Liquid chromatography-mass spectrometry method for the analysis of the anti-cancer agent capecitabine and its nucleoside metabolites in human plasma

A reversed-phase high-performance liquid chromatography method with electrospray ionization and mass spectral detection is described for the determination of capecitabine, 5'-deoxy-5-fluorocytidine and 5'-deoxy-5-fluorouridine in human plasma with 5-chloro-2'-deoxyuridine as the internal standard. An on-line sample clean-up procedure allows dilution of the plasma sample with the initial mobile phase. The linear dynamic range is 0.0500-10.0 microgram/ml for capecitabine, and 0.0500-25.0 microgram/ml for the metabolites, 5'-deoxy-5-fluorocytidine and 5'-deoxy-5-fluorouridine, respectively. This method has been used to analyze plasma samples from patients receiving capecitabine in combination with oxaliplatin.     

The design and synthesis of a new tumor-selective fluoropyrimidine carbamate, capecitabine

To identify an orally available fluoropyrimidine having efficacy and safety profiles greatly improved over those of parenteral 5-fluorouracil (5-FU: 1), we designed a 5-FU prodrug that would pass intact through the intestinal mucisa and be sequentially converted to 5-FU by enzymes that are highly expressed in the human liver and then in tumors. Among various N4-substituted 5'-deoxy-5-fluorocytidine derivatives, a series of N4-alkoxycarbonyl derivatives were hydrolyzed to 5'-deoxy-5-fluorocytidine (5'-DFCR: 8) specifically by carboxylesterase, which exists preferentially in the liver in humans and monkeys. Particularly, derivatives having an N4-alkoxylcarbonyl moiety with a C4-C6 alkyl chain were the most susceptible to the human carboxylesterase. Those were then converted to 5'-deoxy-5-fluorouridine (5'-DFUR: 4) by cytidine deaminase highly expressed in the liver and solid tumors and finally to 5-FU by thymidine phosphorylase (dThdPase) preferentially located in tumors. When administered orally to monkeys, a derivative having the N4-alkoxylcarbonyl moiety with a C5 alkyl chain (capecitabine: 6) The highest AUC and Cmax for plasma 5'-DFUR. In tests with various human cancer xenograft models, capecitabine was more efficacious at wider dose ranges than either 5-FU or 5'-DFUR and was significantly less toxic to the intestinal tract than the others in monkeys.     

Simple high-performance liquid chromatographic method for the quantitation of 5-fluorouracil in human plasma

A simple, rapid, specific and sensitive high-performance liquid chromatography method has been developed for quantitation of 5-fluorouracil (5-FU) in human plasma. The method involves deproteinization of a small sample volume of plasma (150 μl) followed by HPLC on a cation-exchange resin column, Aminex HPX-87H (300×7.8 mm I.D.), preceded by a similar guard cartridge with UV detection at 265 nm. This method allows a good separation of 5-FU with a retention time of 24 min and a detection limit at 25 ng/ml. The calibration curve was linear from 25 to 2000 ng/ml. The coefficient of variation was ≤4.4% for within-day reproducibility and ≤9.5% for day-to-day reproducibility.     

Isolation of an unknown metabolite of capecitabine,an oral 5-fluorouracil prodrug,and its identification by nuclear magnetic resonance and liquid chromatography-tandem mass spectrometry as a glucuroconjugate of 5'-deoxy-5-fluorocytidine,namely 2'-(beta-D-glucuronic acid)-5'-deoxy-5-fluorocytidine

A new metabolite of capecitabine, a prodrug of 5-fluorouracil, was detected by (19)F NMR in bile and liver of rats treated with this anticancer drug. Crude bile and perchloric acid extract of liver was subjected to liquid-liquid separation followed by a pre-purification step on a preparative octadecyl silane column (C(18)). The compound was purified by HPLC optimised to allow the detection of the unknown metabolite and its assumed precursor 5'-deoxy-5-fluorocytidine (5'-DFCR). Treatment with beta-glucuronidase from three sources showed that it was a glucuroconjugate of 5'-DFCR. HPLC-TIS-MS-MS and (1)H NMR allowed identification of the unknown metabolite as 2'-(beta-D-glucuronic acid)-5'-deoxy-5-fluorocytidine.     

