0.9, and 9.0ng/L and the maximum concentrations detected were 27, 38, and 840ng/L, respectively. Along with the method development, retention mechanisms on the hydrophilic interaction chromatography (HILIC) stationary phase were studied. Both partitioning and adsorption play a role in the retention on the tested sulfoalkylbetaine modified silica HILIC column material. The contribution of these two processes is changing over the 1.6-40% range water in the mobile phase. Although the specific break point is difficult to determine, adsorption becomes more significant as the fraction of water in the mobile phase decreases below approximately 16%.

Rapid and simultaneous determination of capecitabine and its metabolites in mouse plasma, mouse serum, and in rabbit bile by high-performance liquid chromatography.

A rapid high-performance liquid chromatography method has been developed for simultaneous determination of capecitabine and its metabolites: 5'-deoxy-5-fluorocytidine (5'-DFCR), 5'-deoxy-5-fluorouridine (5'-DFUR) and 5-fluorouracil (5-FU). 5'-DFCR was synthesized by hydrolyzing capecitabine using commercially available carboxyl esterase (CES) and characterized by NMR, mass spectrometry and elemental analysis. Base-line separations between capecitabine, 5'-DFCR, 5'-DFUR and 5-FU were found with symmetrical peak shapes on a Discovery RP-amide C16 column using 10 mM ammonium acetate at pH 4.0 and methanol as the mobile phase. The retention times of capecitabine, 5'-DFCR, 5'-DFUR and 5-FU were 8.9, 5.0, 5.3 and 3.0 min, respectively. Linear calibration curves were obtained for each compound across a range from 1 to 500 microg ml(-1). The intra- and inter-day relative standard deviations (%RSD) were <5%. A single-step protein precipitation method was employed for separation of the analytes from bio-matrices. Greater than 85% recoveries were obtained for capecitabine, 5'-DFCR, 5'-DFUR and 5-FU from bio-fluids including mouse plasma, mouse serum and rabbit bile.

High-performance liquid chromatographic method for the determination of gemcitabine and 2',2'-difluorodeoxyuridine in plasma and tissue culture media.

Gemcitabine, a pyrimidine antimetabolite undergoes metabolism by plasma and liver cytidine deaminase to form the inactive compound, 2',2'-difluorodeoxyuridine (dFdU). The parent molecule is activated by intracellular phosphorylation. To evaluate the population pharmacokinetics in patients receiving gemcitabine, and to test the relation between gemcitabine infusion rate and antitumor activity in an in vitro bioreactor cell culture system, we developed and validated a sensitive and specific HPLC-UV method for gemcitabine and dFdU. Deproteinized plasma is vortexed, centrifuged, and 25 microL of the acidified extract sample is injected onto a Waters Spherisorb 4.6 mm x 250 mm, 5 microm C18 column at 40 degrees C. The mobile phase (flow rate, 1.0 mL/min) consists of 10:90 (v/v) acetonitrile-aqueous buffer (50 mM sodium phosphate and 3.0 mM octyl sulfonic acid, pH 2.9). Gemcitabine, dFdU, and the internal standard, 2'-deoxycytidine (2'dC) were detected with UV wavelength set at 267 nm. The standard curves for gemcitabine in both matrices ranged from 2 to 200 microM, and for dFdU in plasma, from 2 to 100 microM. Within-run and between-run component precision (CV%) was

Simultaneous determination of capecitabine and its metabolites by HPLC and mass spectrometry for preclinical and clinical studies.

A reverse-phase high-performance liquid chromatography method with electrospray ionization and detection by mass spectrometry is described for the simultaneous determination of capecitabine, its intermediate metabolites (DFCR, DFUR) and the active metabolite 5-fluorouracil in mouse plasma, liver and human xenograft tumours. The method was also cross-validated in human plasma and human tumour for clinical application. The method has greater sensitivity than previously published methods with an equivalent accuracy and precision. It uses less biological material (plasma, tissue) and should therefore be applicable to biopsies in patients treated with capecitabine.

Quantification of 2'-fluoro-2'-deoxyuridine and 2'-fluoro-2'-deoxycytidine in DNA and RNA isolated from rats and woodchucks using LC/MS/MS.

