Rapid and sensitive detection of vinorelbine in the urine of tumor patients by capillary electrophoresis with tris(2,2'-bipyridyl)ruthenium(II)-based electrochemiluminescence assay.

A capillary electrophoresis method coupled with electrochemiluminescence detection for the analysis of vinorelbine (VNB) in the urine of tumor patients was established in this research. Complete determination of VNB was achieved in 8 min using a background electrolyte of 50 mmol/L phosphate buffer (pH 9.0) and a separation voltage of 15 kV. The calibration curves showed a linear range from 2.8 × 10(-10) to 1.6 × 10(-8) mol/L. The relative standard derivation for VNB was below 3.4%. The linear relationships were good and the correlation factor of VNB exceeded 0.985. The detection limits were 1.0 × 10(-11) mol/L under the optimal conditions. The developed method was applied to the sensitive determination of VNB in the urine of tumor patients.

Simultaneous quantification of vinblastine and desacetylvinblastine concentrations in canine plasma and urine samples using LC-APCI-MS/MS.

A highly sensitive and specific liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (LC/APCI-MS/MS) method has been developed and validated for simultaneous quantification of vinblastine and its metabolite, desacetylvinblastine, in canine plasma and urine samples. Plasma and urine samples were processed by a solid phase extraction procedure. The optimal chromatographic behavior of these analytes was achieved on pentafluorophenyl (PFP) propyl analytical column (5µm, 50×2.1mm) under isocratic elution of 0.75mL/min with a mobile phase of 5mM ammonium acetate and methanol. The samples were analyzed in positive ion, multiple reaction monitoring mode. The calibration curves were linear over 0.125-2ng/mL (lower calibration curve); 2-100ng/mL (higher calibration curve) and 0.125-5ng/mL for vinblastine and desacetylvinblastine in plasma, and over 1-2000ng/mL and 0.5-100ng/mL for vinblastine and desacetylvinblastine in urine samples, respectively. The limits of quantitation of vinblastine and desacetylvinblastine were 0.125ng/mL in both matrices. The intra and interday accuracy was above 89% and precision below 8.6% for both analytes in both matrices. The developed method was successfully applied to ongoing in vivo vinblastine pharmacokinetic studies in dogs.

Determination of drug residues in urine of dogs receiving anti-cancer chemotherapy by liquid chromatography-electrospray ionization- tandem mass spectrometry: is there an environmental or occupational risk?

Cytotoxic drugs, previously used only in human medicine, are increasingly utilized for cancer treatment in veterinary practice. We developed and validated a liquid chromatography (LC)-electrospray ionization-tandem mass spectrometry (MS-MS) method to determine vincristine, vinblastine, cyclophosphamide, and doxorubicin in canine urine. Sample pretreatment consisted of liquid-liquid extraction, and LC separation was carried out on an RP C(18) column employing a 0.5% formic acid/methanol gradient system. The analytes were detected in positive ion mode using the MS-MS scan mode. The mean recoveries in six different urine samples were between 64.2% and 86.9%. Limits of quantitation were 0.5 microg/L for vincristine and vinblastine, 1 microg/L for cyclophosphamide, and 5 microg/L for doxorubicin; limits of detection were approximately 0.25 microg/L for vincristine, vinblastine, and cyclophosphamide and 0.5 microg/L for doxorubicin. It could be demonstrated that all investigated drugs are found in urine of dogs undergoing chemotherapy. In samples from day 1 after chemotherapy, as much as 63 microg/L vincristine, 111 microg/L vinblastine, and 762 microg/L doxorubicin could be detected. Cyclophosphamide showed only minor concentrations on day 1, but up to 2583 microg/L could be found directly after chemotherapy. These initial data show that there might be a potential contamination risk when administering cytotoxics in veterinary medicine.

Cytotoxic drug residues in urine of dogs receiving anticancer chemotherapy.

