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Analysis Time (analysis + time)
Kinds of Analysis Time Selected AbstractsComparison of immunoradiometric assays for determination of thyroglobulin: a validation studyJOURNAL OF CLINICAL LABORATORY ANALYSIS, Issue 3 2007L.A. Tortajada-Genaro Abstract In this study we compared and validated commercial immunoradiometric assays (IRMA) to determine thyroglobulin (Tg) levels in serum. From a set of 440 samples, 68 were selected to calculate the validation parameters and the clinical performance of the assays. The commercial kits evaluated were the Tg-CTK (DiaSorin), IRMAZenco Tg (ZenTech), and SELco-Tg (Medipan). We found that 21% of the collected samples were in the critical range of concentration. Detection limits were calculated as being below 3,µg/L. Intra- and inter-reproducibility were lower than 3.1% and 9.2%, respectively. Dilution and recovery studies provided quantitative determinations. Correlation regression coefficients from the results of the methods were obtained. The determined concentrations were compared with the clinical evidence of disease. Variation in the 125-iodine-labeled antibody concentration and control charts showed the robustness of the methods. Analysis time and the simplicity of the methods were also evaluated. Reliable Tg determination is important for monitoring patients with differentiated thyroid cancer (DTC), controlling other thyroid diseases, and assessing the quality of imaging techniques. A strategy for verification and comparison based on analytical parameters and clinical performance is proposed. J. Clin. Lab. Anal. 21:147,153, 2007. © 2007 Wiley-Liss, Inc. [source] Fast Liquid Chromatography for High-Throughput Screening of PolymersMACROMOLECULAR RAPID COMMUNICATIONS, Issue 1 2003Harald Pasch Abstract Liquid chromatography of polymers is traditionally a slow technique with analysis times of typically 30 min per sample. For the application of liquid chromatographic techniques to combinatorial materials research the analysis time per sample must be reduced considerably. Analysis time in SEC can be reduced to about 2 min per sample when high-throughput columns are used. For HPLC small columns with improved separation efficiencies can be used. As compared to conventional technology, time savings of more than 80% are achieved. Chromatogram from conventional SEC column compared to high-speed SEC column tested on an identical instrument with polystyrene standards in THF. [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] Electrochemical Detection of Arsenic(III) in the Presence of Dissolved Organic Matter (DOM) by Adsorptive Square-Wave Cathodic Stripping Voltammetry (Ad-SWCSV)ELECTROANALYSIS, Issue 4 2008Tsanangurayi Tongesayi Abstract This study has demonstrated that As(III) can be electrochemically detected and quantified in the presence of fulvic acid (FA) and dissolved organic matter (DOM). This eliminates the need to remove DOM prior to measurement of As(III) in environmental samples. Apart from reducing analysis time and the cost of the analysis, this could be potentially useful for the development of electrochemical methods for the detection and measurement of As(III) onsite. Both synthetic samples in which FA was added and a real sample with 22.16,mg/L total organic carbon (TOC) were analyzed. [source] Evaluation of CE methods for global metabolic profiling of urineELECTROPHORESIS, Issue 14 2010Rawi Ramautar Abstract In this study, the usefulness of noncovalently coated capillaries with layers of charged polymers is investigated to obtain global electrophoretic profiles of urinary metabolites covering a broad range of different compound classes in a highly repeatable way. Capillaries were coated with a bilayer of polybrene (PB) and poly(vinyl sulfonate) (PVS), or with a triple layer of PB, dextran sulfate (DS) and PB. The bilayer and triple layer coatings were evaluated at acidic (pH 2.0) and alkaline (pH 9.0) separation conditions, thereby providing separation conditions for basic and acidic compounds. A representative metabolite mixture and spiked urine samples were used for the evaluation of the four CE methods. Migration time repeatability (RSD<2%) and plate numbers (N, 100,000,400,000) were similar for the test compounds in all CE methods, except for some multivalent ions that may exhibit adsorption to oppositely charged coatings. The analysis of cationic compounds with the PB-DS-PB CE method at low pH (i.e. after the EOF time) provided a larger separation window and number of separated peaks in urine compared to the analysis with the PB-PVS CE method at low pH (i.e. before the EOF time). Approximately, 600 molecular features were detected in rat urine by the PB-DS-PB CE-MS method whereas about 300 features were found with the PB-PVS CE-MS method. This difference can be attributed to reduced comigration of compounds with the PB-DS-PB CE-MS method and a related decrease of ion suppression. With regard to the analysis of anionic compounds by CE-MS, in general analyte responses were significantly lower than that for cationic compounds, most probably due to less efficient ionization and to ion suppression effects caused by the background electrolyte. Hence, further optimization is required for the sensitive CE-MS analysis of anionic compounds in body fluids. It is concluded that the selection of a CE method for profiling of cationic metabolites in urine depends on the purpose of the study. For high-throughput analyses, the