[Determination of thallium in the urine with colloidal palladium as the matrix modifier by graphite furnace atomic absorption Spectrometry].

Objective: To instruct a method of determining thallium in the urine by graphite furnace atomic absorption spectrometry(GF-AAS) with colloidal palladium as the matrix modifier. Methods: Urine samples were first diluted and then determined by GF-AAS with colloidal palladium while using thermal sample injection. Results: The optimum volume of colloidal palladium was 6 µl and the best ashing temperature was 600-800 ℃ while the atomization temperature was 1700-1900 ℃ . This method showed a good linearity relationship when the concentration between 0.33 and 50.0 µg/L while the correlation coefficient of standard curve line was 0.9992, and the detection limit was 0.33 µg/L and the recovery rate was between 92.7% and 102.3% with the intra-day precision in the range of 2.55% to 3.66% and the inter-day precision in the range of 1.77% to 3.85%. Conclusion: This method has the advantages of low detect limit, high sensitivity and good precision, and it can be used in the biological monitoring and emergency detecting of workers exposed to thallium.

Medication contaminants as a potential cause of anaphylaxis to vincristine.

Vincristine (VCR) is a vinca alkaloid and common chemotherapeutic that is used to treat multiple pediatric and adult malignancies. Despite its common use, cases of anaphylaxis to VCR are rare and typically isolated to a single individual. We report a series of eight patients with adverse reactions to VCR over the course of 11 months at a single institution, four of which progressed to anaphylaxis and one of which resulted in cardiac arrest. Mass spectrometry analysis of medication lots was performed to test for possible contaminant(s). Our findings highlight the risk of anaphylaxis during therapy with VCR.

Raman micro spectroscopy study of the interaction of vincristine with A549 cells supported by expression analysis of bcl-2 protein.

Understanding the interaction of anticancer drugs with model cell lines is important to elucidate the mode of action of these drugs as well as to develop cost effective and rapid screening methods. Raman spectroscopy has been demonstrated to be a valuable technique for high throughput, noninvasive analysis. The interaction of vincristine with a human lung adenocarcinoma cell line (A549) was investigated using Raman micro spectroscopy. The results were correlated with parallel measurements from the MTT cytotoxicity assay, which yielded an IC50 value of 0.10 ± 0.03 µM. The Raman spectral data acquired from vincristine treated A549 cells was analysed to understand its interaction with the nucleus in the cell and elucidate DNA intercalation. The dose dependent spectral changes in the nucleus are analysed by PLS-Jack knifing for the identification of the more significant changes associated with the mode of action of the drug. Results are correlated with a similar dose dependent expression analysis of the bcl-2 protein, an anti-apoptotic protein associated with DNA damage, in the vincristine treated A549 cells using flow cytometry. The results indicate the co-existence of two modes of action, microtubule binding at low doses and DNA intercalation at high doses.

Wipe sampling procedure coupled to LC-MS/MS analysis for the simultaneous determination of 10 cytotoxic drugs on different surfaces.

A simple wipe sampling procedure was developed for the surface contamination determination of ten cytotoxic drugs: cytarabine, gemcitabine, methotrexate, etoposide phosphate, cyclophosphamide, ifosfamide, irinotecan, doxorubicin, epirubicin and vincristine. Wiping was performed using Whatman filter paper on different surfaces such as stainless steel, polypropylene, polystyrol, glass, latex gloves, computer mouse and coated paperboard. Wiping and desorption procedures were investigated: The same solution containing 20% acetonitrile and 0.1% formic acid in water gave the best results. After ultrasonic desorption and then centrifugation, samples were analysed by a validated liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) in selected reaction monitoring mode. The whole analytical strategy from wipe sampling to LC-MS/MS analysis was evaluated to determine quantitative performance. The lowest limit of quantification of 10 ng per wiping sample (i.e. 0.1 ng cm(-2)) was determined for the ten investigated cytotoxic drugs. Relative standard deviation for intermediate precision was always inferior to 20%. As recovery was dependent on the tested surface for each drug, a correction factor was determined and applied for real samples. The method was then successfully applied at the cytotoxic production unit of the Geneva University Hospitals pharmacy.