BACKGROUND: The presence of cytotoxic drug residues in urine of dogs may represent an exposure risk for pet owners and other people as well as a potential environmental contaminant. However, studies on cytotoxic drug residues in excretions of clinical patients are lacking in veterinary oncology. HYPOTHESIS: Variable concentrations of cytotoxic residues are present in urine samples, depending on sampling time and substance. ANIMALS: Client-owned dogs with lymphoma or mast cell tumors treated with standard chemotherapy protocols. METHODS: Urine samples were collected before, directly after, and on days after administration of chemotherapy. Measurement of vincristine, vinblastine, cyclophosphamide, and doxorubicin residues in canine urine was performed by a quantitative liquid chromatography tandem mass spectrometry (LC/MS/MS) method. RESULTS: Median cyclophosphamide residue concentration was 398.2 microg/L directly after treatment (d0) and was below the level of detection on days 1-3 (d1, d2, d3). Median vincristine residue concentration was 53.8 microg/L directly after treatment and was 20.2, 11.4, and 6.6 microg/L on days 1, 2, and 3. Median vinblastine residues were 144.9 (d0), 70.8 (d1), 35.6 (d2), and 18.7 microg/L (d3) with low concentrations detectable for 7 days after treatment. Median urine doxorubicin concentrations were 354.0 (d0), 165.6 (d1), 156.9 (d2), and 158.2 microg/L (d3). Low concentrations of doxorubicin were measurable up to 21 days after administration. CONCLUSIONS AND CLINICAL IMPORTANCE: Variable concentrations of chemotherapeutics were measured in urine samples, depending on sampling time point and drug. Findings may inform current chemoprotection guidelines and help minimize exposure risks.

Development of a sensitive liquid chromatography method coupled with a tandem mass spectrometric detection for the clinical analysis of vinflunine and 4-O-deacetyl vinflunine in blood, urine and faeces.

A sensitive and specific liquid chromatographic method coupled with tandem mass spectrometric detection was set up and fully validated for the simultaneous quantification of vinflunine (VFL) and its pharmacologically active metabolite, 4-O-deacetyl vinflunine (DVFL). The two compounds, as well as vinblastine (used as internal standard), were deproteinised from blood and faeces, analysed on a cyano type column and detected on a Micromass Quattro II system in the positive ion mode after ionisation using an electrospray ion source. In blood, linearity was assessed up to 200 ng/ml for vinflunine and 100 ng/ml for 4-O-deacetyl vinflunine. The lower limit of quantification was validated at 250 pg/ml for both compounds. In other biological media, the linearity was assessed within the same range; the limit of quantification was adjusted according to the expected concentration levels of each compound. This method was first developed in order to identify the structures and to elucidate the metabolic pathway of vinflunine. Thanks to its high sensitivity and specificity, the method has enabled the quantification of vinflunine and 4-O-deacetyl vinflunine in blood at trace levels, and has contributed to the knowledge of vinflunine metabolism by monitoring up to 10 metabolites.

A high performance liquid chromatography method for vinorelbine and 4-O-deacetyl vinorelbine: a decade of routine analysis in human blood.

A sensitive high performance liquid chromatographic method was developed and validated for the simultaneous quantification of vinorelbine and its active metabolite, 4-O-deacetyl vinorelbine, in human biological fluids. These two compounds together with vinblastine, used as internal standard, were extracted from blood and urine by a liquid-liquid process using diethyl ether, and followed by a back-extraction in acidic conditions. Then, they were analysed through a cyano column and detected in ultraviolet at 268 nm. The assay linearity was validated up to 2000 ng/ml. The lower limit of quantification was set at 2.5 ng/ml. The between-run precision and accuracy were always higher than 94%. Biological samples were stable when stored at -80 degrees C over 2 years. The long-term reproducibility and the suitability of this analytical method were demonstrated within the last decade through the analysis of about 7000 samples during the clinical development of i.v. and oral formulations of vinorelbine. Because vinorelbine binds mainly to platelets and blood cells and because this binding is rapidly reversible and highly influenced by environmental conditions, drug concentration in plasma may be highly influenced by the sampling conditions and the centrifugation process used to separate blood cells from plasma. Therefore, this method was developed in blood and then used for sample analyses in routine. The major benefit was that it was easy for nurses to directly collect blood instead of plasma and that reduced volume of sampling could be withdrawn from frail patients. Furthermore, the analysis in blood enabled to quantify vinorelbine and 4-O-deacetyl vinorelbine concentrations for a longer period of time, which resulted in a more accurate evaluation of pharmacokinetic parameters.

Pharmacokinetics, tissue distribution and excretion of vinflunine.

