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Buffer Containing (buffer + containing)
Selected AbstractsDevelopment of off-line and on-line capillary electrophoresis methods for the screening and characterization of adenosine kinase inhibitors and substratesELECTROPHORESIS, Issue 12 2006Jamshed Iqbal Abstract Fast and convenient CE assays were developed for the screening of adenosine kinase,(AK) inhibitors and substrates. In the first method, the enzymatic reaction was performed in a test tube and the samples were subsequently injected into the capillary by pressure and detected by their UV absorbance at 260,nm. An MEKC method using borate buffer (pH,9.5) containing 100,mM SDS (method,A) was suitable for separating alternative substrates (nucleosides). For the CE determination of AMP formed as a product of the AK reaction, a phosphate buffer (pH,7.5 or 8.5) was used and a constant current (95,,A) was applied (method,B). The methods employing a fused-silica capillary and normal polarity mode provided good resolution of substrates and products of the enzymatic reaction and a short analysis time of less than 10,min. To further optimize and miniaturize the AK assays, the enzymatic reaction was performed directly in the capillary, prior to separation and quantitation of the product employing electrophoretically mediated microanalysis (EMMA, method,C). After hydrodynamic injection of a plug of reaction buffer (20,mM Tris-HCl, 0.2,mM MgCl2, pH,7.4), followed by a plug containing the enzyme, and subsequent injection of a plug of reaction buffer containing 1,mM,ATP, 100,,M adenosine, and 20,,M,UMP as an internal standard,(I.S.), as well as various concentrations of an inhibitor, the reaction was initiated by the application of 5,kV separation voltage (negative polarity) for 0.20,min to let the plugs interpenetrate. The voltage was turned off for 5,min (zero-potential amplification) and again turned on at a constant current of ,60,,A to elute the products within 7,min. The method employing a polyacrylamide-coated capillary of 20,cm effective length and reverse polarity mode provided good resolution of substrates and products. Dose,response curves and calculated Ki values for standard antagonists obtained by CE were in excellent agreement with data obtained by the standard radioactive assay. [source] Indirect laser-induced fluorescence detection for capillary electrophoresis using a frequency-doubled diode laserELECTROPHORESIS, Issue 3 2003Natalia Ragozina Abstract A blue (452 nm) frequency-doubled diode laser with a quasi-cw optical output power of 10 ,W is used for indirect laser-induced fluorescence detection in combination with the capillary electrophoretic separation of inorganic anions. As fluorescing probe ion the anion of 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) was selected having an absorption maximum of 454 nm in alkaline medium. Employing a capillary coated with linear acrylamide, baseline separation of eight inorganic anions was possible within 5 min. With a separation buffer containing 50 ,mol·L -1 HPTS and 10 mmol·L -1 lysine the limits of detection for sulfate, nitrite, nitrate, azide, thiocyanate, and chlorate were between 0.9 and 4.7 ,mol·L -1. Separation of chloride and sulfate was achieved by adding 0.25 mmol·L -1 calcium hydroxide to the separation buffer. Inorganic anions in several mineral and tap water samples have been determined with the technique developed and results are compared to data obtained by ion chromatography in combination with conductivity detection after conductivity suppression. [source] Capillary Zone Electrophoresis and Micellar Electrokinetic Capillary Chromatography for Determining Water-Soluble Vitamins in Commercial Capsules and TabletsJOURNAL OF FOOD SCIENCE, Issue 1 2001S-C. Su ABSTRACT: A rapid method was developed for simultaneously determining thiamine, riboflavin, pyridoxine, nicotinamide, nicotinic acid, and ascorbic acid. It was tested on 15 samples. The peaks of all components were cleanly separated with good resolution by capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MECC). CZE was performed with 0.02 M borate buffer, and MECC was performed with 4% acetonitrile in 0.02 M borate/phosphate buffer containing 0.1 M sodium dodecyl sulfate. Average recoveries for all components were 80.3% to 103.7% with coefficients of variation being less than 5%. Thiamine, nicotinic acid, and pyridoxine contents were consistent with those labeled on the packages, but nicotinamide, riboflavin, and ascorbic acid contents of some samples were less. [source] Determination of aerobic-anaerobic metabolism-related compounds in a Chaoborus flavicans population by infusion ion trap mass spectrometry of extracts of individual larvaeRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 6 2006Maria da Graça Gama Melão In a daily migration, the aquatic larvae of Chaoborus flavicans (a phantom midge) alternate oxygen-saturated and anoxic lake strata. To investigate this cycle, larvae were collected at a natural environment, and acetate, propionate, pyruvate, lactate, glycerol, phosphate, maleate, succinate, glucose and citrate were determined. Each larva was homogenized with 200,µL water and deproteinized with a spin-filter; 50,µL aliquots were mixed with 50,µL of a buffer containing 80,mM propylamine, 20,mM HCl and 0.06,mM 2,4-dihydroxybenzoic acid (internal standard) in methanol. The extracts