Development of an analytical methodology for simultaneous determination of vincristine and doxorubicin in pharmaceutical preparations for oncology by HPLC-UV.

A high-performance liquid chromatography-UV methodology (lambda=230 nm) was developed and validated for the simultaneous determination of vincristine and doxorubicin in pharmaceutical preparations used in oncology. The chromatography was carried out on a C18 column using acetonitrile 90% in water-potassium hydrogenphosphate buffer 50 mM, pH 3.2+/-0.1 (32:68, v/v) as mobile phase at a flow rate of 1.5 mL/min. The method proved to be specific, exact, and accurate, in accordance with the ICH standards, presenting linearity in the 1-5 microg/mL and 5-100 microg/mL intervals, and detection (0.19x0.51 microg/mL) and quantification (0.63x1.7 microg/mL) limits for vincristine and doxorubicin, respectively.

Multivariate optimization of solvent bar microextraction combined with HPLC-UV for determination of trace amounts of vincristine in biological fluids.

In the current work, an efficient method named solvent bar microextraction-high performance liquid chromatography-UV detection (HPLC-UV) was developed for preconcentration and determining the trace amount of vincristine (VCR) in biological samples such as plasma and urine. Briefly, VCR was extracted from an aqueous sample with pH 10.7 (donor phase) into 1-octanol as the supported liquid membrane (SLM) which is inserted into the pores of the hollow fiber and followed by back extraction into an aqueous receiving phase (pH=3.1). Studying the factors affecting the extraction performance in order to achieve a high extraction efficiency, requires the design of experiments (DOE) approach. In this regards, diverse factors' effects including the pH value of donor and acceptor phases, extraction time, extraction temperature, stirring rate and salt content of the donor phase were considered. The optimum experimental condition was as following: pH of the source phase, 10.7; pH of the receiving phase, 3.1; stirring rate, 1000rpm; extraction temperature, 51°C; extraction time, 60min and 11.3% w/v NaCl in the sample solution. Under the optimal; extraction condition, a favorable preconcentration factor equal to 98.5 was achieved. The linearity range was obtained in the domain of 0.05-5mgL-1. The limits of detection and quantification were 0.015 and 0.05mgL-1. Within-day and between-day RSDs of the proposed SBME method were 4.1% and 12.5%, respectively. Finally, the applicability of the implemented SBME method was evaluated by the extraction and quantification of VCR from biological samples such as urine and plasma and satisfactory results were obtained.

Effect of a copper gradient on plant community structure.

Vegetation data including plant cover, biomass, species richness, and vegetation height was sampled on a copper-contaminated field with total copper contents varying from 50 to almost 3,000 mg/kg soil. The field was covered by early succession grassland dominated by Agrostis stolonifera. Plant cover, biomass, species richness, and vegetation height generally decreased with increasing copper content, although the highest biomass was reached at intermediate copper concentrations. Multivariate statistical analyses showed that plant community composition was significantly correlated with soil copper concentration and that community composition at soil copper concentrations above 200 mg/kg differed significantly from community composition at lower copper levels. Comparison of single-species (Black Bindweed, Fallopia convolvulus) performance at the field site and in laboratory tests involving field soil and spiked soil indicates that the laboratory tests conventionally applied for risk assessment purposes do not overestimate copper effects. Interaction between copper and other stressors operating only in the field probably balance the higher bioavailability in spiked soil.

Multidrug resistance and the lung resistance-related protein in human colon carcinoma SW-620 cells.