PB-PVS CE-MS method is favored whereas the PB-DS-PB CE-MS method provides a more information-rich metabolic profile, but at the cost of prolonged analysis time. [source] Microstructure of microemulsion in MEEKCELECTROPHORESIS, Issue 4 2010Yuhua Cao Abstract The influences of the composition of microemulsion on the microstructure including dimensions and , potentials of microdroplets were measured in details. The average dynamic dimension of microdroplets was measured by dynamic laser light scattering, and , potential was determined to characterize average surface charge density of microdroplets. The experiment results showed that increase of the amount of surfactant resulted in decrease of microdroplet size but almost invariant , potential, which would enlarge migration time of the microdroplet in MEEKC. With increment of cosurfactant concentration, the microdroplet size had an increasing trend, whereas the , potential decreased. Thus, observed migration velocity of microdroplets increased, which made the separation window in MEEKC shortened. Neither dimension nor , potential of microdroplets changed by varying both the type and the amount of the oil phase. Adding organic solvent as modifier to microemulsion did not change the microdroplet size, but lowered , potential. The migration time of microdroplet still became larger, since EOF slowed down owing to organic solvent in capillary. So, besides increment of surfactant concentration, organic additive could also enlarge the separation window. Increase of cosurfactant concentration was beneficial for separation efficiency thanks to the looser structure of swollen microdroplet, and the peak sharpening might compensate for the resolution and peak capacity owing to a narrow separation window. Except the oil phase, tuning the composition of microemulsion would change the microstructure, eventually could be exploited to optimize the resolution and save analysis time in MEEKC. [source] Online CIEF-ESI-MS in glycerol,water media with a view to hydrophobic protein applicationsELECTROPHORESIS, Issue 23 2009Meriem Mokaddem Abstract A new online coupling of CIEF with ESI-MS has been developed in glycerol,water media. This improved protocol provides: (i) the electric continuity during the whole analysis by a discontinuous filling of the capillary with 60:40 (cm/cm) catholyte/proteins,ampholyte mixture; (ii) the use of an anticonvective medium, i.e. 30:70 glycerol/water, v/v, compatible with MS detection and as an aid to hydrophobic protein solubilization and (iii) the use of unmodified bare fused-silica capillaries, as the glycerol/water medium strongly reduces EOF. Focusing was performed in positive polarity and cathodic mobilization was achieved by both voltage and pressure application. The setup was optimized with respect to analysis time, sensitivity and precision on pI determination. The optimized anolyte and catholyte were composed of 50,mM formic acid/1,mM glutamic acid (pH 2.35) and 100,mM NH3/1,mM lysine (pH 10.6), respectively. The effects of ampholyte concentration, focusing time and ESI parameters were presented for model proteins and discussed. This new integrated protocol should be an easy and effective additional tool in the field of proteome analysis, providing a means for the characterization of a large number of hydrophilic and hydrophobic proteins. [source] Use of coated capillaries for the electrophoretic separation of stereoisomers of a growth hormone secretagogueELECTROPHORESIS, Issue 21 2009Reine Nehmé Abstract The diastereoisomeric separation of peptidomimetics of hexarelin, a strong growth hormone secretagogue, in CE has been studied. Highly sulfated-,-CD was found to be an appropriate selector for the separation of the stereoisomers. However, non-repeatable analyses were obtained on bare fused silica capillary due to the progressive adsorption of the analytes on the capillary wall. Two types of polyelectrolyte coating agents were tested to prevent this phenomenon. Coating with neutral polyethylene oxide was found to be efficient but resulted in a very long analysis time (about 40,min). Coating with cationic poly(diallyldimethylammonium) chloride was found both to prevent analyte adsorption, reduce analysis time and alter separation selectivity. EOF measurement revealed that the highly sulfated-,-CDs were strongly adsorbed on the poly(diallyldimethylammonium) chloride coating surface yielding a stable strong cathodic EOF, which considerably reduced analysis time (about 12,min). Very good repeatability of analysis was obtained (RSDmigration time<1%). [source] Organic solvents in CEELECTROPHORESIS, Issue S1 2009Ernst Kenndler Abstract In this contribution some fundamental aspects are discussed serving for a critical reflection and elucidation of the role of organic solvents in CE. The implications of the solvent on the parameters governing peak resolution are discussed based on the concepts describing migration and zone broadening in capillary zone electrophoresis. This discussion includes the solvent-dependent influence of the ionic strength on the mobility. The role of the solvent on the plate number in case of the inevitable diffusional peak dispersion is outlined, and its effect on other peak broadening contributions is briefly examined. This paper also deals with the problems of conductance, applicable voltage and analysis time upon application of organic solvents, and tries to clarify some misunderstandings common in the literature. [source] Enhanced