Rapid determination of 5-fluorouracil in plasma using capillary electrophoresis

A rapid, simple and sensitive capillary electrophoresis (CE) method used for the determination of 5-fluorouracil in rabbit plasma is described in the present paper. In this method, samples were simply pretreated by a solvent extraction procedure prior to injection. With a running buffer composed of 30 mM Tris-H(3)PO(4) (pH 7.0) and 5% isopropanol, 5-fluorouracil was easily separated from the external standard alpha-phenethylol as well as other substances existed in the plasma. A linearity of 5-fluorouracil was determined in the range from 0.17 to 42.50 microg/ml with a correlation coefficient of 0.999. A limit of quantitation (LOQ) corresponding to signal-to-noise ratio of 10 was obtained (LOQ=0.08 microg/ml). The method was successfully used for determining the 5-fluorouracil in real plasma samples from rabbits.     

Rapid and sensitive high-performance liquid chromatographic analysis of halogenopyrimidines in plasma

Recent studies have stressed the need for individual adjustment of 5-fluorouracil (5-FU) dosage. Most of the techniques previously reported are not well adapted to routine application. We describe a sensitive, selective and simple HPLC technique under isocratic conditions for the quantitation of 5-FU and other halogenopyrimidines. The proportion of reagents and internal standard were optimised to allow the use of minitubes, particularly adapted to large series of plasma assays. High extraction yield, 75% for 5-FU and 90% for 5-bromouracil and 5-chlorouracil, was obtained using 1.2 ml isopropanol–ethyl acetate (15:85, v/v) following precipitation of plasma proteins with 300 mg ammonium sulfate. The mobile phase was 0.01 M phosphate buffer (pH 3.0). Uracil and 5-fluorouracil were fully resolved with Spherisorb ODS2 column. The limits of quantitation and detection in human plasma were 6 ng ml⁻¹ and 3 ng ml⁻¹, respectively, for all compounds studied. The total analysis time required for each run was 25 min. Final results could be given within 90 min of blood sampling. At least 50 plasma samples could be analysed per day. This method has been successfully used for monitoring 5-FU-based treatments.     

Protective effect of anthocyanin-rich extract from bilberry (Vaccinium myrtillus L.) against myelotoxicity induced by 5-fluorouracil

The toxicities associated with 5-fluorouracil (5-FU), a potent broad-spectrum chemotherapeutic agent, can not only affect the morbidity and the efficacy of chemotherapy but also limit its clinical use. The objective of this study is to investigate the effects of a commercial anthocyanin-rich extract from bilberry (AREB) against 5-FU-induced myelotoxicity in vivo, and against chemosensitivity to 5-FU in vitro. A single injection of 5-FU at 200 mg/kg induced severe peripheral erythrocytopenia, thrombocytopenia and leucopenia as well as hypocellularity of the spleen and bone marrow in C57BL/6 mice. Oral administration of 500 mg/kg of AREB for 10 days significantly increased the number of red blood cells, neutrophils, and monocytes in peripheral blood to 1.2-fold, 9-fold, and 6-fold, respectively, compared with those seen after treatment with 5-FU alone (p< 0.05-0.001). The hypocellularity of the spleen and bone marrow caused by 5-FU was also distinctly alleviated in the AREB-treated group. Furthermore, AREB treatment with 50 and 100 microg/ml as a monomeric anthocyanin did not interfere with, but rather enhanced the chemotherapeutic efficacy of 5-FU in vitro. These results suggest that AREB may have protective potential against 5-FU-induced myelotoxiciy and/or the ability to enhance the chemotherapeutic effectiveness of 5-FU.     

