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Rapid Determination (rapid + determination)
Selected AbstractsA Contactless Impedance Probe for Simple and Rapid Determination of the Ratio of Liquids with Different Permittivities in Binary MixturesELECTROANALYSIS, Issue 1 2009Franti, ek Opekar Abstract Simple contactless cells with planar or tubular electrodes have been designed for measurement of the permittivity of solutions. The cells, connected to an integrated circuit of astable multivibrator, respond primarily to the capacitance component of the cell impedance, the multivibrator frequency depends in a defined manner on the solution permittivity and is readily used as the analytical signal in determinations of the ratios of components in binary liquid mixtures; water solution of methanol, ethanol and dioxane have been tested. The response of the cell with planar electrodes satisfies well the simple theoretical model and both the cells provide results with a sufficient sensitivity, a low LOD value (units of %vol) and a good precision (around 1%rel). The cell simplicity, small dimensions, long-term stability and the possibility of powering them from a battery make them suitable for hand-held meters. As an example of application in practice, the content of ethanol was determined in the car fuel petrol. [source] Rapid Determination of Gallamine Triethiodide (Flaxedil®) and Pancuronium Bromide (Pavulon®) in Pharmaceutical and Urine Matrices by Means of Modified-Carbon-Paste Ion-Selective ElectrodesHELVETICA CHIMICA ACTA, Issue 4 2005A new analytical method for the determination of gallamine triethiodide (Flaxedil®; 1) and pancuronium bromide (Pavulon®; 2), two muscle relaxants used in surgical operations and in pain relief, has been developed. Our approach relies on rapid, precise, and sensitive potentiometric sensors based on modified-carbon-paste ion-selective electrodes (CP-ISEs). Linear calibration graphs in the working ranges of ca. 4.5,892 and 7.3,733,,g/ml (in H2O, pH,7.0, T=25°) were established for 1 and 2, respectively; and Nernst slopes corresponding to three- or two-electrons transfers, respectively, were obtained. The method works best in a pH range of 7,9. Average relative errors of 2.12 and 2.14%, with average standard deviations of 1.98,2.47 and 2.64,3.45, respectively, were obtained for urine samples of 1 and 2. The corresponding relative errors for the pharmaceutical samples were 1.59 and 1.64%, with standard deviations of 0.54,1.34 and 0.52,1.67, respectively. Statistical Student and F tests were applied to the data, and satisfactory results were obtained. [source] Rapid Determination of Invert Cane Sugar Adulteration in Honey Using FTIR Spectroscopy and Multivariate AnalysisJOURNAL OF FOOD SCIENCE, Issue 6 2003J. Irudayaraj ABSTRACT: Fourier transform infrared spectroscopy with an attenuated total reflection sampling accessory was combined with multivariate analysis to determine the level (1% to 25%, wt/wt) of invert cane sugar adulteration in honey. On the basis of the spectral data compression by principal component analysis and partial least squares, linear discriminant analysis (LDA), and canonical variate analysis (CVA), models were developed and validated. Two types of artificial neural networks were applied: a quick back propagation network (BPN) and a radial basis function network (RBFN). The prediction success rates were better with LDA (93.75% for validation set) and BPN (93.75%) than with CVA (87.50%) and RBFN (81.25%). [source] Direct, Electronic MicroRNA Detection for the Rapid Determination of Differential Expression Profiles,ANGEWANDTE CHEMIE, Issue 45 2009Hong Yang Die Mittagspause reicht: Ein elektronischer Chip mit nanostrukturierten Mikroelektroden (NMEs) ermöglicht die Analyse der Expressionsprofile von mikroRNAs in nur 30,min , und das ganz ohne enzymatische Verstärkung oder Sequenzmarkierung! Der Chip detektiert die Hybridisierung von mikroRNA-Analyten an NME-Oberflächen und erzeugt eine starke elektrokatalytische Signalverstärkung durch Verwendung eines ultraempfindlichen Redoxreportersystems (siehe Bild). [source] Rapid determination of genomic DNA length for new bacteriophagesELECTROPHORESIS, Issue 12 2007Philip Serwer Professor Abstract dsDNA viruses with long genomes (>200,kb) are expected to be a major source of novel genes. To rapidly characterize the genomes of newly isolated dsDNA bacteriophages, we develop here a procedure for the PFGE of intact long DNA genomes from bacteriophage particles in unfractionated, infected cell lysates of either liquid or gelled cultures. The DNA used for PFGE is suitable for sequencing after extraction with phenol. The PFGE is tuned to the range of expected DNA lengths. This procedure bypasses the isolation of bacteriophage particles and is useful for PFGE analysis of DNA from dissected zones