pH-mediated stacking of anions for CE incorporating a dynamic pH junctionELECTROPHORESIS, Issue 20 2007Stacy D. Arnett Abstract A technique has been developed to enhance analyte focusing for CE for the analysis of physiological samples. High-ionic-strength samples are titrated to low-ionic-strength on-line using pH-mediated sample stacking in conjunction with a dynamic pH junction. This method concentrates analytes by reducing their electrophoretic mobility during field-amplification. Parameters responsible for enhanced focusing were investigated, and an enhanced pH-mediated stacking method was optimized for anionic nucleosides. The process results in ultra-narrow peak widths, for example, 0.28,s for thymidine with a 10,min analysis time. Peak width and resolution with the enhanced stacking method were also compared to normal base stacking and electrokinetic injection. With this technique, mass-loading capacity can be increased without degradation in peak shape and resolution is dramatically improved. [source] On-line sample stacking and short-end injection CE for the determination of fluoxetine and norfluoxetine in plasma: Method development and validation using experimental designsELECTROPHORESIS, Issue 18 2007Chia-Chia Lu Abstract A short-end injection CE method combining field-amplified sample stacking (FASS) is presented for the analysis of fluoxetine (FL) and norfluoxetine in plasma. In this study, FASS enhanced the sensitivity about 1100-fold, while short-end injection reduced the analysis time to less than 4,min. Parameters involved in the separations were investigated using a central composite design (CCD) and response surface methodology to optimize the separation conditions in a total of only 32 runs. Samples injected into the capillary for 99.9,s at a voltage of ,5,kV were stacked in a water plug (0.5,psi, 9,s). Baseline resolution of FL and its major metabolite was achieved using a BGE formulation consisting of phosphate,triethanolamine at low pH, and a separation voltage of ,10,kV. Five percent methanol was added as organic modifier to enhance selectivity and resolution. The linear range was between 10 and 500,ng/mL (r >0.9946), covering the expected plasma therapeutic ranges. The LOD in plasma were 4,ng/mL (S/N,=,3), a value comparable to that obtained using LC-MS, showing the success of the on-line stacking technique. Our method was also successfully validated in quantification and pharmacokinetic studies with three volunteer plasma samples and could be applied to pharmacogenetic studies. [source] Development 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] Capillary electrophoresis-mass spectrometry characterisation of secondary metabolites from the antihyperglycaemic plant Genista teneraELECTROPHORESIS, Issue 11 2006Emma L. Edwards Abstract Genista tenera is endemic to the Portuguese island of Madeira, where an infusion of the aerial parts of the plant is used in folk medicine as an antidiabetic agent. Consequently the medicinal properties of the secondary metabolites of this plant have been the subject of an ongoing study. A recently reported LC-MS method using a 100,min separation allowed identification of five flavonoid components in an extract of the aerial parts of this plant. In order to obtain additional information on the range and complexity of the plant's secondary metabolite components a CE-MS method has been developed and applied for the analysis of an extract of G.,tenera. Twenty-six different components are distinguished in an analysis time of only 10,min. Results demonstrate that CE-MS/MS rapidly generates data complementary to those obtainable by LC-MS/MS and is particularly suited to the analysis of plant metabolites where concentration is not limiting. [source] Chiral capillary electrophoresis applied to the determination of phenylglycidol enantiomers obtained from cinnamyl alcohol by asymmetric epoxidation using new titanium(IV) alkoxide compounds as catalystsELECTROPHORESIS, Issue 16 2004Sonia Morante-Zarcero Abstract A capillary electrophoresis method for the simultaneous determination of phenylglycidol enantiomers in the presence of an excess of cinnamyl alcohol was developed. The effects of the nature, pH and concentration of the buffer, the nature and concentration of chiral selector, the addition of methanol or acetonitrile, and the capillary temperature on the chiral resolution of phenylglycidol enantiomers were studied. Separations were achieved using 20 mM succinylated ,-cyclodextrin dissolved in a 10 mM borate buffer (pH 10.0). Chiral resolution for the phenylglycidol enantiomers in the optimized electrophoretic conditions was higher than 2.0 with an analysis time less than 7 min. The method developed was validated in terms of selectivity, linearity, precision (instrumental repeatability, method repeatability, intermediate precision), the limits of detection and quantitation, and accuracy. Limits of detection of 6.5 mg/L and 8.3 mg/L for (2S,3S)-(,)-3-phenylglycidol ((S,S)-PG) and (2R,3R)-(+)-3-phenylglycidol ((R,R)-PG), respectively, were obtained. The method was applied to study the asymmetric epoxidation of cinnamyl alcohol with titanium(IV) alkoxide compounds as catalysts in order to evaluate their catalytic activity and stereoselectivity of the epoxidation processes. [source] Capillary electrochromatography with monolithic silica column:,I.ELECTROPHORESIS, Issue 3 2003Preparation of silica monoliths having surface-bound octadecyl moieties, applications