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Sample records for absolute quantification method

  1. Simple absolute quantification method correcting for quantitative PCR efficiency variations for microbial community samples.

    PubMed

    Brankatschk, Robert; Bodenhausen, Natacha; Zeyer, Josef; Bürgmann, Helmut

    2012-06-01

    Real-time quantitative PCR (qPCR) is a widely used technique in microbial community analysis, allowing the quantification of the number of target genes in a community sample. Currently, the standard-curve (SC) method of absolute quantification is widely employed for these kinds of analysis. However, the SC method assumes that the amplification efficiency (E) is the same for both the standard and the sample target template. We analyzed 19 bacterial strains and nine environmental samples in qPCR assays, targeting the nifH and 16S rRNA genes. The E values of the qPCRs differed significantly, depending on the template. This has major implications for the quantification. If the sample and standard differ in their E values, quantification errors of up to orders of magnitude are possible. To address this problem, we propose and test the one-point calibration (OPC) method for absolute quantification. The OPC method corrects for differences in E and was derived from the ΔΔC(T) method with correction for E, which is commonly used for relative quantification in gene expression studies. The SC and OPC methods were compared by quantifying artificial template mixtures from Geobacter sulfurreducens (DSM 12127) and Nostoc commune (Culture Collection of Algae and Protozoa [CCAP] 1453/33), which differ in their E values. While the SC method deviated from the expected nifH gene copy number by 3- to 5-fold, the OPC method quantified the template mixtures with high accuracy. Moreover, analyzing environmental samples, we show that even small differences in E between the standard and the sample can cause significant differences between the copy numbers calculated by the SC and the OPC methods.

  2. MARQUIS: A Multiplex Method for Absolute Quantification of Peptides and Post-Translational Modifications

    PubMed Central

    Curran, Timothy G; Zhang, Yi; Ma, Daniel J.; Sarkaria, Jann N.; White, Forest M

    2014-01-01

    Absolute quantification of protein expression and post-translational modifications by mass spectrometry has been challenging due to a variety of factors, including the potentially large dynamic range of phosphorylation response. To address these issues, we have developed MARQUIS — Multiplex Absolute Regressed Quantification with Internal Standards — a novel mass spectrometry-based approach using a combination of isobaric tags and heavy-labeled standard peptides to construct internal standard curves for peptides derived from key nodes in signal transduction networks. We applied MARQUIS to quantify phosphorylation dynamics within the EGFR network at multiple time points following stimulation with several ligands, enabling a quantitative comparison of EGFR phosphorylation sites and demonstrating that receptor phosphorylation is qualitatively similar but quantitatively distinct for each EGFR ligand tested. MARQUIS was also applied to quantify the effect of EGFR kinase inhibition on glioblastoma patient derived xenografts. MARQUIS is a versatile method, broadly applicable and extendable to multiple mass spectrometric platforms. PMID:25581283

  3. Comparison and applications of label-free absolute proteome quantification methods on Escherichia coli.

    PubMed

    Arike, L; Valgepea, K; Peil, L; Nahku, R; Adamberg, K; Vilu, R

    2012-09-18

    Three different label-free proteome quantification methods--APEX, emPAI and iBAQ--were evaluated to measure proteome-wide protein concentrations in the cell. All the methods were applied to a sample from Escherichia coli chemostat culture. A Pearson squared correlation of approximately 0.6 among the three quantification methods was demonstrated. Importantly, the sum of quantified proteins by iBAQ and emPAI corresponded with the Lowry total protein quantification, demonstrating applicability of label-free methods for an accurate calculation of protein concentrations at the proteome level. The iBAQ method showed the best correlation between biological replicates, a normal distribution among all protein abundances, and the lowest variation among ribosomal protein abundances, which are expected to have equal amounts. Absolute quantitative proteome data enabled us to evaluate metabolic cost for protein synthesis and apparent catalytic activities of enzymes by integration with flux analysis. All the methods demonstrated similar ATP costs for protein synthesis for different cellular processes and that costs for expressing biomass synthesis related proteins were higher than those for energy generation. Importantly, catalytic activities of energy metabolism enzymes were an order or two higher than those of monomer synthesis. Interestingly, a staircase-like protein expression was demonstrated for most of the transcription units.

  4. Absolute quantification of myocardial blood flow.

    PubMed

    Yoshinaga, Keiichiro; Manabe, Osamu; Tamaki, Nagara

    2016-07-21

    With the increasing availability of positron emission tomography (PET) myocardial perfusion imaging, the absolute quantification of myocardial blood flow (MBF) has become popular in clinical settings. Quantitative MBF provides an important additional diagnostic or prognostic information over conventional visual assessment. The success of MBF quantification using PET/computed tomography (CT) has increased the demand for this quantitative diagnostic approach to be more accessible. In this regard, MBF quantification approaches have been developed using several other diagnostic imaging modalities including single-photon emission computed tomography, CT, and cardiac magnetic resonance. This review will address the clinical aspects of PET MBF quantification and the new approaches to MBF quantification.