of bacteriophage plaques. [source] Rapid determination of acyclovir in plasma and cerebrospinal fluid by micellar electrokinetic chromatography with direct sample injection and its clinical applicationELECTROPHORESIS, Issue 4 2006Hsin-Hua Yeh Abstract A simple MEKC with UV detection at 254,nm for analysis of acyclovir in plasma and in cerebrospinal fluid (CSF) by direct injection without any sample pretreatment is described. The separation of acyclovir from biological matrix was performed at 25°C using a BGE consisting of Tris buffer with SDS as the electrolyte solution. Several parameters affecting the separation of the drug from biological matrix were studied, including the pH and concentrations of the Tris buffer and SDS. Using dyphylline as an internal standard, the linear ranges of the method for the determination of acyclovir in plasma and in CSF all exceeded the range of 2,50,,g/mL; the detection limit of the drug in plasma and in CSF (S/N = 3; injection 3.45,kPa, 5,s) was 1.0,,g/mL. The applicability of the proposed method for determination of acyclovir in plasma and CSF collected at 8,h after intravenous administration of 500,mg acyclovir (Zovirax®) in two patients with herpes simplex encephalitis was demonstrated. [source] Rapid determination of aliphatic amines in water samples by pressure-assisted monolithic octadecylsilica capillary electrochromatography-mass spectrometryELECTROPHORESIS, Issue 18-19 2004Bricio Santos Abstract A pressure-assisted capillary chromatography-mass spectrometry method based on the use of a monolithic octadecylsilica (ODS) capillary is proposed for the determination of aliphatic amines. A 25 mM citric acid buffer containing 10% methanol is used as running electrolyte. Separation is achieved by simultaneously applying a capillary electrophoresis (CE) voltage of 13 kV and an overimposed pressure of 8 bar. The use of pressure is required to ensure stable electrospray conditions. Analysis times are reduced by using a capillary column consisting of a 30 cm long monolithic silica capillary column bound with ODS and a fused-silica capillary column also 30 cm long. The proposed method was successfully applied to the determination of low-molecular-weight aliphatic amines in tap and river water. The analysis of real samples requires cleanup and preconcentration, which can be performed automatically by inserting a minicolumn in the replenishment system of the commercial instrument. [source] Rapid determination of stress factors and absolute residual stresses in thin filmsJOURNAL OF APPLIED CRYSTALLOGRAPHY, Issue 6 2006E. Eiper A methodology is presented that allows the determination of experimental stress factors in thin films on the basis of static diffraction measurements. The approach relies on the characterization of thin films deposited on a monocrystalline substrate serving as a mechanical sensor. Rocking-curve measurements of the symmetrical reflections of the substrate are used to determine the substrate curvature and subsequently the macroscopic stress imposed on the film. The elastic strain in the film is determined by lattice-spacing measurement at different sample tilt angles. The calculated experimental stress factors are applied to thin films deposited on other types of substrates and are used to determine the absolute magnitude of the residual stress. The approach is applied to nanocrystalline TiN and CrN thin films deposited on Si(100) and steel substrates, characterized using a laboratory-type ,/, goniometer. [source] Rapid determination of rice seed vigour by spontaneous chemiluminescence and singlet oxygen generation during early imbibitionLUMINESCENCE: THE JOURNAL OF BIOLOGICAL AND CHEMICAL LUMINESCENCE, Issue 1 2003Wenli Chen Abstract Using a highly sensitive single photon counter, a spontaneous chemiluminescence (CL) study on rice (Oryza sativa L.) seeds stored in different years was carried out. We first observed that the degree of ageing in rice seeds was related to the intensity of spontaneous CL during early imbibition (0,30 min). Rice seeds stored for a shorter time had a stronger intensity of CL in early imbibition. The germination rate of rice seeds showed an obvious positive correlation with the intensity of spontaneous CL. Singlet oxygen (1O2) in rice seeds during early imbibition was investigated by a CL method using a cypridina luciferin analogue, 2-methyl-6-(p -methoxyphenyl)-3,7-dihydroimidazo [1,2,] pyrazin-3-one (MCLA), as a selective CL probe. Additional experimental evidence for the formation of 1O2 came from the quenching effect of sodium azide (NaN3) on MCLA-mediated rice seeds' CL. Analysis based on the experimental results demonstrated that spontaneous CL in rice seeds during early imbibition was mainly contributed by singlet oxygen (1O2). Copyright © 2002 John Wiley & Sons, Ltd. [source] Rapid determination