Apatmers are synthesized using 2'-fluoropyrimdines in place of normal pyrmidines to stabilize them against enzymatic degradation, and thereby improve their therapeutic efficacy. Despite this stabilizing effect, the apatmers can still be degraded by nucleases in the blood. Primer template extension studies have demonstrated that mammalian DNA polymerases can incorporate these 2'-fluoropyrimidines into growing strands of DNA. The toxicologic effects of these compounds have been examined in rats and woodchucks, animals known to be susceptible to the toxic effects of other modified pyrimidines. Whether these nucleosides can be incorporated into DNA in vivo has not been established. These studies report the development of methodologies and the results of studies designed to determine if and to what extent 2'-fluoropyrimidines are incorporated into tissue DNA following long-term treatment. Rats were dosed intravenously with either 2'-fluorouridine (2'-FU) or 2'-fluorocytidine (2'-FC) at doses of 5, 50, and 500 mg/kg/day for 90 days. Woodchucks were dosed intravenously with either 2'-FU or 2'-FC at doses of 0.75 or 7.5 mg/kg/day for 90 days. The amounts of 2'-FU or 2'-FC in DNA and RNA were quantified using newly developed LC/MS/MS methodologies. Administration of 2'-FU to rats and woodchucks resulted in incorporation of the compound into DNA from liver, spleen, testis, muscle, and kidney. Incorporation also occurred in RNA from rat liver (only tissue examined). Similarly, administration of 2'-FC to rats and woodchucks resulted in incorporation into liver DNA (only tissue examined). These data demonstrate that 2'-fluoropyrimidines are incorporated into DNA and RNA of various tissues of rats and woodchucks following long-term administration.

[Study on brain targeting 3',5'-dioctanoyl-5-fluoro-2'-deoxyuridine pharmacosomes].

AIM: To investigate the specific brain drug targeting of 5-fluoro-2'-deoxyuridine (FUdR) by synthesis of 3', 5'-dioctanoyl-5-fluoro-2'-deoxyuridine (DO-FUdR) and incorporation into DO-FUdR pharmacosomes (DO-FUdR-PS). METHODS: DO-FUdR-PS was prepared by thin-layer ultrasonication technique. In vitro drug release was studied in pH 7.4 phosphate-buffered saline containing 0.3% pancreatic enzyme at 37 degrees C using bulk-equilirium reverse dialysis bag technique. The concentration of FUdR in various organs were determined by reversed phase HPLC after i.v. administration of DO-FUdR-PS and FUdR. RESULTS: The mean particle size of DO-FUdR-PS was 76 nm with drug loading of 29.02% and entrapment efficiency of 96.62%. The in vitro drug release kinetics could be characterized by bioexponential equation. Compared with FUdR injection, DO-FUdR-PS showed high concentration in tested organs. The brain AUC ratio of DO-FUdR-PS to FUdR was 10.97. CONCLUSION: DO-FUdR-PS showed a good targeting efficiency in vivo. PS can improve the ability of drug to cross blood-brain barrier and is a promising drug targeting system for the treatment of central nervous system disorders.

Reporter gene imaging: effects of ganciclovir treatment on nucleoside uptake, hypoxia and perfusion in a murine gene therapy tumour model that expresses herpes simplex type-1 thymidine kinase.

Perfusion, hypoxia and nucleoside uptake during ganciclovir therapy were determined in a murine HSV-1 TK-expressing tumour model (KBALB-STK). HSV-1 TK mRNA transcription in this cell line was confirmed by RT-PCR. BALB/c mice bearing KBALB-STK tumours accumulated (E)-5-(2-[125I]iodovinyl)-2'-fluoro-2'-deoxyuridine ([125I]IVFRU) (2.54% injected dose.g-1) and could be readily detected with planar imaging following administration of [131I]IVFRU. However, a single dose of ganciclovir (100 mg.kg-1 intraperitoneally) decreased tumour uptake of [125I]IVFRU to 0.33% injected dose.g-1. Subsequent single daily doses of ganciclovir over 3 consecutive days had a negligible effect on [125I]IVFRU uptake, which remained low. Tumour perfusion during 3 days of ganciclovir treatment was monitored with intravenous [99Tcm]HMPAO. Tumour perfusion increased from day 0 (no ganciclovir treatment) with 1.83% injected dose.g-1 tumour, to a maximum at day 2 (3.77% injected dose.g-1). In the same animals, accumulation of [3H]misonidazole decreased from 0.70% injected dose.g-1 at day 0 to a minimum at day 3 (0.24% injected dose.g-1), indicating that tumour tissue had become less hypoxic over the ganciclovir regimen. The uptake of [125I]IVFRU into the acid insoluble fraction of KBALB-STK cells in vitro in the presence of ganciclovir (2.0 microM) was completely inhibited, leading to a 57% decrease in total cellular accumulation of radioactivity. However, cytosolic entrapment of [125I]IVFRU was not affected by the presence of ganciclovir. These results indicate that the mechanisms leading to IVFRU exclusion during ganciclovir treatment of HSV-1 TK-expressing tumours can be attributed, at least partially, to inhibition of [125I]IVFRU-nucleotide incorporation into DNA.

Immunocytochemical localization of an immunoconjugate (antibody IgG against prostatic acid phosphatase conjugated to 5-fluoro-2'-deoxyuridine) in human prostate tumors.

