Bioanalytical method development for a generation 5 polyamidoamine folic acid methotrexate conjugated nanoparticle

Generation 5 poly(amidoamine) dendrimer nanoparticles conjugated with folic acid and methotrexate (G5-MTX-FA) for targeted treatment of cancer are of recent interest. The increased efficacy of these nanodevices over the free methotrexate has been shown in vitro and in vivo. The heterogeneous nature of this nanoparticle together with possible release of active compounds complicated the method development. This work presents a bioanalytical assay for the detection of nanoparticle-conjugated methotrexate, released methotrexate, and its main plasma metabolite 7-hydroxymethotrexate in rat plasma. Determination of G5-MTX-FA-associated methotrexate occurred by a reductive cleavage of the C9–N10 bond in methotrexate, resulting in a highly fluorescent 2,4-diamino-6-methylpteridine reporter molecule that could be measured by reversed-phase chromatography and fluorescence detection. It was found that reduction should occur directly in the plasma matrix to avoid irreversible adsorption of the nanodevice during sample preparation. The method was linear over a range from 50 to 10,000 nM G5-MTX-FA utilizing 100 μL of plasma. Nanoparticle-released methotrexate and its metabolite 7-hydroxymethotrexate were determined by reversed-phase chromatography followed by online post-column photochemical derivatization and fluorescence detection. The method was specific for these analytes irrespective of nanoparticle concentration. Sample preparation consisted of perchloric acid protein precipitation followed by a strong anion exchange solid-phase extraction. Limits of quantification were about 50 nM for methotrexate and 10 nM for 7-hydroxymethotrexate. Preliminary pharmacokinetic profiles of intravenous and subcutaneous administered G5-MTX-FA in rats were obtained. These data indicated that less than 0.1% of the methotrexate mass is released from the nanoparticle in plasma.     

Dried plasma spot–based liquid chromatography–tandem mass spectrometry for the quantification of methotrexate in human plasma and its application in therapeutic drug monitoring

Methotrexate, a folic acid antitumor drug, is widely used to treat childhood acute lymphoblastic leukemia. Therapeutic drug monitoring is crucial for adjusting the dosage of methotrexate according to its plasma concentration and reducing adverse effects. Micro-sampling strategies, like dried plasma spot, is an attractive but underutilized method that has the desired features of easy collection, storage, and transport, and overcomes known hematocrit issues in dried blood spot analysis. This study describes a dried plasma spot–based liquid chromatography–tandem mass spectrometry method for quantification of methotrexate. The assay showed good linearity over 30–2000 ng/mL (R² ≥ 0.995) as well as excellent precision (0.6–9.3%) and accuracy (89.2–108.3%). Methotrexate was extracted from dried plasma spot and wet plasma samples with recoveries greater than 92.1%, and no significant matrix effect was observed. A comparison of dried plasma spot and wet plasma concentrations was assessed in 27 patients treated with methotrexate and Passing–Bablok regression coefficients showed that no significant difference between the two methods. The Bland–Altman plots showed similar agreement between the methods, indicating that the proposed dried plasma spot sampling method is an effective way to monitor the concentration of methotrexate in human plasma.     

Determination of methotrexate and its metabolites 7-hydroxymethotrexate and 2,4-diamino-N10-methylpteroic acid in biological fluids by liquid chromatography with fluorimetric detection

A high-performance liquid chromatographic method involving post-column cleavage and fluorimetric detection has been developed for the determination of methotrexate and its metabolites in biological fluids. The cleavage is based on photooxidative reaction of methotrexate and its metabolites to highly fluorescent products. The photoreaction occurs during the flow of eluate containing a certain amount of hydrogen peroxide through a PTFE capillary irradiated by UV light. The method allows the determination of methotrexate, 7-hydroxymethotrexate and 2,4-diamino-N10-methylpteroic acid in plasma, urine and ascitic fluid samples at concentrations as low as 2 X 10(-8)M.     

A new ultrafast and high-throughput mass spectrometric approach for the therapeutic drug monitoring of the multi-targeted anti-folate pemetrexed in plasma from lung cancer patients

An analytical assay has been developed and validated for ultrafast and high-throughput mass spectrometric determination of pemetrexed concentrations in plasma using matrix assisted laser desorption/ionization–triple quadrupole–tandem mass spectrometry. Patient plasma samples spiked with the internal standard methotrexate were measured by multiple reaction monitoring. The detection limit was 0.4 fmol/μL, lower limit of quantification was 0.9 fmol/μL, and upper limit of quantification was 60 fmol/μL, respectively. Overall observed pemetrexed concentrations in patient samples ranged between 8.7 (1.4) and 142.7 (20.3)?pmol/μL (SD). The newly developed mass spectrometric assay is applicable for (routine) therapeutic drug monitoring of pemetrexed concentrations in plasma from non-small cell lung cancer patients.     