to the separation of neutral, charged species, their chromatographic characterization Abstract Monolithic silica columns with surface-bound octadecyl (C18) moieties have been prepared by a sol-gel process in 100 ,m ID fused-silica capillaries for reversed-phase capillary electrochromatography of neutral and charged species. The reaction conditions for the preparation of the C18-silica monoliths were optimized for maximum surface coverage with octadecyl moieties in order to maximize retention and selectivity toward neutral and charged solutes with a sufficiently strong electroosmotic flow (>,2 mm/s) to yield rapid analysis time. Furthermore, the effect of the pore-tailoring process on the silica monoliths was performed over a wide range of treatment time with 0.010 M ammonium hydroxide solution in order to determine the optimum time and conditions that yield mesopores of narrow pore size distribution that result in high separation efficiency. Under optimum column fabrication conditions and optimum mobile phase composition and flow velocity, the average separation efficiency reached 160,000 plates/m, a value comparable to that obtained on columns packed with 3 ,m C18-silica particles with the advantages of high permeability and virtually no bubble formation. The optimized monolithic C18-silica columns were evaluated for their retention properties toward neutral and charged analytes over a wide range of mobile phase compositions. A series of dimensionless retention parameters were evaluated and correlated to solute polarity and electromigration property. A dimensionless mobility modulus was introduced to describe charged solute migration and interaction behavior with the monolithic C18-silica in a counterflow regime during capillary electrochromatography (CEC )separations. The mobility moduli correlated well with the solute hydrophobic character and its charge-to-mass ratio. [source] Opportunities for ultra-high resolution analysis of essential oils using comprehensive two-dimensional gas chromatography: a reviewFLAVOUR AND FRAGRANCE JOURNAL, Issue 3 2003Robert Shellie Abstract In comprehensive 2D gas chromatography, the entire sample is simultaneously subjected to analysis on two capillary columns. By using a suitable modulation interface between the primary and secondary columns, hundreds of fast, second-dimension chromatograms are produced. The data from these chromatograms are treated such that a 3D surface plot or a 2D contour plot of the components' individual retention times, on each column, as well as peak responses, are represented. In a properly tuned comprehensive 2D chromatogram, the individual sample components are spread throughout a 2D separation space, providing a signi,cant increase in the probability of resolving a greater number of sample components without increasing the analysis time. Comprehensive 2D,GC has proved useful for high-resolution conventional essential oil analysis as well as high-resolution enantioselective essential oil analysis. Combining comprehensive 2D,GC with either a quadrupole or time-of-,ight mass spectrometer gives a powerful 3D analysis technique, which is extremely effective for complex sample analysis. The present status and opportunities arising from these ultra-high resolution approaches are discussed herein. Copyright © 2003 John Wiley & Sons, Ltd. [source] Efficient implicit finite element analysis of sheet forming processesINTERNATIONAL JOURNAL FOR NUMERICAL METHODS IN ENGINEERING, Issue 8 2003A. H. van den Boogaard Abstract The computation time for implicit finite element analyses tends to increase disproportionally with increasing problem size. This is due to the repeated solution of linear sets of equations, if direct solvers are used. By using iterative linear equation solvers the total analysis time can be reduced for large systems. For plate or shell element models, however, the condition of the matrix is so ill that iterative solvers do not reach the huge time-savings that are realized with solid elements. By introducing inertial effects into the implicit finite element code the condition number can be improved and iterative solvers perform much better. An additional advantage is that the inertial effects stabilize the Newton,Raphson iterations. This also applies to quasi-static processes, for which the inertial effects finally do not affect the results. The presented method can readily be implemented in existing implicit finite element codes. Industrial size deep drawing simulations are executed to investigate the performance of the recommended strategy. It is concluded that the computation time is decreased by a factor of 5 to 10. Copyright © 2003 John Wiley & Sons, Ltd. [source] Semen quality in fertile US men in relation to geographical area and pesticide exposureINTERNATIONAL JOURNAL OF ANDROLOGY, Issue 1 2006Shanna H. Swan Summary We conducted the first US study to compare semen quality among study centres using standardized methods and strict quality control. We present data on semen quality in partners of 493 pregnant women recruited through prenatal clinics in four US cities during 1999,2001. Sperm concentration, semen volume and motility were determined at the centres and morphology was assessed at a central laboratory. While between-centre differences in sperm morphology and sample volume were small, sperm concentration and motility were significantly reduced in Columbia, MO (MO) relative to men in New York, NY, Minneapolis, MN and Los