Current chemotherapeutic and/or endocrine treatments for adenocarcinoma of the prostate (CaP) do not selectively target neoplastic prostate cells. Therefore, new approaches are needed to improve treatment for prostate tumors. We hypothesized that because of the specific binding of antibody immunoglobulin G (IgG) against human prostatic acid phosphatase (PAcP), PAcP-IgG could function as a carrier protein for the conjugated chemotherapeutic drugs and that the immunoconjugate would then selectively localize (bind) to epithelial cells of human prostate tumors, but not to epithelial cells of other solid organs. Our objective was to test this hypothesis using human prostate, colon, and kidney tissue samples and human prostate pieces incubated in short-term organ culture. We used derivatives of 5-fluorouracil labeled with fluorescein isothiocyanate (FITC) and rabbit anti-PAcP-IgG tagged with CY3/rhodamine alone or as an immunoconjugate. Localization of PAcP-IgG alone and the immunoconjugate in prostate produced similar and specific immunostaining in prostate epithelial cells and their tumors, but not in epithelia of colon and kidney tissue sections or in prostate sections-treated with normal rabbit serum. Confocal microscopy showed co-localization of CY3 and FITC of the immunoconjugate in the same group of prostate epithelial cells and their tumors. Organ culture studies showed that human prostate tissue samples incubated with normal rabbit serum did not show any fluorescence whereas those cultured with PAcP-IgG immunoconjugate showed fluorescence in glandular epithelial cells. The later study also showed that in organ culture the immunoconjugate had penetrated and labeled prostate glands internal to the cut surfaces. Drug labeled with FITC did not localize specifically in the prostatic epithelium. Analysis of our data has shown that PAcP-IgG was needed for specific localization of the immunoconjugate in prostate glands. We conclude that PAcP-IgG was essential for delivery and binding of the drug in human prostate. This is the first report to show that PAcP-IgG-5-Fu-2'-d-based immunoconjugate was selective and specific to epithelial cells of human prostate and its tumors, as revealed by organ culture, immunocytochemical, and confocal microscopic techniques.

The influence of BIBW22BS, a dipyridamole derivative, on the antiproliferative effects of 5-fluorouracil, methotrexate and gemcitabine in vitro and in human tumour xenografts.

Dipyridamole is known as a potent inhibitor of facilitated diffusion-mediated nucleoside transport as well as a modulator of 'classical' multidrug resistance. BIBW22BS, a derivative of dipyridamole, has been found to be 20- to 100-fold more potent in the reversal of multidrug resistance when compared to the parent compound. In parallel, we studied the efficacy of BIBW22BS in the modulation of the antiproliferative effects of 5-fluorouracil, methotrexate and gemcitabine in human cancer cell lines. BIBW22BS, at non-toxic concentrations up to 1.0 microM, increased the antiproliferative effects of 5-fluorouracil 2- to 6-fold in seven of the eight colon cancer cell lines tested in a dose-dependent manner. The addition of 1.0 microM BIBW22BS to methotrexate resulted in a slight increase in the antiproliferative effects, but inhibited the activity of gemcitabine 30- to 100-fold in various cancer cell lines. In vitro, no notable difference was found between BIBW22BS and dipyridamole in their capacity to modulate the activity of the antimetabolites studied. BIBW22BS did not affect the growth inhibition induced by 5-fluorouracil or gemcitabine in human tumour xenografts grown subcutaneously in nude mice. We confirmed the higher potency of BIBW22BS when compared to dipyridamole in the reversal of drug resistance in the Pgp-positive COLO 320 cell line.

Liquid chromatographic study of the stability of 5-halogeno-2'-deoxyuridines.

The stability of a series of 5-halogeno-2'-deoxyuridines was investigated using liquid chromatography as the analytical technique. Characteristics and profiles of the acidic, neutral and alkaline degradation are described, together with Arrhenius relationships and activation parameters for weakly acidic media.

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.

High-performance liquid chromatographic method for the simultaneous measurement of floxuridine and fluorouracil in human body fluids.

A method for the simultaneous measurement of floxuridine (5-fluorodeoxyuridine) and fluorouracil in human plasma and peritoneal fluid has been developed. This method utilizes high-performance liquid chromatographic analysis with a Zorbax RX column (25 cm x 4.6 mm I.D.) plus a guard cartridge of the same material. The sensitivity limits for this assay are 0.25 mumol/l in a 20-microliters sample. The detection limit at a signal-to-noise ratio of 3 is 2.5 nmol/l. This procedure has been used to quantitatively measure concentration versus time profiles of floxuridine and fluorouracil in plasma and peritoneal fluid of human patients after receiving intraperitoneal administration of floxuridine.