Paired-Ion High Pressure Liquid Chromatography of Methotrexate and Metabolites in Biological Fluids

Abstract

A paired-ion high pressure liquid chromatography procedure (HPLC) is described for the separation of methotrexate and its major metabolites (7-hydroxymethotrexate, 4-amino-4-deoxy-N^lO-methylpteroic acid, methotrexate diglutamate, methotrexate triglutamate), and therapy-related compounds (Diamox and 5-methyltetrahydrofolate). The mobile phase consisted of a 70% solution of 5 mM hexanesulfonic acid, pH 3.75, and 30% methanol eluted on a reverse phase column or columns and monitored at 305 nm or 280 nm UV absorption. The lower limit of sensitivity for methotrexate and 7-hydroxymethotrexate in plasma was 2 ng/ml.     

The Study of Electrochemical Characteristics of Methotrexate

In this paper, the electrochemical characteristics of methotrexate are studied by using different electrochemical methods at the mercury drop electrode. In Britton‐Robinson buffer solution (pH 9.20), a pair of redox peaks of methotrexate controlled by adsorption is obtained by cyclic voltammetry. The differential pulse voltammetry peak currents have a linear relationship with methotrexate concentrations in the range of 1.0 × 10^?;5 mol/L ～ 1.0 × 10^?;8 mol/L with a detection limit of 2.0 × 10^?;9 mol/L, which has been used in real sample analysis with satisfactory result. Moreover, the electrode reaction mechanism of the system was studied and the kinetic parameters were obtained too. The electrode reduction of MTX is a quasi‐reversible process with two electrons and two protons.     

Pharmacokinetic application of a bio‐analytical LC‐MS method developed for 5‐fluorouracil and methotrexate in mouse plasma,brain and urine

In the past we have reported significant cognitive deficits in mice receiving 5‐fluorouracil in combination with low‐dose methotrexate. To explain such interactions, a pharmacokinetic study was designed. A sensitive bio‐analytical method was therefore developed and validated for 5‐fluorouracil and methotrexate in mouse plasma, brain and urine with liquid chromatography coupled to a single quadrupole mass spectrometer. Chromatographic separation was accomplished by Agilent® Zorbax® SB‐C₁₈ column, with isocratic elution (5 mM ammonium acetate and methanol, 70:30, %v/v) at a flow rate of 300 μL/min. The limit of quantitation for both drugs was 15.6 ng/mL (plasma and brain) and 78.1 ng/mL (urine), with interday and intraday precision and accuracy ≤15% and a total run time of 6 min. This bio‐analytical method was used for the pharmacokinetic characterization of 5‐fluorouracil and methotrexate in mouse plasma, brain and urine over a period of 24 h. This method allowed characterization of the brain concentrations of 5‐fluorouracil over a period of 24 h. Copyright © 2013 John Wiley & Sons, Ltd.     

Automatic search for maximum similarity between molecular electrostatic potential distributions

Summary A new computer program has been developed to automatically obtain the relative position of two molecules in which the similarity between molecular electrostatic-potential distributions is greatest. These distributions are considered in a volume around the molecules, and the similarity is measured by the Spearman rank coefficient. The program has been tested using several pairs of molecules: water vs. water; phenylethylamine and phenylpropylamine vs. benzylamine; and methotrexate vs. dihydrofolic acid.     

高效液相色谱二极管阵列检测器测定化妆品中的甲氨蝶呤

建立了高效液相色谱(HPLC)/二极管阵列检测器(DAD)测定化妆品中甲氨蝶呤的方法. 乳液、膏霜、化妆水、散粉、唇膏类等不同类型的化妆品样品经超声提取后, 高效液相色谱DAD 扫描检测, 并在302 nm进行分析. 用保留时间结合紫外光谱定性, 外标法定量. 甲氨蝶呤在0.05～100 mg/L的范围内线性关系良好(r＝0.9999). 在添加质量浓度为0.05～1.0 mg/L范围时, 回收率在87.8%～103.4%之间, 相对标准偏差在0.42%～6.8%, 检出限为0.02 mg/L, 定量限为0.05 mg/L. 该法可用于化妆品中甲氨蝶呤的检测.     