Determination of 5-fluorouracil in microvolumes of human plasma by solvent extraction and high-performance liquid chromatography

In the present study, a new reversed-phase HPLC method has been developed and validated for the quantitative determination of 5-fluorouracil (5-FU) in human plasma using only 100-μl samples. The sample extraction and clean-up procedure involved a simple liquid–liquid extraction after addition of 5-chlorouracil (5-CU), used as internal standard, with 5 ml ethyl acetate. Chromatographic separations were performed on an Inertsil ODS-3 column (250×4.6 mm ID; 5 μM particle size), eluted with a mobile phase composed of acidified water (pH 2.0). The column effluent was monitored by UV absorption measurement at a wavelength of 266 nm. The calibration curves were constructed over a range of 0.20–50.0 μM and were fitted by weighted (1/x) linear regression analysis using the ratio of peak heights of 5-FU and 5-CU versus concentrations of the nominal standards. Extraction recoveries over the total range averaged 92 and 93% for 5-FU and 5-CU, respectively. The lower limit of quantitation was established at 0.20 μM (26 ng/ml), with within-run and between-run precisions of 4.2 and 7.0%, respectively, and an average accuracy of 109.3%. The within-run and between-run precisions at four tested concentrations analyzed in quintuplicate over a time period of four days were <1.4 and <4.4%, respectively. The accuracy at the tested concentrations ranged from 98.4 to 102.3%. Compared to previously described validated analytical methods for 5-FU, our present assay provides equivalent to superior sensitivity using only microvolumes of sample.     

A possible role of thymidine phosphorylase expression and 5-fluorouracil increased sensitivity in oropharyngeal cancer patients

Thymidine Pi deoxyribosyltransferase (TP) is an enzyme involved in DNA synthesis up-regulated in tumours and it is also a pro-angiogenic factor. TP cannot activate capecitabine, because capecitabine first needs conversion by carboxylesterase and cytidine deaminase into 5-deoxy-fluorouridine. This compound can be activated by TP to 5-fluorouracil (5-FU). Although TP is not necessary for 5-FU toxicity, experimental data suggest that high levels of TP correlate with an enhanced response to 5-FU therapy. In this study, we have analysed by immunohistochemistry CD34, CD68 and TP positive cells in bioptic samples from 53 patients with T(1-3) N(0-1) M(0) oropharyngeal squamous cell carcinoma (OSC) and from 24 patients with non-dysplastic oropharyngeal leukoplakia (NDOLP). Results showed that the mean of TP-positive cells, CD68 positive macrophages and CD34 positive endothelial cells eval-uated as microvessel density (MVD) was significantly higher in OSC than in NDOLP. Moreover, at a median follow-up of 19 months, patients with TP expression and higher MVD showed a better survival rate as compared to those with low MVD, probably as a consequence of 5-FU-based therapy.We hypothesized a role for TP in oropharyngeal tumourigenesis and 5-FU activation in the adjuvant setting of OSC patients.     

Gas chromatographic—mass spectrometric method for routine monitoring of 5-fluorouracil in plasma of patients receiving low-level protracted infusions

A gas chromatographic—mass spectrometric (GC—MS) method is described which quantitates 5-fluorouracil (5-FU) plasma levels ranging from 0.5 to 50 ng/ml. The analysis uses two internal standards, 1,3-[¹⁵N₂]-5-fluorouracil and 5-chlorouracil. Extraction and derivatization of the pyrimidine bases were accomplished in a single step using acetonitrile. Compounds were analyzed as their 1,3-dipentafluorobenzyl derivatives by electron-impact MS, and the GC—MS analysis was automated with respect to sample injection and data reduction. Stability of the analysis was demonstrated by continuous unattended analysis of 5-FU in human plasma for periods of up to three days with no deterioration of the quantitative results. The method is applicable to quantitating 5-FU plasma levels in patients receiving protracted infusions of the drug for colorectal cancer or other malignancies.     

Design and synthesis of the tumor-activated prodrug of dihydropyrimidine dehydrogenase (DPD) inhibitor,RO0094889 for combination therapy with capecitabine

A series of tumor-activated prodrugs of the inhibitors of dihydropyrimidine dehydrogenase (DPD), an enzyme catabolizing 5-fluorouracil (5-FU: 4g), has been designed and synthesized. RO0094889 (11c) is a prodrug of 5-vinyluracil (4c), a known DPD inhibitor, and was designed to generate 4c selectively in tumor tissues by sequential conversion of 11c by three enzymes: esterase, cytidine deaminase and thymidine phosphorylase, the latter two of which are known to be highly expressed in various tumor tissues. When capecitabine (1), a tumor-activated prodrug of 5-FU, was co-administered orally with 11c, 5-FU in tumor tissues was significantly increased with only a slight increase of 5-FU in plasma as compared with oral capecitabine alone.     