of three anticoagulant rodenticides in whole blood by liquid chromatography coupled with electrospray ionization mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 18 2006Mi-cong Jin A rapid, sensitive and selective method for the simultaneous determination of bromadiolone, flocoumafen and brodifacoum in whole blood using warfarin as internal standard (IS) by high-performance liquid chromatography coupled with electrospray ionization mass spectrometry (HPLC/ESI-MS) has been developed and validated. The target compounds were extracted from the whole blood with ethyl acetate and separated on an XDB C18 column (150,mm,×,2.1,mm i.d.,×,5,µm) by using a mobile phase consisting of 0.2% acetic acid/methanol (12/88, v/v) at a constant flow rate of 0.50 mL/min. The analytes were detected using negative ESI-MS in the selected ion monitoring (SIM) mode. The molecular ions [MH], of m/z 527, 541,523 and 307 were selected for the quantification for bromadiolone, flocoumafen, brodifacoum and the IS, respectively. The calibration curves were linear (r2,>,0.995) in the concentration range of 0.50,100.00 ng/mL. The method showed a satisfactory sensitivity (0.05,0.5 ng/mL using 200 µL blood), precision (RSD,<,11.9%), accuracy (recovery: 82.0,96.1%) and selectivity. This method was successfully applied to the determination of the analytes for the diagnoses of poisoned human beings and animals. Copyright © 2006 John Wiley & Sons, Ltd. [source] Rapid determination of rifaximin in rat serum and urine by direct injection on to a shielded hydrophobic stationary phase by HPLCBIOMEDICAL CHROMATOGRAPHY, Issue 6 2009R. Nageswara Rao Abstract A simple and rapid reversed-phase HPLC method for determination of rifaximin in rat serum and urine was developed. Separation of rifaximin from biological matrix was achieved by direct injection of rat serum and urine onto a restricted-access medium, Supelco LC-Hisep, a shielded hydrophobic stationary phase, using acetonitrile:water:acetic acid (18:82:0.1 v/v/v) as a mobile phase. The linear range was 0.10,20 µg/mL (r2 > 0.999, n = 6), intraday and interday variation was <6.10%. The limits of detection and quantification were 0.03 (signal-to-noise ratio >3) and 0.10 µg/mL (signal-to-noise ratio >10), respectively. The method was successfully applied to pharmacokinetic studies of rifaximin after an oral administration to rats. Copyright © 2008 John Wiley & Sons, Ltd. [source] Rapid determination of short-chain fatty acids in colonic contents and faeces of humans and rats by acidified water-extraction and direct-injection gas chromatographyBIOMEDICAL CHROMATOGRAPHY, Issue 8 2006Guohua Zhao Abstract Short-chain fatty acids (SCFAs) have attracted much attention recently because of their positive physiological effects. In this work, a rapid and reliable gas chromatographic method for determination of eight SCFAs, in colonic and faecal samples from rats and humans has been developed and validated. The methodology involves extraction of the SCFAs in water before a direct injection procedure on a FFAP capillary column. A stock standard solution containing acetic acid, propionic acid, n -butyric acid, i -butyric acid, n -valeric acid, i -valeric acid, n -caproic acid and n -heptanoic acid was prepared and used. A high line-arity (r2 > 0.9990), low quantification limit (2.38,30.14 µm) and high recovery for most acids were obtained. Acidification of faecal samples was found to be crucial for quantitative determination of the SCFAs, and adjustment of pH to 2,3 was regarded as necessary. Glass wool inserted in the glass liner of the injection port proved effective in preventing the contamination of the column by non-volatiles, and 12% formic acid reduced the ghost peak that appeared gradually after several injections. After validation, the methodology was applied on two faecal samples from rats fed diets containing different amount of dietary fibre and one faecal sample from human fed a normal diet to test the accuracy of the developed method. Copyright © 2005 John Wiley & Sons, Ltd. [source] Rapid determination of enzyme activities of recombinant human cytochromes P450, human liver microsomes and hepatocytesBIOPHARMACEUTICS AND DRUG DISPOSITION, Issue 9 2003Anima Ghosal Abstract Cytochrome P450 (CYP) substrates that yield fluorescent metabolites were used for rapid screening of drug metabolism activities of 13 recombinant human cytochromes P450, human liver microsomes and human hepatocytes. Reproducible results were obtained using a fluorescent plate reader (CytoFluor) more expediently than those generated using conventional HPLC methods. Typically, results for 96 samples were obtained with the plate reader in less than 10 min as opposed to 15,35 min/sample required by conventional HPLC. The fluorescent substrates used to measure CYP activities were as follows: 