Anisotropic membranes with carboxypeptidase G1

Anisotropic polysulfone membranes were prepared with carboxypeptidase G₁ embedded in the polymer structure. The enzymatically active flat and hollow-fiber membranes were obtained by precipitating the polymer from solution in an organic mixture in which an aqueous solution of the enzyme had been dispersed. The process has been found to be particularly suitable for the immobilization of enzymes in anisotropic hollow fibers that exhibited no detectable enzyme leakage upon perfusion. The pH profiles measured with the enzyme in free solution and in the embedded form were similar. Kinetic parameters of multitubular enzyme reactors were investigated by measuring the rate of hydrolysis of glutamate from folic acid or methotrexate at different flow rates and substrate concentrations. The relatively slow mass transfer in such reactors was found to affect strongly the observed kinetics. The results of in vitro experiments with 5000 fiber reactors suggest that hollow fiber cartridges prepared with such membranes have clinical potential for the extracorporeal removal of methotrexate from blood.     

Stable and sensitive method for the simultaneous determination of N5-methyltetrahydrofolate, leucovorin, methotrexate and 7-hydroxymethotrexate in biological fluids

A high-performance liquid chromatographic method for the determination of N5-methyltetrahydrofolic acid, leucovorin, methotrexate and 7-hydroxymethotrexate in plasma and liquor samples is presented. Gradient elution is used to increase the sensitivity. Four sample preparation methods were compared with respect to the stability of the injectable sample. Samples can be pretreated with a simple deproteinization method. For enhanced selectivity a solid-phase extraction procedure is described.     

PHOTOSENSITIZATION BY METHOTREXATE PHOTOPRODUCTS

Abstract— Photoproducts induced upon excitation of methotrexate by UV light have been separated by ion exchange chromatography. They include 2,4-diamino-6-pteridinecarboxylic acid, 2,4-diamino-6-pteridine-carboxaldehyde and other unidentified pteridine derivatives. The same photoproducts can be also formed upon photodynamic reaction using hematoporphyrin as photosensitizer. In oxygen saturated aqueous solutions (pH∼7), methotrexate photoproducts sensitize the oxidation of histidine and tryptophan by UV light by a process involving singlet oxygen. In aqueous solutions containing albumin or in human serum, the same photoproducts are formed from free methotrexate but not from albumin-bound methotrexate. In the latter case the results may suggest that methotrexate covalently binds to albumin upon excitation with UV light either in absence or in presence of oxygen. These results could explain the photosensitization accompanying cancer chemotherapy with high dose methotrexate and also the synergistic effects of PUVA + low dose methotrexate in psoriasis therapy.     

Molecularly imprinted solid-phase extraction combined with electrochemical oxidation fluorimetry for the determination of methotrexate in human serum and urine

The method of synthesis and evaluation of molecularly imprinted polymers was reported. As a selective solid-phase extraction sorbent, the polymers were coupled with electrochemical fluorimetry detection for the efficient determination of methotrexate in serum and urine. Methotrexate was preconcentrated in the molecularly imprinted solid-phase extraction microcolumn packed with molecularly imprinted polymers, and then eluted. The eluate was detected by fluorescence spectrophotometer after electrochemical oxidation. The conditions of preconcentration, elution, electrochemical oxidation and determination were carefully studied. Under the selected experimental conditions, the calibration graph of the fluorescence intensity versus methotrexate concentration was linear from 4x10(-9) g mL(-1) to 5x10(-7) g mL(-1), and the detection limit was 8.2x10(-10) g mL(-1) (3sigma). The relative standard deviation was 3.92% (n=7) for 1x10(-7) g mL(-1) methotrexate. The experiments showed that the selectivity and sensitivity of fluorimetry could be greatly improved by the proposed method. This method has been successfully applied to the determination of methotrexate. At the same time, the binding characteristics of the polymers to the methotrexate were evaluated by batch and dynamic methods.     

Some characteristics of a protein-coated ODS column and its use for the determination of drugs by the direct injection analysis of plasma samples

Summary It was found that an ODS column of small pore (120?) which was coated with denatured plasma proteins (protein-coated ODS) no longer adsorbed plasma proteins from aqueous solution but retained the characteristics of native ODS for small hydrophobic molecules. Elemental analysis and nitrogen desorption (BET) analysis showed that the protein-coated ODS contained ca 25 mg proteing⁻¹ dry resin and that the pore diameter or pore volume was similar to that of native ODS. The coated denatured proteins, which seemed to be adsorbed on the external surface of the porous resin, were not eluted under usual reverse-phase elution conditions. Operating as either an analytical column or a pre-column, this protein-coated ODS column was used to analyse spiked-drugs in plasma. The recovery of all the spiked-drugs (such as doxorubicin, methotrexate) was almost quantitative (98–102%) with good reproducibilities (c.v., less than 2%). The present method was useful for the determination of total, that is, free + bound-to-plasma-proteins, hydrophobic drugs in plasma in view of its accuracy and simplicity. Presented at the 15th International Symposium on Chromatography, Nürnberg, October 1984     

The Effect of a New Glucose–Methotrexate Conjugate on Acute Lymphoblastic Leukemia and Non-Hodgkin’s Lymphoma Cell Lines