BACKGROUND: Lung resistance-related protein (LRP), the major vault protein in humans, is sometimes overexpressed in multidrug-resistant cells. Because cells transfected with the LRP gene did not express the multidrug-resistant phenotype, we investigated whether LRP is involved in multidrug resistance. METHODS: SW-620 cells, a human colon carcinoma cell line, alone or transfected with an expression vector carrying a LRP-specific ribozyme or with an empty vector, were treated with sodium butyrate to induce differentiation. Expression of P-glycoprotein, multidrug resistance protein, and LRP in the cells was examined by northern and western blotting, and the efflux of doxorubicin in the cells or isolated nuclei was examined by fluorescence microscopy. RESULTS: A 2-week treatment with sodium butyrate induced LRP and conferred resistance to doxorubicin, vincristine, etoposide, gramicidin D, and paclitaxel (Taxol) in SW-620 cells. Insertion of either of two LRP-specific ribozymes into SW-620 cells inhibited these activities. Levels of drugs accumulating in the cells were not decreased by sodium butyrate, suggesting that the adenosine triphosphate-binding cassette transporter is not involved in sodium butyrate-induced multidrug resistance. Doxorubicin was mainly located in the nuclei of untreated cells and in the cytoplasm of sodium butyrate-treated cells. Isolated nuclei from untreated cells or sodium butyrate-treated cells incubated with anti-LRP polyclonal antibodies contained more doxorubicin than the nuclei of sodium butyrate-treated cells alone. Efflux of doxorubicin was greater from the nuclei of sodium butyrate-treated cells than the nuclei of untreated cells or of sodium butyrate-treated cells transfected with a LRP-specific ribozyme and was inhibited by an anti-LRP polyclonal antibody. CONCLUSIONS: LRP is involved in resistance to doxorubicin, vincristine, etoposide, paclitaxel, and gramicidin D and has an important role in the transport of doxorubicin from the nucleus to the cytoplasm.

Reliability of preparation procedure for sphingosomal vincristine.

PURPOSE: The reliability of the preparation procedure for sphingosomal vincristine was studied. The effect of minor variations in the constitution conditions on the integrity of the drug product was also examined. METHODS: Two studies were conducted. The laboratory study, which had two parts, was conducted to determine the effects of deliberately varying the constitution conditions, including such key parameters as incubation time and incubation temperature. In the field study, 20 pharmacists unfamiliar with sphingosomal vincristine were asked to constitute the product using the written instructions provided in the package insert as their sole guidance. All samples in both studies were evaluated by measuring key product characteristics, including free (un-encapsulated) vincristine sulfate, total vincristine sulfate, vincristine degradation products, and in vitro release rate. Vincristine loading into sphingosomes was considered acceptable if the percentage of free vincristine sulfate did not exceed 10%. RESULTS: In the laboratory study, samples that were incubated at 60-75 degrees C for 5-60 minutes met all the acceptance criteria. However, acceptable loading was not achieved for samples that were incubated at 55 degrees C for 10 minutes or less. In the field study, all the pharmacist-prepared samples met the acceptance criteria, with the results for free vincristine sulfate demonstrating a high degree of statistical confidence in the reliability of the loading procedure. CONCLUSION: The recommended constitution procedure of sphingosomal vincristine from a three-vial kit can be reliably performed in pharmacies with a high degree of confidence. Small variations in temperature and incubation time had no effects on the quality of the product.

Simultaneous determination of vincristine, vinblastine, catharanthine, and vindoline in leaves of catharanthus roseus by high-performance liquid chromatography.

A simple reversed-phase liquid chromatographic method is developed for the simultaneous quantitation of the anticancerous drugs vincristine, vinblastine, and their precursors catharanthine and vindoline using a Merck Chromolith Performance reversed-phase high-performance liquid chromatography column. A better resolution is obtained in comparison with available particulate-type C18 columns. The column provides good reproducibility and peak symmetry. Chromatography is carried isocratically with a mobile phase of acetonitrile-0.1M phosphate buffer containing 0.5% glacial acetic acid (21:79, v/v; pH 3.5) at a flow rate of 1.2 mL/min and UV detection at 254 nm. Parameters such as linearity, limits of quantitation (LOQ) and detection (LOD), precision, accuracy, recovery, and robustness are studied. The method is selective and linear for alkaloid concentration in the range 0.25 microg-25 microg/mL. The LOQ and LOD are 25, 46, 56, and 32 microg/mL and 8, 14, 18, and 10 microg/mL, respectively. The results of accuracy studies are good. Values for coefficient of variation are 2.50, 1.82, 1.33, and 1.13, respectively. The percent recovery of the alkaloids was found to be 96%, 97%, 98%, and 98%, respectively. Peak purity and homogeneity of these compounds in plant extract is studied using a photodiode-array detector. This simple and rapid method of analysis is applied for the determination of these alkaloids in a large number of leaf extracts of Catharanthus roseus..

[A study on the hairy root culture and antitumor alkaloids production of Catharanthus roseus].