3-cyano-7-ethoxycoumarin (CEC) for CYP1A1, CYP1A2, CYP2C9 and CYP2C19; 7-ethoxyresorufin (7-ER) for CYP1A1, CYP1A2 and CYP1B1; 3-[2-(N,N -diethyl- N -methylammonium)ethyl]-7-methoxy-4-methylcoumarin (AMMC) for CYP2D6; dibenzylfluorescein (DBF) for CYP3A4, CYP3A5 and CYP2C8; 7-methoxy-4-trifluoromethylcoumarin (7-MFC) for CYP2E1, CYP2B6 and CYP2C18; and coumarin for CYP2A6. The chemical inhibition and correlation data indicated that the following substrates can be used as specific functional probes for individual cytochrome P450 present in human liver microsomes: coumarin for CYP2A6 (r=0.82), AMMC for CYP2D6 (r=0.83) and DBF for CYP3A4 (r=0.92). The fluorescent plate reader was found to be useful for the rapid assessment of CYP activities (positive control) in both intact cells and subcellular fractions. Copyright © 2003 John Wiley & Sons, Ltd. [source] A method for establishing the critical threshold for aeolian transport in the fieldEARTH SURFACE PROCESSES AND LANDFORMS, Issue 10 2004John E. Stout Abstract A basic feature of any wind-eroding surface is its threshold , the wind speed at which sediment transport is initiated. A new method was developed and tested that allows for the rapid determination of threshold under natural wind conditions in the ,eld. A mathematical expression that relates saltation activity and relative wind strength was reformulated so that threshold may be calculated from measurements of saltation activity and the mean and standard deviation of wind speed. To test the new method and determine its usefulness, a ,eld experiment was performed within a region of low-relief dunes on the Southern High Plains of West Texas. The experimental system consisted of a 2-m meteorological tower and a piezoelectric saltation sensor. It was found that during periods of active aeolian activity, threshold values could be calculated every 5 minutes. This new method allows for routine monitoring of surface threshold conditions in the ,eld. Example threshold calculations are presented and they demonstrate that the method works well. Published in 2004 by John Wiley & Sons, Ltd. [source] A Tubulin-Based Quantitative Assay for Taxol (Paclitaxel) with Enzyme Channeling SensingELECTROANALYSIS, Issue 8 2004Shin-ichiro Suye Abstract We report the development and characterization of a biosensor for sensitive and rapid determination of the anticancer agent Taxol (paclitaxel). The sensor is based on the interaction of Taxol with its receptor protein tubulin in conjunction with the separation-free immunosensor technique of enzyme channeling. The sensor system consisted of a three electrode electrochemical cell containing a graphite carbon electrode modified with glucose oxidase and tubulin as working electrode poised at +40,mV (vs. Ag/AgCl). Addition of Taxol, horseradish peroxidase labeled Taxol, glucose and potassium iodide to the cell generated a cathodic current response that was proportional to the concentration of Taxol in the range of 10 to 1,000,pM. [source] Analysis of aristolochic acids by CE-MS with carboxymethyl chitosan-coated capillaryELECTROPHORESIS, Issue 10 2009Xiaofang Fu Abstract A CE-MS method for rapid determination of aristolochic acid-I and aristolochic acid-II (AA-II) in traditional Chinese medicines and biological samples was described in the present paper. AA-I and AA-II can be baseline separated within 6,min by CE-MS with carboxymethyl-chitosan-coated capillary. CZE conditions including pH, concentration of buffer, applied voltage, and capillary temperature were systematically investigated, and the composition and flow rate of sheath liquid were also optimized for CE-MS. Furthermore, the CE-UV method without any additives in BGE solution was established and compared with the CE-MS method. The results showed that the two methods could achieve satisfactory separation efficiency, repeatability, and linearity, while the LOD was 0.6,,g/mL for CE-UV and 0.05,,g/mL for CE-MS. Compared with the CE-UV method, the sensitivity of CE-MS was significantly improved, in addition to the structure information provided by MS detection at the same time. As an application example, a spiked sample in human serum was analyzed by the CE-MS method, indicating that the new CE-MS method can be applied to analyze AAs in biological samples. [source] SPE and large-volume sample stacking in MEKC for determination of doxycycline in biological fluids: Comparison of direct injection to SPE-MEKCELECTROPHORESIS, Issue 21 2008Rade Injac Abstract A novel and simple method has been developed for the determination of doxycycline (DOX) in biological fluids. The method is based on SPE, large-volume sample stacking (LVSS) and MEKC with UV-DAD detection. Six SPE cartridges have been used in investigation for sample clean up and pre-concentration (Supelco® LC-8, LC-18, LC-SCX, and LC-WCX, as well as StrataŌ-X