The plasma pharmacokinetics, tissue distribution, excretion and binding to plasma proteins of vinflunine, were investigated after intravenous (iv) administration. We obtained plasma profiles after iv administration of vinflunine at the doses of 3.5, 7 and 14 mg/kg in rats. The t1/2 values for vinflunine were estimated to be 18.38+/-1.20, 17.05+/-0.77, 18.35+/-1.57 h, and the mean AUC0-t values were 3.48+/-0.38, 6.54+/-0.68, 12.79+/-2.93 microg x h/ml, respectively. Of the various tissues tested, vinflunine was widely distributed into tissues, with the highest concentrations of vinflunine being found in well perfused organs. Maximal concentration of vinflunine was reached at 0.5 h postdose in the majority of tissues. In tumor-bearing mice, the similar pattern of tissue distribution was observable, except that vinflunine can be distributed into tumor. The binding of vinflunine in human and rat plasma proteins were 39.6% and 58.4% respectively. Within 96 h after administration, 9.58%, 15.36% and 0.71% of the given dose was excreted in urine, feces and bile, respectively. In conclusion, Vinflunine had a longer terminal half-life, a wide tissue distribution and less than 25% of the given dose was excreted as unchanged drug, suggesting metabolism as a major style of elimination.

New sensitive liquid chromatography method coupled with tandem mass spectrometric detection for the clinical analysis of vinorelbine and its metabolites in blood, plasma, urine and faeces.

A new sensitive and specific liquid chromatographic method coupled with tandem mass spectrometric detection was set up and validated for the simultaneous quantitation of vinorelbine, its main metabolite, 4-O-deacetylvinorelbine and two other minor metabolites, 20'-hydroxyvinorelbine and vinorelbine 6'-oxide. All these compounds, including vinblastine (used as internal standard) were deproteinised from blood, plasma and faeces (only diluted in urine), analysed on a cyano column and detected on a Micromass Quattro II system in the positive ion mode after ionisation, using an electrospray ion source. Under tandem mass spectrometry conditions, the specific product ions led one to accurately quantify vinorelbine and its metabolites in all biological fluids. In whole blood, linearity was assessed up to 200 ng/ml for vinorelbine and up to 50 ng/ml for the metabolites. The limit of quantitation was validated at 250 pg/ml for both vinorelbine and 4-O-deacetylvinorelbine. In the other biological media, the linearity was assessed within a same range and the limit of quantitation was adjusted according to the expected concentrations of each compound. This method was initially developed in order to identify the metabolite structures and to elucidate the metabolic pathway of vinorelbine. Thanks to its high sensitivity, this method has enabled the quantitation of vinorelbine and all its metabolites in whole blood over 168 h (i.e., 4-5 elimination half lives) whilst the previous liquid chromatographic methods allowed their measurement for a maximum of 48-72 h. Therefore, using this method has improved the reliability of the pharmacokinetic data analysis of vinorelbine.

Tissue disposition, excretion and metabolism of vinblastine in mice as determined by high-performance liquid chromatography.

We have developed and validated a selective analytical procedure, based on ion-exchange normal-phase liquid chromatography with fluorescence detection and liquid-liquid extraction, for the analysis of vinblastine (VBL) in biological matrices. The assay is suitable for the determination of the parent compound and its metabolites in plasma, tissue, faeces and urine specimens. Pharmacokinetics studies were performed in male FVB mice receiving VBL by intravenous (i.v.) bolus injection at a dose of 6 mg/kg. Plasma concentrations were monitored until 48 h after drug administration. Urine and faeces samples were collected in 24-h portions for up to 72 h and tissue samples were obtained at 4, 24, 72 and 168 h after drug administration. To facilitate a comparison between the findings we obtained by high-performance liquid chromatography (HPLC) and the results of previous studies using radiolabeled drug monitoring, some of the animals were also given radiolabeled drug. Large discrepancies were observed between the results obtained by the two methods. Excretion of the radiolabel in faeces and urine was 85% of the dose within 72 h. HPLC revealed that only 18% of the dose was excreted as unchanged drug and 19%, as measurable metabolites [O4-deacetylvinblastine (DVBL) and two unknown compounds]. In most of the tissues taken at 4 h after drug administration, virtually all of the radioactivity represented VBL or DVBL. In all tissues taken at 72 h after drug administration, however, only very little of the radioactivity remained in the form of these compounds. Following the administration, VBL and DVBL were distributed extensively to most tissues. Many tissues appeared to possess effective means of extruding the cytotoxic drug with decreasing plasma levels. However, in some organs, including those from the genital tract and lymphatic tissues, VBL and DVBL were retained for prolonged periods. Our studies confirm previous indications that selective retention may be the basis of the activity of VBL against malignant transformations derived from these tissues.