OBJECTIVE: To establish transformation system and obtain alkaloids from the hairy root of Catharanthus roseus. METHOD: Hairy roots were obtained by infecting the different explants of C. roseus. Culture conditions of hairy root were optimized. RESULT: The best transformation condition was leaf infected by two-day's pre-culture and two-day's co-culture and additional A(S) (hydroxyacetosyringone) 100 mg x L(-1). The inducing rate of hairy root was up to 86.25%. The best condition of hairy root culture was MS medium with sucrose as carbon material and lactalbumin as nitron material. The analysis result showed that the contents of total alkaloids in hairy roots were higher than explants and calli. CONCLUSION: Hairy root of C. roseus will be useful for the production of active components in C. roseus.

Penetration of intra-arterially administered vincristine in experimental brain tumor.

Vincristine is an integral part of the "PCV" regimen that is commonly administered to treat primary brain tumors. The efficacy of vincristine as a single agent in these tumors has been poorly studied. This study was designed to determine whether vincristine enters normal rat brain or an intracranially or subcutaneously implanted glioma and to assess the presence of the efflux pump P-glycoprotein (P-gp) on tumor and vascular endothelial cells. The 9L rat gliosarcoma was implanted intracranially and subcutaneously in three Fischer 344 rats. On day 7, [3H]vincristine (50 microCi, 4.8 microg) was injected into the carotid artery, and the animals were euthanized 10 or 20 min later. Quantitative autoradiography revealed that vincristine levels in the liver were 6- to 11-fold greater than in the i.c. tumor, and 15- to 37-fold greater than in normal brain, the reverse of the expected pattern with intraarterial delivery. Vincristine levels in the s.c. tumor were 2-fold higher than levels in the i.c. tumor. P-gp was detected with JSB1 antibody in vascular endothelium of both normal brain and the i.c. tumor, but not in the tumor cells in either location, or in endothelial cells in the s.c. tumor. These results demonstrate that vincristine has negligible penetration of normal rat brain or i.c. 9L glioma despite intra-arterial delivery and the presence of blood-brain barrier dysfunction as demonstrated by Evan's blue. Furthermore, this study suggests that P-gp-mediated efflux from endothelium may explain these findings. The lack of penetration of vincristine into brain tumor and the paucity of single-agent activity studies suggest that vincristine should not be used in the treatment of primary brain tumors.

Probenecid reverses multidrug resistance in multidrug resistance-associated protein-overexpressing HL60/AR and H69/AR cells but not in P-glycoprotein-overexpressing HL60/Tax and P388/ADR cells.

PURPOSE: To determine whether probenecid, an inhibitor of organic anion transport, is able to reverse multidrug resistance (MDR) through modulation of the drug transport function of MDR-associated protein (MRP) and P-glycoprotein (P-gP). METHODS: Two MRP-overexpressing cell lines (HL60/AR and H69/AR) and two P-gP-overexpressing cell lines (HL60/Tax and P388/ADR) were cultured with different concentrations of daunorubicin (DNR) or vincristine (VCR) in the presence or absence of various concentrations of probenecid (0.01-10 mM). Drug sensitivity was determined using an MTT assay. DNR accumulation and subcellular distribution were determined by flow cytometry and confocal microscopy respectively. VCR accumulation was determined by scintillation spectrometry. RESULTS: Probenecid, in a concentration-dependent manner, reversed resistance to DNR and VCR in HL60/AR and H69/AR tumor cell lines. This effect of probenecid on MDR was associated with an increased accumulation of DNR and VCR and correction of the altered subcellular distribution of DNR. The concentrations of probenecid that reversed MDR are clinically achievable in vivo. In contrast, probenecid did not reverse MDR in either HL60/Tax or P388/ADR tumor cell lines that overexpress P-gP. CONCLUSION: These results suggest that probenecid is an effective chemosensitizer of MRP-associated MDR tumor cells and is a potential candidate for clinical use to reverse MDR.

Continuous intravenous infusion of vinca alkaloid using a subcutaneously implanted pump in a canine model.