and X-C). DOX was determined on a 56,cm (effective length 50,cm)×50,,m id fused-silica capillary. The BGE was 20,mM borate buffer, pH 9.3, containing 80,mM SDS and 7.5%,v/v of methanol (30,s×50,mbar), and the temperature and voltage were 25°C and 30,kV, respectively. The analytical wavelength was set at 210,nm. Under optimized conditions it is possible to determine DOX in human serum, urine, semen, tears and saliva with recovery of 97.5% (RSD 2.5%). The method was shown to be sensitive (LOD is 1,,g/L) and precise (intra-day RSD 0.2 and 2.4%; inter-days 0.4 and 3.5% for migration time and peak area, respectively). Results for developed SPE-LVSS-MEKC were compared with LVSS-MEKC method with direct sample injection. The new LVSS-MEKC method is presented as a useful technique for rapid determination without extraction procedure of DOX in human urine and serum, using 80,mM of SDS, 10%,v/v of methanol and 40,mM borate buffer (pH 9.3; 30,s×50,mbar; 25°C; 30,kV; 350,nm), but not for the other biological fluids, according to lower sensitivity of the method and because of the sample composition. [source] A novel and sensitive test for rapid determination of water toxicityENVIRONMENTAL TOXICOLOGY, Issue 3 2002S. Ulitzur Abstract The performance of a novel, rapid, and sensitive test for detecting chemical toxicants in water is described in this article. The bioassay utilizes a highly sensitive variant of the luminescent bacterium Photobacterium leiognathi that allows the detection in water at levels below milligrams per liter of diverse groups of toxicants, including heavy metals, pesticides, PCBs, polycyclic aromatic hydrocarbons, and fuel traces. For most toxic agents reported in this study, the new assay was markedly more sensitive than the MicrotoxŌVibrio fischeri assay according to the bacterial bioluminescence toxicity data reported in the literature. Additional features of the new bioassay include the ability to discriminate between cationic heavy metals and organic toxicants and the option of being run at ambient temperatures (18°C,27°C), thereby enabling on-site testing with low-cost luminometers. In addition, the stability of the freeze-dried bacterial reagent preparation at ambient temperatures precludes the need for refrigeration or freezing during shipment, which contributes to further reducing overall operational costs. © 2002 Wiley Periodicals, Inc. Environ Toxicol 17: 291,296, 2002; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/tox.10060 [source] Efficient modal analysis of systems with local stiffness uncertaintiesINTERNATIONAL JOURNAL FOR NUMERICAL METHODS IN ENGINEERING, Issue 6-7 2009S. F. Wojtkiewicz Abstract The characterization of the uncertainty in modal quantities of an uncertain linear structural system is essential to the rapid determination of its response to arbitrary loadings. Although the size of many computational structural models used is extremely large, i.e. thousands of equations, the uncertainty to be analyzed is oftentimes localized to very small regions of the model. This paper addresses the development of an efficient, computational methodology for the modal analysis of linear structural systems with local stiffness uncertainties. The newly developed methodology utilizes an enriched basis that consists of the sub-spectrum of a nominal structural system augmented with additional basis vectors generated from a knowledge of the structure of the stiffness uncertainty. In addition, methods for determining bounds on the approximate modal frequencies and mode shapes are discussed. Numerical results demonstrate that the algorithm produces highly accurate results with greatly reduced computational effort. Copyright © 2009 John Wiley & Sons, Ltd. [source] Application of solid phase microextraction for the determination of soil fumigants in water and soil samplesJOURNAL OF SEPARATION SCIENCE, JSS, Issue 1 2005Sonia Fuster Abstract The potential of solid phase microextraction (SPME) for the determination of the soil fumigants 1,3-dichloropropene (1,3-DCP) and methyl isothiocyanate (MITC) in environmental samples such as soil and water samples has been investigated. Direct immersion SPME followed by GC/ECD/NPD analysis allowed the rapid determination of the two fumigants in water samples, with very little sample manipulation, giving an LOD of 0.5 ,g L,1. Precision, calculated as relative standard deviation (RSD) for six replicates at three concentration levels, was found to be lower than 20% at the concentration levels tested. For the analysis of soil samples, headspace (HS)-SPME combined with GC/ECD/NPD analysis has been applied. Quantification using matrix-matched calibration curves allowed determination of both analytes (MITC and 1-3-DCP) with a LOD of 0.1 ,g kg,1 (RSD <10%) for the two concentration levels assayed (0.02 and 0.2 mg kg,1). The HS-SPME