Angeles, CA; total number of motile sperm was 113 × 106 in MO and 162, 201 and 196 × 106 in CA, MN and NY respectively. Differences among centres remained significant in multivariate models that controlled for abstinence time, semen analysis time, age, race, smoking, history of sexually transmitted disease and recent fever (all p -values <0.01). We hypothesized that poorer sperm concentration and motility in MO men relative to other centres might be related to agricultural pesticides that are commonly used in the mid-west. We investigated this hypothesis by conducting a nested case,control study within the MO cohort. We selected 25 men in this cohort for whom all semen parameters (concentration, % normal morphology and % motile) were low as cases and an equal number of men for whom all semen parameters were within normal limits as controls. We measured metabolites of eight non-persistent, current-use pesticides in urine samples the men had provided at the time of semen collection. Pesticide metabolite levels were elevated in cases compared with controls for the herbicides alachlor and atrazine, and for the insecticide diazinon (2-isopropoxy-4-methyl-pyrimidinol) (p -values for Wilcoxon rank test = 0.0007, 0.012, and 0.0004 for alachlor, atrazine and diazinon respectively). Men with higher levels of alachlor or diazinon were significantly more likely to be cases than men with low levels [odds ratios (OR) = 30.0, 16.7 for alachlor and diazinon respectively], as were men with atrazine over the limit of detection (OR = 11.3). These associations between current-use pesticides and reduced semen quality suggest that agricultural chemicals may have contributed to the reduced semen quality seen in fertile men from mid-Missouri. [source] Generalization of multivariate optical computations as a method for improving the speed and precision of spectroscopic analysesJOURNAL OF CHEMOMETRICS, Issue 6 2008Marc K. Boysworth Abstract Multivariate optical computations (MOCs) offer improved analytical precision and increased speed of analysis via synchronous data collection and numerical computation with scanning spectroscopic systems. The improved precision originates in the redistribution of integration time from spurious channels to informative channels in an optimal manner for increasing the signal-to-noise ratio with multivariate analysis under the constraint of constant total analysis time. In this work, MOCs perform the multiplication and addition steps of spectral processing by adjusting the integration parameters of the optical detector or adjusting the scanning profile of the tunable optical filter. Improvement in the precision of analysis is achieved via the implicit optimization of the analytically useful signal-to-noise ratio. The speed improvements are realized through simpler data post-processing, which reduces the computation time required after data collection. Alternatively, the analysis time may be significantly truncated while still seeing an improvement in the precision of analysis, relative to competing methods. Surface plasmon resonance (SPR) spectroscopic sensors and visible reflectance spectroscopic imaging were used as test beds for assessing the performance of MOCs. MOCs were shown to reduce the standard deviation of prediction by 15% compared to digital data collection and analysis with the SPR and up to 45% for the imaging applications. Similarly, a 30% decrease in the total analysis time was realized while still seeing precision improvements. Copyright © 2008 John Wiley & Sons, Ltd. [source] Development of a single-tube PCR-pyrosequencing method for the simultaneous and rapid detection of four variant alleles of CYP2C9 gene polymorphismJOURNAL OF CLINICAL PHARMACY & THERAPEUTICS, Issue 2 2008Y. Okada MS Summary Background and Objective:, CYP2C9 is a polymorphic enzyme that has been reported to metabolize several clinically useful drugs such as warfarin, phenytoin and non-steroidal anti-inflammatory drugs. We designed a rapid single-tube multiplex assay to detect four variant alleles of the CYP2C9 in a single polymerase chain reaction (PCR) and a single pyrosequencing reaction. Methods:, A multiplex PCR was designed to amplify two fragments simultaneously, one containing 430C>T (CYP2C9*2) polymorphism and other containing 1075A>C (CYP2C9*3), 1076T>C (CYP2C9*4) and 1080C>G (CYP2C9*5) polymorphisms. Results:, Four variants of the CYP2C9 gene could be simultaneously detected using only two varieties of pyrosequencing primers in a single-tube. The success rate for the four SNPs (*2, *3,*4 and *5) was high. Genotypes obtained by the multiplex reaction were 100% concordant with genotypes obtained using direct DNA sequencing (n = 96). The analysis time was halved, compared with existing simplex pyrosequencing. The system allowed high-throughput analysis of over 384 samples per hour. Discussion:, Our method reduces running cost and halves analysis time, compared to simplex pyrosequencing. Another advantage of this method is that it analyses and determines multiple bases around the polymorphic site thereby reducing the possibility of scoring a truncated PCR product. [source] Methodology Optimization for Quantification of Total Phenolics and Individual Phenolic Acids in Sweetpotato (Ipomoea batatas L.) RootsJOURNAL OF FOOD SCIENCE, Issue 7 2007M.S. Padda ABSTRACT:, Phenolic acids are one of the several classes of naturally occurring antioxidant compounds found in sweetpotatoes. Simplified, robust, and rapid methodologies