Patients with hematologic malignancies require intensive therapies, including high-dose chemotherapy. Antimetabolite–methotrexate (MTX) has been used for many years in the treatment of leukemia and in lymphoma patients. However, the lack of MTX specificity causes a significant risk of morbidity, mortality, and severe side effects that impairs the quality of patients’ life. Therefore, novel targeted therapies based on the malignant cells’ common traits have become an essential treatment strategy. Glucose transporters have been found to be overexpressed in neoplastic cells, including hematologic malignancies. In this study, we biologically evaluated a novel glucose–methotrexate conjugate (Glu–MTX) in comparison to a free MTX. The research aimed to assess the effectiveness of Glu–MTX on chosen human lymphoma and leukemia cell lines. Cell cytotoxicity was verified by MTT viability test and flow cytometry. Moreover, the cell cycle and cellular uptake of Glu–MTX were evaluated. Our study reveals that conjugation of methotrexate with glucose significantly increases drug uptake and results in similar cytotoxicity of the synthesized compound. Although the finding has been confined to in vitro studies, our observations shed light on a potential therapeutic approach that increases the selectivity of chemotherapeutics and can improve leukemia and lymphoma patients’ outcomes.     

Simple and sensitive HPLC method for the fluorometric determination of methotrexate and its major metabolites in human plasma by post-column photochemical reaction

A simple and sensitive HPLC method has been developed for the determination of methotrexate (MTX) and its major metabolites, 7‐hydroxymethotrexate (7‐OH‐MTX) and 2,4‐diamino‐N^10‐methylpteroic acid (DAMPA), in human plasma. After deproteinization of the plasma with 5% aqueous acetonitrile solution containing 5% trichloroacetic acid, MTX, 7‐OH‐MTX, DAMPA and 2,4‐diaminopteroic acid (DAPA) as an internal standard were separated on a reversed‐phase column, and the eluent was subsequently irradiated with UV light (245 nm), producing fluorescent photolytic degradation products. The analytes were then detected spectrofluorometrically at 452 nm with excitation at 368 nm. The extraction efficiencies of MTX, 7‐OH‐MTX and DAMPA from plasma at 100 pmol/mL were 81.5 ± 5.4, 82.5 ± 5.3 and 56.2 ± 7.0%, respectively. The limits of quantification for MTX, 7‐OH‐MTX and DAMPA in plasma were 5 pmol (2.3 ng), 0.8 pmol (0.38 ng) and 10 pmol (3.4 ng)/mL, respectively. The within‐ and between‐day variations for MTX, 7‐OH‐MTX and DAMPA were reliable (each was lower than 6.3%). This method was also used to monitor the concentrations of MTX and its metabolites in a patient on MTX therapy. Copyright © 2011 John Wiley & Sons, Ltd.     

Microanalysis of methotrexate by high-performance liquid chromatography using a fluorescence detector

Methotrexate (N¹⁰-methyl-4-aminofolic acid) has been determined by HPLC after oxidizing it with permanganate to a fluorogenic derivative. The detection limit has been 0.01 g/ml. The method has been applied for the determination of methotrexate in blood serum of rats.     

BUILDER v.2: Improving the chemistry of a de novo design strategy

Summary Significant improvements have been made to the de novo drug design program BUILDER. The BUILDER strategy is to find molecule templates that bind tightly to hot spots in the target receptor, and then generate bridges to join these templates. In this paper, the bridging algorithm has been further developed to improve the chemical sense and diversity of the bridges, as well as the robustness of the technique. The improved algorithm is then applied to rebuild known bridges in methotrexate and HIV protease. Finally, the entire BUILDER approach is tested by rebuilding methotrexate de novo.     

Effects of cold plasma and UV-C radiation on isotonic solution

The possibility of hypochlorite formation by treating a 0.9% NaCl aqueous solution (isotonic solution) with pulsed UV-C radiation from a weakly ionized plasma of air spark discharge, continuous UV-C radiation from a low-pressure mercury lamp, or cold plasma of flash corona discharge has been explored in flow and batch modes. The mechanisms of reverse reactions in which the product hypochlorite is consumed have been analyzed. It has been shown that the reverse reaction is interrupted in the case of short-term contact of the processed liquid with flash discharge plasma; as a result, hypochlorite accumulates.     

Interpretation of ion-acoustic solitons of unusual form in experiments in SF6-Ar plasma

The emergence in SF₆-Ar plasma of ion-acoustic solitons with angular profiles or profiles with several maxima has been explained. It has been shown that the cause of these profiles is that the phase trajectories of the solitons of this type in the phase portrait cover one or more separatrices, which in turn can appear only in plasma with a complex chemical composition.