procedure developed in this paper was applied to soil samples from experimental green house plots treated with metham-Na, a soil disinfestation agent that decomposes in soil to MITC. The absence of sample manipulation as well as the low solvent consumption in SPME methodology are among the main advantages of this analytical approach. [source] Evaluation of lignans and free and linked hydroxy-tyrosol and tyrosol in extra virgin olive oil after hydrolysis processesJOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 5 2006Nadia Mulinacci Abstract We describe chemical hydrolytic procedures to evaluate the total amount of tyrosol and hydroxy-tyrosol free and/or linked to secoiridoidic molecules (acid hydrolysis). At the same time a rapid determination of the lignans in complex minor polar compound (MPC) extracts is proposed (alkaline hydrolysis). High-performance liquid chromatography/diode array detection (HPLC/DAD) and HPLC/MS were applied as reference methods to evaluate the quantitative results from the hydrolysis experiments. The optimized acid hydrolysis procedures were first applied to an oleuropein standard and then to MPC fractions extracted from several commercial extra virgin olive oils. The results confirm the applicability of the method, consisting in the acid hydrolysis of complex mixtures of secoiridoidic derivatives, to determine the antioxidant potential in terms of MPC. These data can contribute to forecasting the potential ageing resistance of an extra virgin olive oil in terms of antioxidant potency. Finally, alkaline hydrolysis allows confirmation and easy determination of the amount of lignans, especially in those MPC fractions which are particularly complex. Copyright © 2006 Society of Chemical Industry [source] An enzyme-based method for the rapid determination of sucrose, glucose and fructose in sugar beet roots and the effects of impact damage and postharvest storage in clampsJOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 1 2002T Spackman, Victoria M Abstract A high-throughput enzyme-coupled assay is described for the determination of sucrose, glucose and fructose in sugar beet roots. This method is sensitive, rapid and inexpensive and has been used to highlight the increases in sucrose loss following root stresses such as freezing or aggressive harvesting. Sugar beet roots lose 12.5% of their sucrose following an episode of impact damage greater than 2,J, rising to 19.7% loss after 8,J, with concurrent increases in glucose and fructose. Increases in glucose and fructose are particularly pronounced following a period of clamp storage (up to 2.3 and 3.3,µg,mg,1 fresh weight, respectively). © 2001 Society of Chemical Industry [source] Analytical strategies for identifying drug metabolitesMASS SPECTROMETRY REVIEWS, Issue 3 2007Chandra Prakash Abstract With the dramatic increase in the number of new chemical entities (NCEs) arising from combinatorial chemistry and modern high-throughput bioassays, novel bioanalytical techniques are required for the rapid determination of the metabolic stability and metabolites of these NCEs. Knowledge of the metabolic site(s) of the NCEs in early drug discovery is essential for selecting compounds with favorable pharmacokinetic credentials and aiding medicinal chemists in modifying metabolic "soft spots". In development, elucidation of biotransformation pathways of a drug candidate by identifying its circulatory and excretory metabolites is vitally important to understand its physiological effects. Mass spectrometry (MS) and nuclear magnetic resonance (NMR) have played an invaluable role in the structural characterization and quantification of drug metabolites. Indeed, liquid chromatography (LC) coupled with atmospheric pressure ionization (API) MS has now become the most powerful tool for the rapid detection, structure elucidation, and quantification of drug-derived material within various biological fluids. Often, however, MS alone is insufficient to identify the exact position of oxidation, to differentiate isomers, or to provide the precise structure of unusual and/or unstable metabolites. In addition, an excess of endogenous material in biological samples often suppress the ionization of drug-related material complicating metabolite identification by MS. In these cases, multiple analytical and wet chemistry techniques, such as LC-NMR, enzymatic hydrolysis, chemical derivatization, and hydrogen/deuterium-exchange (H/D-exchange) combined with MS are used to characterize the novel and isomeric metabolites of drug candidates. This review describes sample preparation and introduction strategies to minimize ion suppression by biological matrices for metabolite identification studies, the application of various LC-tandem MS (LC-MS/MS) techniques for the rapid quantification and identification of drug metabolites, and