were optimized to quantify total and individual phenolic acids in sweetpotato roots. Total phenolic acid content was quantified spectrophotometrically using both Folin,Denis and Folin,Ciocalteu reagents. The Folin,Ciocalteu reagent gave an overestimation of total phenolic acids due to the absorbance of interfering compounds (that is, reducing sugars and ascorbic acid). Individual phenolic acids were quantified by high-performance liquid chromatography (HPLC) using the latest in column technology. Four reversed-phase C18 analytical columns with different properties (dimensions, particle size, particle shape, pore size, and carbon load) were compared. Three different mobile phases using isocratic conditions were also evaluated. A column (4.6 × 150 mm) packed with 5-,m spherical silica particles of pore size 110 Ĺ combined with 14% carbon load provided the best and fast separation of individual phenolic acids (that is, chlorogenic acid, caffeic acid, and 3 isomers of dicaffeoylquinic acid) with a total analysis time of less than 7 min. Among the 3 mobile phases tested, a mobile phase consisting of 1% (v/v) formic acid aqueous solution: acetonitrile: 2-propanol, pH 2.5 (70:22:8, v/v/v) gave adequate separation. Among the solvents tested, aqueous mixtures (80:20, solvent:water) of methanol and ethanol provided higher phenolic acid extraction efficiency than the aqueous mixture of acetone. [source] A sequence optimization strategy for chromatographic separation in reversed-phase high-performance liquid chromatographyAICHE JOURNAL, Issue 2 2010Xueling Du Abstract A sequence optimization strategy combining an artificial neural network (ANN) and a chromatographic response function (CRF) for chromatographic separation in reversed-phase high-performance liquid chromatography has been proposed. Experiments were appropriately designed to obtain unbiased data concerning the effects of varying the mobile phase composition, flow-rate, and temperature. The ANN was then used to simultaneously predict the resolution and analysis time, which are the two most important features of chromatographic separation. Subsequently, a CRF consisting of resolution and analysis time was used to predict the optimum operating conditions for different specialized purposes. The experimental chromatograms were consistent with those predicted for given conditions, which verified the applicability of the method. Furthermore, the proposed optimization strategy was applied to literature data and very good agreement was obtained. The results show that a strategy of sequential combination of ANN and CRF can provide a more flexible and efficient optimization method for chromatographic separation. © 2009 American Institute of Chemical Engineers AIChE J, 2010 [source] Comparison of segmentation methods for MRI measurement of cardiac function in ratsJOURNAL OF MAGNETIC RESONANCE IMAGING, Issue 4 2010Johannes Riegler MSc Abstract Purpose To establish the accuracy, intra- and inter-observer variabilities of four different segmentation methods for measuring cardiac functional parameters in healthy and infarcted rat hearts. Materials and Methods Six Wistar rats were imaged before and after myocardial infarction using an electrocardiogram and respiratory-gated spoiled gradient echo sequence. Blinded and randomized datasets were analyzed by various semi-automatic and manual segmentation methods to compare their measurement bias and variability. In addition, the accuracy of these methods was assessed by comparison with reference measurements acquired from high-resolution three-dimensional (3D) datasets of a heart phantom. Results Relative inter- and intra-observer variability were found to be similar for all four methods. Semi-automatic segmentation methods reduced analysis time by up to 70%, while yielding similar measurement bias and variability compared with manual segmentation. Semi-automatic methods were found to underestimate the ejection fraction for healthy hearts compared with manual segmentation while overestimating them in infarcted hearts. However, semi-automatic segmentation of short axis slices agreed better with 3D reference scans of a heart phantom compared with manual segmentation. Conclusion Semi-automatic segmentation methods are faster than manual segmentation, while offering a similar intra- and inter-observer variability. However, a potential bias has been observed between healthy and infarcted hearts for different methods, which should also be considered when selecting the most appropriate analysis technique. J. Magn. Reson. Imaging 2010;32:869,877. © 2010 Wiley-Liss, Inc. [source] Chemical cross-linking with NHS esters: a systematic study on amino acid reactivitiesJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 5 2009Stefanie Mädler Abstract Structure elucidation of tertiary or quaternary protein structures by chemical cross-linking and mass spectrometry (MS) has recently gained importance. To locate the cross-linker modification, dedicated software is applied to analyze the mass or tandem mass spectra (MS/MS). Such software requires information on target amino acids to limit the data analysis time. The most commonly used homobifunctional N-hydroxy succinimide (NHS) esters are often described as reactive exclusively towards primary amines, although side reactions with tyrosine and serine have been reported. Our goal was to systematically study the reactivity of NHS esters and derive some general rules for their attack of nucleophilic amino