were infused in an electrospray ionization ion-trap mass spectrometer. The limits of detection for the [M,H], peaks ranged from 2,µM for pyruvate and lactate to 200,µM for acetate and glycerol. The MS2 ion-trap spectra obtained at pH 7 (ammonium acetate buffer) were used to distinguish maleate (cis -2-butenedioic), which gave [M,CO2,H], (m/z 71), from fumarate (trans -2-butenedioic), which showed first a loss of water yielding an instable peak at m/z 97. The compounds involved in the aerobic-anaerobic adjustment of the metabolism were revealed by linear discriminant analysis. Acetate, citrate, glucose, maleate (which decreased during the daytime), and particularly succinate (which increased), showed the maximal discrimination power between the day- and night-time samples. Copyright © 2006 John Wiley & Sons, Ltd. [source] Expression, crystallization and preliminary X-ray crystallographic studies of Arthrobacter globiformis inulin fructotransferaseACTA CRYSTALLOGRAPHICA SECTION D, Issue 12 2003Mitsuru Momma A recombinant form of Arthrobacter globiformis inulin fructotransferase (DFAIII-producing) has been overexpressed in Escherichia coli and purified to homogeneity. Crystals were obtained at 293,K by the hanging-drop vapour-diffusion technique using 0.1,M Na HEPES pH 7.5 buffer containing 1.5,M lithium sulfate as a precipitant. Crystals of the recombinant wild-type enzyme diffracted to better than 1.5,Å at 100,K using a synchrotron-radiation source at the Photon Factory. The crystal belonged to space group R32, with unit-cell parameters a = b = 92.02, c = 229.82,Å in the hexagonal axes. Assuming the presence of one molecule in the asymmetric unit, the VM value for the crystal was 2.15,Å3,Da,1, indicating a solvent content of 42.8%. Selenomethionine-derivative crystals belonged to a different space group, C2, with unit-cell parameters a = 159.32, b = 91.92, c = 92.58,Å, , = 125.06. Matthews coefficient calculations suggested that the C2 selenomethionine-derivative crystal contained three molecules per asymmetric unit. [source] Inhibitory Effects of Silibinin on Cytochrome P-450 Enzymes in Human Liver MicrosomesBASIC AND CLINICAL PHARMACOLOGY & TOXICOLOGY, Issue 6 2000Svane Beckmann-Knopp Silibinin, the main constituent of silymarin, a flavonoid drug from silybum marianum used in liver disease, was tested for inhibition of human cytochrome P-450 enzymes. Metabolic activities were determined in liver microsomes from two donors using selective substrates. With each substrate, incubations were carried out with and without silibinin (concentrations 3.7,300 ,M) at 37° in 0.1 M KH2PO4 buffer containing up to 3% DMSO. Metabolite concentrations were determined by HPLC or direct spectroscopy. First, silibinin IC50 values were determined for each substrate at respective KM concentrations. Silibinin had little effect (IC50>200 ,M) on the metabolism of erythromycin (CYP3A4), chlorzoxazone (CYP2E1), S(+)-mephenytoin (CYP2C19), caffeine (CYP1A2) or coumarin (CYP2A6). A moderate effect was observed for high affinity dextromethorphan metabolism (CYP2D6) in one of the microsomes samples tested only (IC50=173 ,M). Clear inhibition was found for denitronifedipine oxidation (CYP3A4; IC50=29 ,M and 46 ,M) and S(,)-warfarin 7-hydroxylation (CYP2C9; IC50=43 ,M and 45 ,M). When additional substrate concentrations were tested to assess enzyme kinetics, silibinin was a potent competitive inhibitor of dextromethorphan metabolism at the low affinity site, which is not CYP2D6 (Ki,c=2.3 ,M and 2.4 ,M). Inhibition was competitive for S(,)-warfarin 7-hydroxylation (Ki,c=18 ,M and 19 ,M) and mainly non-competitive for denitronifedipine oxidation (Ki,n=9 ,M and 12 ,M). With therapeutic silibinin peak plasma concentrations of 0.6 ,M and biliary concentrations up to 200 ,M, metabolic interactions with xenobiotics metabolised by CYP3A4 or CYP2C9 cannot be excluded. [source] An HPLC assay for the lipophilic camptothecin analog AR-67 carboxylate and lactone in human whole bloodBIOMEDICAL CHROMATOGRAPHY, Issue 10 2010Eleftheria Tsakalozou Abstract AR-67 (7-t-butyldimethylsilyl-10-hydroxycamptothecin, DB-67) is a camptothecin analog currently in early stage clinical trials. The lactone moiety of camptothecins hydrolyzes readily in blood to yield the pharmacologically inactive carboxylate form. However the lactone form of third-generation lipophilic congeners, such as AR-67, is more stable, possibly due to partitioning into red cell membranes. This prompted us to develop a reverse-phase HPLC method with fluorescence detection (excitation 380,nm/emission 560,nm), which could quantitate the concentration of AR-67 lactone and carboxylate in whole blood. Samples were prepared by red cell lysis, protein precipitation with methanol and centrifugation to remove denatured materials. Recovery was estimated to be >85%. Analytes were eluted isocratically with 0.15,m ammonium acetate buffer containing 10,mm TBAP (pH 6.5) and acetonitrile (65:35, v/v) on a Nova-Pak C18 column (4,µm; 3.9 × 150,mm). The assay was linear in the ranges 0.5,300 and 2.5,300,ng/mL for carboxylate and lactone, respectively. Accuracy and precision were acceptable. AR-67 forms were stable in whole blood and in methanolic supernatants. This assay has been successfully applied to measure AR-67 concentrations in whole blood of patients enrolled in a phase I study. Copyright © 2010 John Wiley & Sons, Ltd. [source] | ||||||||||