future trends in this field. © 2007 Wiley Periodicals, Inc., Mass Spec Rev [source] In silico mining of EST databases for novel pre-implantation embryo-specific zinc finger protein genes,MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 3 2001Kong-Bung Choo Abstract Progress in the understanding of early mammalian embryo development has been severely hampered by scarcity of study materials. To circumvent such a constraint, we have developed a strategy that involves a combination of in silico mining of new genes from expressed sequence tags (EST) databases and rapid determination of expression profiles of the dbEST-derived genes using a PCR-based assay and a panel of cDNA libraries derived from different developmental stages and somatic tissues. We demonstrate that in a random sample of 49 independent dbEST-derived zinc finger protein genes mined from a mouse embryonic 2-cell cDNA library, more than three-quarters of these genes are novel. Examination of characteristics of the human orthologues derived from these mouse genes reveals that many of them are associated with human malignancies. Expression studies have further led to the identification of three novel genes that are exclusively expressed in mouse embryos before or up to the 8-cell stage. Two of the genes, designated 2czf45 and 2czf48 (2czf for 2 -cell zinc finger), are zinc finger protein genes coding for a RBCC protein with a RFP domain and a protein with three C2H2 fingers, respectively. The third gene, designated 2cpoz56, codes for a protein with a POZ domain that is often associated with zinc finger proteins. These three genes are candidate genes for regulatory or other functions in early embryogenesis. The strategy described in this report should generally be applicable to rapid and large-scale mining of other classes of rare genes involved in other biological and pathological processes. Mol. Reprod. Dev. 59:249,255, 2001. © 2001 Wiley-Liss, Inc. [source] Validation of a real-time monitoring method for aniline in freshwater by high-performance liquid chromatography on porous graphitic carbon/electrospray ionization tandem mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 14 2004Raphaėl Delépée Aniline is an anthropogenic organic compound widely used in polymer, rubber, pharmaceutical and dye industries but also used in biodegradability assays of chemical compounds as a positive biodegradation standard. By the two approaches, the rapid determination of aniline is necessary because of the high toxicity of aniline on hemoglobin. A high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) method for the determination of aniline in water is described here. This method, using benzylamine as internal standard, was validated. No time-consuming sample preparation was needed. A rapid separation (7,min between two chromatographic runs) of aniline and benzylamine was performed on a Hypercarb porous graphitic carbon column using a gradient of methanol and 100,mM formic acid. The obtained limits of detection and quantification were 10 and 1,ng/mL, respectively. The response for aniline was quadratic. We show that this problem could be circumvented by showing that the [calculated concentration,=,f(introduced concentration)] function was linear. The linearity range was 10,1000,ng/mL. An example of an application consisting of an aniline 42-day degradation kinetic in water was demonstrated. Copyright © 2004 John Wiley & Sons, Ltd. [source] Validation of the FACSCount AF System for Determination of Sperm Concentration in Boar SemenREPRODUCTION IN DOMESTIC ANIMALS, Issue 6 2002C Hansen Contents A flow cytometric method has been developed for rapid determination of sperm concentration in semen from various mammalian species., All cells containing DNA are stained with SYBR-14 or propidium iodide (PI) and sperm concentration is determined in relation to an internal standard of fluorescent microspheres (beads). Satisfactory staining can be achieved within 2,3 min and the following flow cytometric analysis on the FACSCount AF System rapidly provides the user with a precise and accurate assessment of the sperm concentration. In this study, the FACSCount AF System and Sperm Counting Reagent (BD Biosciences) was compared with microscopic counting using a Bürker,Türk haemocytometer. In addition, sperm concentration was determined using the Corning 254 spectrophotometer which is used routinely by Danish artificial insemination stations for boars. The results show that the agreement between flow cytometry and microscopic counting is very high. The slope for the regression line was 1.12 (SE = 0.03) with an estimated intercept with the Y-axis of 22 × 106sperm/ml (SE = 10 × 106 sperm/ml) and an estimated error of the model of 10 × 106 sperm/ml. For the spectrophotometer, the slope of the regression line was 1.09 (SE = 0.07) with an