acid side chains in peptides. We therefore studied the cross-linking reactions of synthesized and commercial model peptides with disuccinimidyl suberate (DSS). The first reaction site in all cases was expectedly the ,-NH2 -group of the N -terminus or the ,-NH2 -group of lysine. As soon as additional cross-linkers were attached or loops were formed, other amino acids were also involved in the reaction. In addition to the primary amino groups, serine, threonine and tyrosine showed significant reactivity due to the effect of neighboring amino acids by intermediate or permanent Type-1 cross-link formation. The reactivity is highly dependent on the pH and on adjacent amino acids. Copyright © 2009 John Wiley & Sons, Ltd. [source] A rapid and sensitive liquid chromatography/positive ion tandem mass spectrometry method for the determination of cimetropium in human plasma by liquid,liquid extractionJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 7 2006Heon-Woo Lee Abstract We have developed and validated a simple detection system with high-performance liquid chromatography (HPLC) with positive ion electrospray ionization tandem mass spectrometry (ESI-MS/MS) for determining cimetropium levels in human plasma using scopolamine butyl bromide as an internal standard (I.S.). The acquisition was performed in the multiple reaction monitoring (MRM) mode, by monitoring the transitions: m/z 357.9 > 103.1 for cimetropium and m/z 359.9 > 103.1 for butyl-scopolamine. The method involves a simple single-step liquid,liquid extraction with dichloromethane. The analyte was chromatographed on an YMC C18 reversed-phase chromatographic column by isocratic elution with 10 mM ammonium formate buffer,methanol (19 : 81, v/v; adjusted to pH 4.0 with formic acid). The results were linear over the studied range (0.2,100 ng ml,1), with r2 = 1.0000, and the total analysis time for each run was 2 min. Intra- and interassay precisions were 0.70,8.54% and 1.08,4.85%, respectively, and intra- and interassay accuracies were 97.56,108.23% and 97.48,103.91%, respectively. The lower limit of quantification (LLOQ) was 0.2 ng ml,1. At this concentration, mean intra- and interassay precisions were 8.54% and 4.85%, respectively, and mean intra- and interassay accuracies were 97.56% and 98.91%, respectively. The mean recovery ranged from 62.71 ± 4.06 to 64.23 ± 2.32%. Cimetropium was found to be stable in plasma samples under typical storage and processing conditions. The devised assay was successfully applied to a pharmacokinetic study of cimetropium bromide administered as a single oral dose (150 mg) to healthy volunteers. Copyright © 2006 John Wiley & Sons, Ltd. [source] Accessible proteomics space and its implications for peak capacity for zero-, one- and two-dimensional separations coupled with FT-ICR and TOF mass spectrometryJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 3 2006Jennifer L. Frahm The number and wide dynamic range of components found in biological matrixes present several challenges for global proteomics. In this perspective, we will examine the potential of zero-dimensional (0D), one-dimensional (1D), and two-dimensional (2D) separations coupled with Fourier-transform ion cyclotron resonance (FT-ICR) and time-of-flight (TOF) mass spectrometry (MS) for the analysis of complex mixtures. We describe and further develop previous reports on the space occupied by peptides, to calculate the theoretical peak capacity available to each separations-mass spectrometry method examined. Briefly, the peak capacity attainable by each of the mass analyzers was determined from the mass resolving power (RP) and the m/z space occupied by peptides considered from the mass distribution of tryptic peptides from National Center for Biotechnology Information's (NCBI's) nonredundant database. Our results indicate that reverse-phase-nanoHPLC (RP-nHPLC) separation coupled with FT-ICR MS offers an order of magnitude improvement in peak capacity over RP-nHPLC separation coupled with TOF MS. The addition of an orthogonal separation method, strong cation exchange (SCX), for 2D LC-MS demonstrates an additional 10-fold improvement in peak capacity over 1D LC-MS methods. Peak capacity calculations for 0D LC, two different 1D RP-HPLC methods, and 2D LC (with various numbers of SCX fractions) for both RP-HPLC methods coupled to FT-ICR and TOF MS are examined in detail. Peak capacity production rates, which take into account the total analysis time, are also considered for each of the methods. Furthermore, the significance of the space occupied by peptides is discussed. Copyright © 2006 John Wiley & Sons, Ltd. [source] Direct exposure electron ionization mass spectrometry and gas chromatography/mass spectrometry techniques to study organic coatings on archaeological amphoraeJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 5 2005Maria Perla Colombini Abstract Two different analytical approaches, direct exposure electron ionization mass spectrometry (DE-MS) and gas chromatography/mass spectrometry (GC/MS), were compared in a study of archaeological resinous materials. DE-MS was found to be an efficient fingerprinting tool for the fast screening of organic archaeological samples and for providing information on the major components. GC/MS appeared to be more efficient in unravelling the sample composition at a molecular level, despite the long analysis time and the need for a wet chemical pretreatment. Both procedures were applied to characterize the organic material