Biological monitoring of occupational exposure to 5-fluorouracil: Urinary α-fluoro-β-alanine assay by high performance liquid chromatography tandem mass spectrometry in health care personnel

A new sensitive and specific HPLC–MS/MS method for the determination of α-fluoro-β-alanine (FBAL), the main metabolite of the antineoplastic drug 5-fluorouracil (5-FU), in urine for the biological monitoring survey of health care workers exposed to 5-FU is described. This procedure is characterized by a pre-column FBAL derivatization by 2,4-dinitrofluorobenzene followed by solid phase extraction sample clean-up. The chromatographic separation was achieved by hydrophilic interaction chromatography (HILIC) on a ZIC HILIC column (Sequant) and the quantification was performed by tandem mass spectrometry. The method offers high sensitivity with a quantification limit of 1 μg/l, which is an improvement on those previously reported. The within- and between-day precisions were less than 13% and 15% respectively at the LOQ and no significant relative matrix effect was observed for FBAL. The validated method was applied to the biological monitoring of occupational exposure to 5-FU in a French hospital. Pre- and post-shift urine samples were collected from 19 workers in a hospital pharmacy and an oncology ward over a period of 5 days. On a total of 121 analysed samples, measurable amounts of FBAL were detected in up to 29%, the concentrations range from LOQ to 22.7 μg/l, yielding evidence of occupational exposure to 5-FU. Such data are scarce and represent a step forward in assessing the occupational health risks associated with handling antineoplastic drugs.     

Gas-liquid chromatographic analysis of fluoropyrimidine nucleosides and fluorouracil in plasma and urine

A gas-liquid chromatographic method employing on-column alkylation and a nitrogen-sensitive detector was developed for the analysis of 5-fluoro-2'-deoxyuridine, 5-fluorouridine, and 5-fluorouracil in plasma and urine. Samples (0.72 ml) containing the fluoropyrimidine and internal standard (5-chloro-2'-deoxyuridine for nucleoside analyses and 6-methyluracil for 5-fluorouracil analyses) were prepared for gas-liquid chromatography by sequential cation-exchange and anion-exchange column chromatography. Recoveries of fluoropyrimidines were 71-95% over the concentration ranges studied. The dried eluate from the anion-exchange column was dissolved in p-tolyltrimethylammonium hydroxide in methanol before gas-liquid chromatographic analysis. Columns packed with either 3% SP-2100 on Supelcoport or 3% OV-1 on Gas-Chrom Q were suitable for nucleoside analyses; a column packed with 0.75% Carbowax 20M-5% KOH on Chromsorb G was used for 5-fluorouracil analyses. The fluoropyrimidine nucleosides were well separated from each other and from the potentially interfering endogenous compounds 2'-deoxyuridine and uridine; 5-fluorouracil was well separated from uracil. Linear standard curves (peak area ratio method) were obtained for plasma containing 0.025 to 20 micrograms FdUrd (0.1 to 81 microM) or 0.05 to 1.0 microgram FUrd (0.2 to 3.8 microM), and for urine containing 0.2 to 1.0 microgram (0.8 to about 4 microM) of the nucleosides. Standard curves for 5-fluorouracil (1.5 to 7.9 microM) and 2'-deoxyuridine (0.9 to 4.4 microM) were also linear. A measurable amount of 5-fluorouracil, equivalent to 4 to 7% of the 5-fluoro-2'-deoxyuridine injected, was formed from the nucleoside on the gas-liquid chromatographic column, requiring correction of 5-fluorouracil concentrations measured in the presence of 5-fluoro-2'-deoxyuridine.     

Simultaneous determination of 5-fluorouracil and uracil by high-performance liquid chromatography using four serial columns

A sensitive assay was developed for the quantitation of 5-fluorouracil (5-FU) and uracil using liquid–liquid extraction (LLE) and HPLC with UV detection. Analyses were performed with four μBondapak C₁₈ columns connected in series using 20 mM acetic acid with 1% ACN as mobile phase. The calibration curves were linear across the range of 26–1000 ng ml⁻¹ (0.21–7.8 μM) for 5-FU and 1.0–14.0 μg ml⁻¹ (0.01–110 μM) for uracil. This assay has been implemented to determine the plasma concentrations for pharmacokinetic studies for 5-FU and uracil in conjunction with clinical trials.     