Biodetermination of N-(deacetyl-O-4-vinblastoyl-23)-L-tryptophan, a metabolite of vintriptol, by high-performance liquid chromatography with fluorescence detection.

The determination of N-(deacetyl-O-4-vinblastoyl-23)-L-tryptophan (vintriptol acid, VtrpA), a metabolite of the investigational semi-synthetic vinca alkaloid vintriptol [N-(deacetyl-O-4-vinblastoyl-23)-L-ethyltryptophan, VtrpE], in plasma and urine samples is described. Sample pretreatment included liquid-liquid extraction of the buffered (pH 5.0) biological samples with chloroform-2-propanol (95:5, v/v). The analyses were performed by ion-exchange high-performance liquid chromatography on normal-phase silica with fluorescence detection. The assay was applied to the analysis of samples from cancer patients who had been treated with VtrpE in a phase I clinical study. VtrpA was found to be a principal metabolite of VtrpE with up to 1.2% of the administered dose excreted in the urine.

High-performance liquid chromatographic determination of vinblastine, 4-O-deacetylvinblastine and the potential metabolite 4-O-deacetylvinblastine-3-oic acid in biological fluids.

Procedures for the determination of vinblastine (VBL), 4-O-deacetylvinblastine (DVBL) and 4-O-deacetylvinblastine-3-oic acid (DVBLA) in biological samples using high-performance liquid chromatography (HPLC) combined with selective sample clean-up are presented. VBL and DVBL were determined in plasma and urine using ion-exchange normal-phase HPLC with fluorescence detection. The limit of detection was 1 microgram/l for both compounds using a 500-microliter sample. Successful chromatographic analyses of DVBLA were achieved by using a glass column packed with 5-microns Hypersil ODS and acetonitrile-0.05 M phosphate buffer (pH 2.7) (23:77, v/v). Positive identification was supported by the use of diode-array detection. The limit of detection (at 270 nm) was 10 micrograms/l using 1-ml samples.

Pharmacokinetics and disposition of the KS1/4 monoclonal antibody-desacetylvinblastine hydrazide conjugate LY203725 in rats and monkeys.

The plasma pharmacokinetics of the monoclonal antibody-vinca conjugate KS 1/4-desacetylvinblastine hydrazide (DAVLB-hyd; [3H]LY203725) have been evaluated in rats (17 mg/kg) and monkeys (15 mg/kg) after i.v. dosing. Plasma concentrations of radioactivity 1 hr after dosing were higher in monkeys than in rats. The biphasic elimination of radioactivity in rats was characterized by half-lives (T1/2) of 10 and 143 hr, whereas the elimination of radioactivity in monkeys was characterized by T1/2 values of 11 and 66 hr. Plasma total antibody and radioactivity concentrations were similar within 6 (rat) and 24 (monkey) hr after dosing; however, total antibody concentrations were greater than radioactivity concentrations thereafter, indicating the presence of free antibody in the plasma. Plasma elimination T1/2 values and areas under the curve of total antibody in rats and monkeys were greater than those of radioactivity. The presence of free antibody implies the presence of free DAVLB-hyd; however, plasma concentrations of free DAVLB-hyd were at least 3 orders of magnitude less than those of radioactivity in both species. The T1/2 of free DAVLB-hyd in plasma of LY203725 dosed monkeys was 16 hr. Hydrolysis of the conjugate to yield free DAVLB-hyd was observed upon incubation of conjugate with rat plasma in vitro. Administration of DAVLB-hyd to rats resulted in a rapid initial decrease in plasma DAVLB-hyd followed by a slower (T1/2 = 1.4 hr) elimination rate. Fecal excretion was the predominant mode of elimination of radioactivity in both rats and monkeys dosed with [3H]LY203725.