present as coatings in Roman and Egyptian amphorae. DE-MS successfully identified abietanic compounds, hence a diterpenic resinous material could be identified and its degree of oxidation assessed. GC/MS enabled us to identify dehydroabietic acid, 7-oxodehydroabietic acid, 15-hydroxy-7-oxodehydroabietic acid, 15-hydroxydehydroabietic acid, retene, tetrahydroretene, norabietatriene, norabietatetraene and methyl dehydroabietate. These oxidized and aromatized abietanes provided evidence that the amphorae examined were waterproofed with a pitch produced from resinous wood of plants from the Pinaceae family. The chemometric evaluation of the GC/MS data highlighted significant chemical differences between the pitches found in the two archaeological sites, basically related to differences in the production techniques of the materials and in their degradation pathways. Copyright © 2005 John Wiley & Sons, Ltd. [source] Measurement of caffeine and five of the major metabolites in urine by high-performance liquid chromatography/tandem mass spectrometryJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 3 2005Allan Weimann Abstract Analysis of caffeine and its metabolites is of interest with respect to caffeine exposure, for kinetic and metabolism studies and for opportunistic in vivo estimation of drug metabolizing enzyme activity in humans and animals. For the latter, analysis is usually done by high-performance liquid chromatography (HPLC) with UV detection. However, this method is close to the detection limit for certain of the metabolites and requires very long chromatography, 30,60 min. We have developed a fast method for the quantification of caffeine and its metabolites 1-methylxanthine, 1-methyluric acid, 1,7-dimethyluric acid, 5-acetylamino-6-amino-3-methyluracil (AAMU) and 5-acetylamino-6-formylamino-3-methyluracil (AFMU) by HPLC tandem mass spectrometry (MS/MS) in urine that requires only its dilution with buffer and centrifugation before injection into the HPLC/MS/MS system. The chromatography lasts 7 min and is followed by 4.5 min for re-equilibration of the HPLC column, giving a total analysis time of 11.5 min. The method provides a great sensitivity improvement with detection limits for all analytes ,25 nM in real samples. Also, the analysis provides much improvement in capacity to ,125 samples per 24 h. Intra- and inter-day coefficients of variation of a single analysis are <6.5% for all the analytes. The inter-day coefficient of variation of duplicate analyses is <4.8% for all analytes. The method is automated, including automated integration, and it is fast, robust and suitable for large-scale investigations in humans and animals. Copyright © 2005 John Wiley & Sons, Ltd. [source] TO DIVE OR NOT TO DIVE: SCUBA VERSUS ROV SAMPLING OF MACROALGAE AT 30M DEPTHJOURNAL OF PHYCOLOGY, Issue 2001Article first published online: 24 SEP 200 Spalding, H. L. Moss Landing Marine Laboratories, 8272 Moss Landing, Rd., Moss Landing, CA 95039 USA Remotely Operated Vehicles (ROVs) and enriched air Nitrox SCUBA diving have recently become available to researchers for studying the deep-water environment. Each use a different technique for collecting macroalgal abundance data: ROVs use collections and high-resolution digital video which can be quantified using an integrative laser and computer imagery program (high tech), while divers often count the densities of individuals and use a point contact method for sampling percent (%) cover in situ (low tech). While the types of data collected by both techniques are the same, the effects of the different sampling methods on data resolution are unknown. As part of a larger study on deep-water macroalgae in central California, I compared the abundance of common macroalgae (% cover of macroalgal groups and individuals/m2) collected by divers and the ROV Ventana at a depth of 30m at 3 locations in central California. Generally, there were no significant differences between diver and ROV data in the % cover of coralline Rhodophyta, non-coralline Rhodophyta, and Pleurophycus gardneri/m2. The use of a laser-calibrated computer imagery program and an ROV with user-controlled lighting greatly decreased lab analysis time, and a method for sampling macroalgal layers with the ROV was developed. Thus, ROVs with high-resolution digital video and supplemental macroalgal collections can be used to quantify deep-water algae as accurately as in situ divers, but without the limited dive time, depth limits, and physical demands of the latter. [source] Methacrylate ester-based monolithic columns for nano-LC separation of tocopherols in vegetable oilsJOURNAL OF SEPARATION SCIENCE, JSS, Issue 17-18 2010María Jesús Lerma-García Abstract The separation and determination of tocopherols (Ts) in vegetable oils by nano-LC chromatography with UV,vis detection using lauryl methacrylate ester-based monolithic columns has been developed. The separation of Ts was optimized in terms of mobile phase composition on the basis of the best compromise among efficiency, resolution and analysis time. Using a mobile phase composed of ACN/methanol/water, an excellent resolution between Ts was achieved within 18,min. The LODs were lower than 0.26,,g/mL, being repeatability values of retention time and peak area below 0.15 and 3.1%, respectively. The method was applied to the quantification of Ts and tocotrienols present in several vegetable oils from different botanical origins. [source] | ||||||||||||||||||||||||||||||||||