estimated intercept of 137 × 106 sperm/ml (SE = 25 × 106 sperm/ml). The average error made by the spectrophotometer was 55 × 106 sperm/ml. In addition, the results obtained using flow cytometry was highly repeatable (CV = 2.7%) in comparison with the spectrophotometric method (CV = 6.3%). These results indicate that the FACSCount AF System is a valuable tool for precise and accurate assessment of sperm concentration in boar semen and that use of this system may lead to production of more uniform insemination doses containing a specific number of sperm per dose. [source] Simple and rapid determination of 1-deoxynojirimycin in mulberry leavesBIOFACTORS, Issue 1-4 2004Toshiyuki Kimura Abstract A simple and rapid method for determining 1-deoxynojirimycin (DNJ), a potent glucosidase inihibitor present in mulberry leaves (Morus alba and Morus bombysis), by high performance liquid chromatography coupled to an evaporative light scattering detector (ELSD) has been developed. DNJ was separated from extract of mulberry leaves on TSK gel Amide-80 column, which is a representative column for hydrophilic interaction chromatography. During post column detection, DNJ was detected by ELSD and concurrently identified by mass spectrometry. The detection limit was 100 ng. This method is sufficiently sensitive for determining DNJ in mulberry leaves and other related products. [source] High-performance liquid chromatographic method for simple and rapid determination of linezolid in human plasmaBIOMEDICAL CHROMATOGRAPHY, Issue 8 2006Lauren M. Boak Abstract A simple high-performance liquid chromatographic (HPLC) method was developed and validated for rapid quantification of linezolid in human plasma. Protein precipitation using a mixture of 5% trichloroacetic acid and methanol (3:1, v/v) provided a straightforward method of sample preparation and the internal standard eperezolid was employed. A concentration range from 0.20 to 40.0 mg/L was utilized to construct calibration curves, and analysis of low- (0.40 mg/L), medium- (7.50 mg/L) and high-quality (25.0 mg/L) control samples revealed excellent reproducibility (,3.88%) and accuracy (,4.20%). The recovery of both linezolid and eperezolid was approximately 100%. No interference was observed at the retention times of linezolid and eperezolid from blank plasma or eight commonly used antibiotics. Tests confirmed the stability of linezolid in plasma during three freeze,thaw cycles, on the bench during 24 h and during long-term storage of frozen plasma for up to 12 weeks; in extracts it was stable in the HPLC autosampler over 12 h. Overall, this assay offers a highly simplistic approach to quantifying linezolid in plasma, and would be well suited to clinical pharmacokinetic, pharmacodynamic and toxicodynamic analyses. Copyright © 2005 John Wiley & Sons, Ltd. [source] An ESI-MS/MS Method for Screening of Small-Molecule Mixtures against Glycogen Synthase Kinase-3, (GSK-3,)CHEMBIOCHEM, Issue 7 2008Ivan Partserniak Abstract Glycogen synthase kinase-3, (GSK-3,) is involved in the hyperphosphorylation of previously phosphorylated (primed) substrates, and is currently assayed using an approach based on the incorporation of ,- 32P-radiolabelled isotopes into substrate peptides. The requirement to detect hyperphosphorylation of a primed substrate poses a particular challenge for development of a high-throughput screening assay, as many current kinase assays are designed to produce a signal in the presence of any phosphorylation site, and thus are only suitable for ,-unphosphorylated substrates. Herein, we have developed an electrospray-ionization tandem mass spectrometry (ESI-MS/MS) assay to allow for direct detection of a hyperphosphorylated product which is formed in a solution reaction involving a primed peptide substrate (GSM peptide) and GSK-3,. Optimum reaction conditions (level of Mg2+, buffer type, ionic strength, pH, enzyme concentration, and reaction time) were established to both maintain the activity of GSK-3, and allow for substrate and product quantification through ESI/MS/MS. We show that the MS-based assay allows for rapid determination of GSK-3, activity from reaction volumes of ,40 ,L and that it can be used to assess IC50 values and the site of action of known inhibitors. It also can be used for automated screening of small-molecules mixtures to identify inhibitors of GSK-3,. [source] Emerging methods for the rapid determination of enantiomeric excessCHIRALITY, Issue 7 2002M.G. Finn Abstract Methods for enantiomeric excess determination using a variety of spectroscopic techniques are summarized. Particular attention is paid to techniques that have promise for application to problems of combinatorial catalyst discovery but have not yet been so employed. Chirality 14:534,540, 2002. © 2002 Wiley-Liss, Inc. [source] | |||||||||||||||||||||||||||||||