Analysis of vinca alkaloids in plasma and urine using high-performance liquid chromatography with electrochemical detection.

The reversed-phase high-performance liquid chromatography with electrochemical detection was used to quantify plasma and urine levels of vinblastine, vincristine, vindesine and a metabolite of vinblastine, desacetylvinblastine. Sample clean-up consisted of solid-phase extraction with a Bond Elut CN column. The extracts were separated on a Hypersil ODS column. The mobile phase consisted of a mixture of methanol and 10 mM phosphate buffer (pH 7.0). The limit of sensitivity using electrochemical detection was 100 pg on-column for all compounds with a signal-to-noise ratio of 3. Quantification of the compounds in human plasma and urine was possible down to 1 ng/ml (ca. 1 pmol). Pharmacokinetic results show that the sensitivity of the method is adequate for drug monitoring in clinical research.

Solid-phase extraction of vinblastine and vincristine from plasma and urine: variable drug recoveries due to non-reproducible column packings.

A sensitive and selective high-performance liquid chromatographic (HPLC) method for the determination of vinblastine and vincristine in plasma and urine is described. The drugs are isolated from 1.0 ml of the biological fluid with a solid-phase extraction column (Bond-Elut Diol). The HPLC method was combined with electrochemical detection at +850 mV versus an Ag/AgCl reference electrode. The detection limit is 100 pg for vinblastine and 250 pg for vincristine with a signal-to-noise ratio of 3, which permits the determination of these compounds in biological fluids at the nanogram level. Evaluation of the isolation method revealed that the drug recoveries and the reproducibility of the extraction procedure depend on the batch number of the solid-phase extraction column used.

Clinical pharmacokinetics of vindesine: repeated treatments by intravenous bolus injections.

Vindesine was administered intravenously to 12 patients with advanced cancer. Treatment was repeated after 2 weeks or more with a 1.5-to 2-fold increased dose of vindesine. Five patients received one or two additional injections at the higher dose level. One patient was given 0.4, 1 and 4 mg of vindesine on days 1, 3 and 6 and then 8 mg on days 19 and 34. Plasma samples and urine were collected over 3 days after injection and monitored for vindesine by radioimmunoassay. Significant time-dependence of vindesine plasma concentration decay kinetics at a constant dose was observed in four patients out of six. The comparison of the kinetics after administration of different doses to the same patient revealed frequent deviations from linearity with no obvious general trend. Urinary excretion was very low (1-12% of the dose), and urinary excretion rates correlated with plasma concentrations. Renal clearances were variable from one patient to another and also for the same patient from one injection to another. These data were interpreted in terms of time- and dose-dependence of vindesine pharmacokinetics.

Pharmacokinetics and metabolism of vinblastine in humans.

The pharmacokinetics of vinblastine in humans was examined using a radioimmunoassay specific for both the Vinca alkaloids and aromatic ring [3H]vinblastine. The data were consistent with a three-compartment open model system with the following values, alpha phase: t1/2=3.90+/-1.46 min; Vc=16.8+/-7.1 liters. beta phase:t1/2=53.0+/-13.0 min; Vbeta=79.0+/-52.0 liters; gamma phase:t1/2=1173.0+/-65.0 min; Vgamma=1656.0+/-717.0 liters. Most significant was the finding that vinblastine is metabolized to deacetylvinblastine and that this compound is more biologically active on a weight basis than the parent. No other biologically active metabolites appeared to be present in urine or in stool.

Pharmacological studies with vinblastine in the dog.

Tritiated vinblastine was prepared by catalytic exchange and its metabolism was studied in dogs. Plasma levels of drug fell in biphasic mode with initial and secondary phase half-lives of 17 to 38 min and 3 to 5 hr, respectively. Between 28.6 and 79.1% of plasma tritium was precipitable with cold trichloroacetic acid and thus was presumably protein bound. Blood leukocytes had levels of intracellular tritium between 2.4 and 11.8 times those of the coincident plasma samples. Over a 9-day period, urinary excretion accounted for 12.1 to 16.8% and fecal excretion accounted for 30.1 to 36.1% of the administered radioactivity. Ratios of biliary to plasma radioactivity varied between 7.3 and 56.9, with unchanged vinblastine being the mamor component (46.8 to 80.7%) in the bile.