Anticancer activities of a chemically sulfated polysaccharide obtained from Grifola frondosa and its combination with 5-Fluorouracil against human gastric carcinoma cells

Chemically sulfated polysaccharide (S-GAP-P) was derived from water-insoluble polysaccharide of Grifola frondosa mycelia. In this research, we investigated the anticancer effects of S-GAP-P and its combination with 5-fluorouracil (5-FU) on human gastric carcinoma SGC-7901 cells. Results showed that S-GAP-P distinctly inhibited SGC-7901 cells growth in a dose-dependent manner and induced cell apoptosis evidenced by characteristic DNA ladder and sub-G₀/G₁ peak. Furthermore, the combination of S-GAP-P (10–50 μg/ml) with 1 μg/ml 5-FU resulted in a significant inhibition on SGC-7901 cells growth, meaning the beneficial interaction between the two drugs. All these results suggested that S-GAP-P has evident anticancer activity through apoptotic induction and could significantly accelerate the anticancer activity of 5-FU.     

Column-switching technique for the sensitive determination of ertapenem in human cerebrospinal fluid using liquid chromatography and ultraviolet absorbance detection

A sensitive reversed-phase high-performance liquid chromatographic (RP-HPLC) assay with on-line extraction was developed for quantifying ertapenem in human cerebrospinal fluid (CSF). This assay is at least five times more sensitive than previously published ertapenem methods with a lower limit of quantitation at 0.025 microg/ml. In this assay, a CSF sample is extracted on-line using a RP extraction column and an aqueous acidic mobile phase (0.1% formic acid) to wash away polar endogenous materials, while ertapenem is retained on the column. Ertapenem is then back-flushed off the extraction column and directed to a RP analytical column using an acidic mobile phase with an organic modifier (acetonitrile/0.1% formic acid, 15:85 (v/v)) and detected using UV absorbance. The acidic mobile phase provided a sharper chromatographic peak and on-line extraction allowed large injection volumes (> or = 150 microl) of buffered CSF to be injected without compromising column integrity. These assay conditions were necessary to quantify ertapenem at levels expected to be found in human CSF (< 0.05 microg/ml). The method was successfully validated and implemented for a clinical study: intraday precision and accuracy of the CSF assay for calibration standards (0.025-10 microg/ml) and quality control samples (0.1, 0.5, and 2.5 microg/ml) were < 6.2% coefficient of variation and 96.8-104.0% of nominal concentration, respectively.     

Development of a sensitive and selective LC-MS/MS method for the determination of α-fluoro-β-alanine, 5-fluorouracil and capecitabine in human plasma

A sensitive and selective quantitative method to determine α-fluoro-β-alanine (FBAL), 5-fluorouracil (5-FU), and capecitabine (Cape) from a single human plasma aliquot (50 μL) has been developed and validated. First, 5-FU and Cape were extracted by liquid–liquid extraction (LLE) using a mixture of acetonitrile and ethyl acetate. This was followed by derivatization with dansyl chloride. The dansyl-derivatives from 5-FU and Cape were further purified using LLE with methyl tertiary-butyl ether (MTBE) and analyzed using a reversed-phase analytical column “Primesep D” (2.1 mm × 50 mm; 5 μm) with embedded basic ion-pairing groups. The remaining aqueous phase containing FBAL was treated with dansyl chloride and the dansyl-FBAL was purified by solid phase extraction. Ultra high pressure liquid chromatography (UPLC) technology on a BEH C18 stationary phase column with 1.7 μm particle size was used for analysis of dansyl-FBAL. The method was validated over the concentration ranges of 10–10,000, 5–5000, and 1–1000 ng/mL for FBAL, 5-FU, and Cape, respectively. The results from assay validation show that the method is rugged, precise, accurate, and well suited to support pharmacokinetic studies where approximately 300 samples can be extracted and analyzed in 1 day.     