A major drawback of infusions of the vinca alkaloids is the lengthy period of hospitalization which is often required for this novel technique of cancer therapy. A potentially useful system to deliver outpatient therapy has been investigated in a preclinical study. A self-contained infusion pump powered by a self-charging fluorocarbon system has been implanted SC in three dogs. The performance of two pumps which had been factory-calibrated to deliver 2.5 and 4.5 ml/day, respectively, was evaluated during 22 infusions of the vinca alkaloids (vincristine, 7; vinblastine, 7; and vindesine, 8). Infusions were given over a 5- to 7-day period and were repeated at 3-week intervals. No malfunctioning of the pumps occurred in over 500 cumulative days of use. The flow rates of the pumps were quite stable except in one animal whose increased flow rate was probably a consequence of fever due to self-induced inflammation about the pump pocket. No local or distant tissue reactions to the pump were observed. Decomposition of vincristine and vinblastine in the infusate at the end of 5- or 7-day infusions was minimal as determined by high-pressure liquid chromatography. The amount of decomposition of vindesine in the infusate was variable. Steady-state concentrations of vincristine during infusion were always greater than 10(-9) M, and were similar to those previously determined in our clinical infusion trials using a dosage of 0.5 mg/m2/day. Clinical evaluation of this system for prolonged infusions of vincristine and other vinca alkaloids appears to be warranted.

Relationship between the cellular accumulation and the cytotoxicity of S12363, a new Vinca alkaloid derivative.

S12363, a new vinca alkaloid derivative, was considerably more cytotoxic to murine L1210 cells and five human tumor cell lines (HL60, HT-29, COLO 320DM, NCI-H460, and PANC-1) than was vincristine (VCR) or vinblastine (VLB). S 12,363 bound to tubulin in crude extracts from brain or L1210 cells with an affinity similar to that of VLB and VCR (apparent Kd value: 1.1-1.6, 1.2-1.7, and 0.6-0.8 microM, respectively). After 1 h exposure, the accumulation of 20 nM [3H]-S 12,363 by L1210 cells was 4- to 18-fold that of [3H]-VLB and [3H]-VCR, respectively. After the cells had been preloaded for 1 h with the labeled drugs and then incubated for 3 h in drug-free medium, 37%-55% of the [3H]-S 12,363 was retained by the cells vs 36%-47% of the [3H]-VCR and less than 6% of the [3H]-VLB. Similar results were obtained for the five human cell lines tested. The accumulation factors (intracellular vs extracellular concentrations) found for [3H]-S 12,363 (54- to 167-fold) were significantly higher than those observed for [3H]-VCR (5- to 14-fold) or [3H]-VLB (19- to 41-fold). Greater than 90% of the radioactivity extracted from L1210 cells that had been treated with [3H]-S 12,363 was recovered as unmodified drug, demonstrating that [3H]-S 12,363 was not metabolized by these cells. S 12,362, which differs from S 12,363 only in the absolute configuration of the asymmetric carbon atom of its alpha-aminophosphonic side chain, was 300 times less cytotoxic, bound to tubulin with a lower affinity (apparent Kd value, 4.9-9.6 microM), and was neither accumulated nor retained by the cells. Taken together, these results demonstrate that the potency of S 12,363 is due at least in part to its cellular accumulation and retention.

High-performance liquid chromatographic determination of vincristine sulfate in preformulation studies.

A fast and simple procedure was developed for the quantitative determination of vincristine sulfate for use in preformulation studies. The procedure involves the use of high-performance liquid chromatography with a reverse-phase column and a mobile phase containing the sodium salt of 1-pentanesulfonic acid for ion-pairing. The procedure has been shown to be specific for vincristine sulfate in the presence of forced degradation products of this substance, vinblastine (a structurally similar Vinca alkaloid), and several possible formula excipients. The procedure is linear from 10-200% of the normal injection concentration, and has an assay precision (relative 2 sigma) of +/- 1.6%. Recovery of known samples averaged 99.7%.

Development of obesity and neurochemical backing in aurothioglucose-treated mice.

To clarify the neurochemical backing of aurothioglucose (ATG)-induced obesity in mice, we investigated lesion sites, hypothalamic neurotransmitters and c-Fos-like immunoreactivity (Fos-IR). At day 2 after ATG, tissue loss or cells death was observed in several parts of the ventral area of the ventromedial hypothalamic nucleus (VMH), and the dorsal area of arcuate nucleus and in the nucleus of the solitary tract (NTS). However, the greater part of the VMH was retained. Body weight began to increase in week 1. Hypothalamic serotonin (5-HT) and the metabolites were increased at day 2. The contents of acetylcholine, norepinephrine and dopamine in the hypothalamus showed no significant change. In week 1, the area shown tissue loss was compacted and plugged up. In the control group, most obvious c-Fos-like immunoreactive region was paraventricular nucleus (PVN). At day 2, Fos-IR was observed around destroyed regions in the hypothalamus and NTS, but few Fos-IR was found in the other regions including PVN. The Fos-IR around destroyed regions diminished after week 1. In week 3, Fos-IR in the PVN increased. These results suggest that the development of ATG-induced obesity cannot be attributed to solely VMH destruction. The restoration processes of the neuronal dysfunction involving PVN seem to play an important role in the development of obesity. NTS lesion and 5-HT system might contribute to decrease in food intake for several days after ATG.

Sensitive and rapid spectrophotometric methods for determination of anticancer drugs.

Sensitive, rapid, and simple spectrophotometric methods were developed for determination of the anticancer drugs vinblastine sulfate (VBS) and vincristine sulfate (VCS), which belong to the class of vinca alkaloids. The first method is based on the reaction of VBS and VCS with diazotized dapsone, forming yellow azo products with absorption maxima at 430 nm. The colored species obey Beer's law in the concentration range of 0.5-24 microg/mL for VBS and 0.5-12 microg/mL for VCS. The second method describes the reaction of VBS and VCS with iron(III) and subsequent reaction with ferricyanide in hydrochloric acid medium to yield blue products with absorption maxima at 750 nm. The Beer's law range for this method is 0.1-4 microg/mL for VBS and 0.5-10 microg/mL for VCS. With both methods, colored species were stable for 1 h. The methods are simple and reproducible and are applied for determination of VBS and VCS in pharmaceutical formulations. Commonly encountered pharmaceuticals added as excipients do not interfere in the analysis and the results obtained in the analysis of dosage forms agree well with the labeled contents.

Structure elucidation of indole-indoline type alkaloids: a retrospective account from the point of view of current NMR and MS technology.

In this review our aim is to look back on how the structure elucidation of bisindoles, especially with focus placed on vinblastine and vincristine analogues, has evolved alongside with the development of MS and NMR over the last 60 years from the perspective of our present-day use of state-of-the-art MS and NMR instrumentation and on the basis of our own accumulated views and experience in the field.

Paraplegia and paraparesis from intrathecal methotrexate and cytarabine contaminated with trace amounts of vincristine in China during 2007.

PURPOSE: The production and administration of drugs used intrathecally requires special care to prevent contamination with neurotoxic agents. In 2007, we investigated a widespread outbreak of paraplegia and paraparesis among Chinese patients who received intrathecal drugs to identify the presumed contaminant and its source to prevent further cases. PATIENTS AND METHODS: We defined a case as onset from January 1 to October 31, 2007, of bilateral flaccid paraparesis or paraplegia or retention and incontinence of stool or urine, in a patient receiving intrathecal drugs. Using a retrospective cohort approach, we selected 12 hospitals from all hospitals that had reported cases. In these hospitals, we identified all 448 patients (including 107 cases) who received intrathecal chemotherapy or chemoprophylaxis in 2007. We calculated attack rates and Mantel-Haenszel adjusted risk ratios for intrathecal drug type and lot. RESULTS: All 12 hospitals used intrathecal methotrexate or cytarabine produced by one pharmaceutical plant. Only two lots of each drug were associated with cases. Lot-specific attack rates ranged from 42% to 100% (risk ratio, ∞; lower confidence bounds, 1.8 to 7.3). Vincristine production had immediately preceded production of the implicated lots on the same equipment. By using ultra performance liquid chromatography, we detected vincristine (0.28 to 18 µg) in unused vials from implicated lots of methotrexate and cytarabine. CONCLUSION: Trace amounts of vincristine that contaminated intrathecal drugs caused a large outbreak of severe neurologic damage. Vincristine and other neurotoxic drugs should not be produced on any equipment that is also used for producing drugs that are to be administered intrathecally.