Effect of lycopene as an adjuvant therapy with 5-florouracil in human colon cancer

Colon cancer (CC) is among the most frequent human cancers. Although, there is improvement in diagnostic techniques and existing treatment possibilities. Still, there is an unmet need for a novel treatment regimen that will improve the patient's quality of life. Here, the role of lycopene as an adjuvant therapy with 5-fluorouracil (5-FU) was explored in Caco2 colon cancer cells. Cells were exposed to a dose (3 µg/ml) of 5-FU and three doses (60, 90, 120 µg/ml) of lycopene either alone or as a mixture with 5-FU. Cytotoxicity, genotoxicity, oxidative stress, gene expression, and apoptotic parameters were investigated in this study. Findings showed that 5-FU or lycopene alone induced a dose-dependent increase in cytotoxicity which was slightly reduced in lycopene mixtures. Apoptotic assays showed that 5-FU induced a significant level of apoptosis but not necrosis. However, a lycopene mixture with 5-FU enhanced 5-FU triggered apoptosis and promoted necrosis. The mixtures were also shown to suppress mitochondrial membrane potential while gene expression analyses showed the induction of Bax expression upon exposure to mix 90 exhibited the highest Bax to Bcl-2 ratio and caspase 3 and 9 gene expression. Furthermore, the mixture treatment also inhibited cell migration in the wound healing assay compared to 5-FU alone. In conclusion, lycopene was found to sensitize Caco 2 cell lines to 5-FU treatment by inducing the expression of apoptotic genes. This, coupled with lycopene suppression of cytotoxicity and cell migration, indicates lycopene may be a promising candidate for adjuvant therapy involving 5-FU in CC.     

Technique validation by liquid chromatography for the determination of acyclovir in plasma

In this research project, a high-performance liquid chromatography (HPLC) method was developed for the determination of acyclovir (ACV) in plasma. The plasma samples, recharged with acyclovir and in presence of 5'-N-methylcarboxyamidoadenosine (MECA) as an internal standard, were purified using a solid-phase extraction technique with Waters Oasis HLB columns. The separation of the components from the extract was carried out in a LiChrospher 100 RP-18 column for further ultraviolet detection at a wavelength range of 250-260 nm. The mobile phase composition was 18% acetonitrile, sodium dodecylsulphate 5 mM and phosphate buffer at pH 2.6 with an analysis time of 13 min per sample. The average retention time for acyclovir was of 5.0 min and for the internal standard 11.2 min. The calibration curve was linear ranging between 0.05 and 1.80 microg/ml. The detection limit was 0.006 microg/ml with a quantification limit of 0.020 microg/ml. The ACV recuperation percentage for 250 microl of plasma was between 94.7 and 109.7% with a coefficient of variation not higher than 5.2%. This method was developed and validated for use in bioavailability and bioequivalence studies.     

Simple and sensitive determination of 5-fluorouracil in plasma by high-performance liquid chromatography: Application to clinical pharmacokinetic studies

5-Fluorouracil (5-FU) is an antineoplastic agent widely employed in the treatment of many types of cancer. Recent studies have proved the need for individual adjustment of 5-FU dosage based on pharmacokinetics. A simple and sensitive high-performance liquid chromatographic method for the determination of 5-FU in plasma and their preliminary clinical pharmacokinetics is described. After sample acidification with 20 μl of orthophosphoric acid (5%), the drug is extracted from plasma using n-propanol–diethyl ether (16:84). The organic layer is evaporated to dryness, the residue dissolved in 100 μl of mobile phase and 20 μl of this mixture is injected into a LiChrospher 100RP-18 (5 μm, 250×4.0 mm) analytical column. Mobile phase consisted of potassium dihydrogenphosphate (0.05 M, adjusted to pH 3). The limit of quantitation was 2 ng/ml. The method showed good precision: the within-day relative standard deviation (RSD) for 5-FU (10–20 000 ng/ml) was 3.75% (2.57–5.93); the between-day RSD for 5-FU, in the previously described range, was 5.74% (4.35–7.20). The method presented here is accurate, precise and sensitive and it has been successfully applied for 5-FU pharmacokinetic investigation and therapeutic drug monitoring.