  5. In vivo absolute quantification for mouse muscle metabolites using an inductively coupled synthetic signal injection method and newly developed 1H/31P dual tuned probe

    PubMed Central

    Lee, Donghoon; Marro, Kenneth; Mathis, Mark; Shankland, Eric; Hayes, Cecil

    2013-01-01

    Purpose To obtain robust estimates of 31P metabolite content in mouse skeletal muscles using our recently developed MR absolute quantification method and a custom-built 1H/31P dual tuned radiofrequency (RF) coil optimized for mouse leg. Materials and Methods We designed and fabricated a probe consisting of two dual tuned 1H/31P solenoid coils: one leg was inserted to each solenoid. The mouse leg volume coil was incorporated with injector coils for MR absolute quantification. The absolute quantification method uses a synthetic reference signal injection approach and solves several challenges in MR absolute quantification including changes of coil loading and receiver gains. Results The 1H/31P dual tuned probe was composed of two separate solenoid coils, one for each leg, to increase coil filling factors and signal-to-noise ratio. Each solenoid was equipped with a second coil to allow injection of reference signals. 31P metabolite concentrations determined for normal mice were well within the expected range reported in the literature. Conclusion We developed an RF probe and an absolute quantification approach adapted for mouse skeletal muscle. PMID:24464912

  6. Implementation of an absolute brain 1H-MRS quantification method to assess different tissue alterations in multiple sclerosis.

    PubMed

    Bagory, Matthieu; Durand-Dubief, Françoise; Ibarrola, Danielle; Comte, Jean-Christophe; Cotton, François; Confavreux, Christian; Sappey-Marinier, Dominique

    2012-10-01

    Magnetic resonance spectroscopy has emerged as a sensitive modality to detect early and diffuse alterations in multiple sclerosis. Recently, the hypothesis of neurodegenerative pathogenesis has highlightened the interest for measurement of metabolites concentrations, to gain specificity, in a large brain volume encompassing different tissue alterations. Therefore, we proposed in this paper the implementation of an absolute quantification method based on localized spectroscopy at short (30 ms) and long (135 ms) echo time of a volume including normal appearing white matter, cortical gray matter, and lesions. First, methodological developments were implemented including external calibration, and corrections of phased-array coil sensitivity and cerebrospinal fluid volume contribution. Second, these improvements were validated and optimized using an original methodology based on simulations of brain images with lesions. Finally, metabolic alterations were assessed in 65 patients including 26 relapsing-remitting, 17 primary-progressive (PP), 22 secondary-progressive (SP) patients, and in 23 normal subjects. Results showed increases of choline, creatine, and myo-inositol concentrations in PP and SP patients compared to controls, whereas the concentration of N-acetyl compounds remained constant. The major finding of this study was the identification of Cho concentration and Cho/tNA ratio as putative markers of progressive onset, suggesting interesting perspectives in detection and followup of neurodegenerative processes.

  7. Application of the standard addition method for the absolute quantification of neutral lipids in microalgae using Nile red.

    PubMed

    Bertozzini, Elena; Galluzzi, Luca; Penna, Antonella; Magnani, Mauro

    2011-10-01

    Microalgae are considered one of the best candidates for biofuel production due to their high content in neutral lipids, therefore, an accurate quantification of these lipids in microalgae is fundamental for the identification of the better candidates as biodiesel source. Nile red is a fluorescent dye widely employed for the quantification of neutral lipids in microalgae. Usually, the fluorescence intensity of the stained samples is correlated to the neutral lipid content determined with standard methods, in order to draw a standard curve and deduce the neutral lipids concentration of the unknown samples positioning their fluorescence intensity values on the curve. Standard methods used for the neutral lipids determination are laborious and often implying solvent extraction and/or other transformation (i.e. saponification or transesterification) of the sample. These methods are also time consuming and may give rise to an underestimation of the lipid content due to variable extraction yields. The approach described in this paper combines the standard addition method and the fluorometric staining using Nile red, avoiding the association of traditional neutral lipids quantification methods to the fluorometric determination. After optimization of instrument parameters and staining conditions, a linear correlation between the fluorescence intensity of each sample stained with the Nile red and its neutral lipids content deduced with the standard addition method was identified. The obtained curve allowed the direct determination of neutral lipids content maintaining a linearity range from 0.12 to 12 μg of neutral lipids per ml of sample, without need of pre-concentration. This curve was then used in the quantification of the neutral lipids content in culture of Skeletonema marinoi (Bacillariophyceae) at different days from the inoculum. This method was also successfully applied on Chaetoceros socialis (Bacillariophyceae) and Alexandrium minutum (Dinophyceae).

  8. Droplet digital PCR for absolute quantification of pathogens.

    PubMed

    Gutiérrez-Aguirre, Ion; Rački, Nejc; Dreo, Tanja; Ravnikar, Maja

    2015-01-01

    The recent advent of different digital PCR (dPCR) platforms is enabling the expansion of this technology for research and diagnostic applications worldwide. The main principle of dPCR, as in other PCR-based methods including quantitative PCR (qPCR), is the specific amplification of a nucleic acid target. The distinctive feature of dPCR is the separation of the reaction mixture into thousands to millions of partitions which is followed by a real time or end point detection of the amplification. The distribution of target sequences into partitions is described by the Poisson distribution, thus allowing accurate and absolute quantification of the target from the ratio of positive against all partitions at the end of the reaction. This omits the need to use reference materials with known target concentrations and increases the accuracy of quantification at low target concentrations compared to qPCR. dPCR has also shown higher resilience to inhibitors in a number of different types of samples. In this chapter we describe the droplet digital PCR (ddPCR) workflow for the detection and quantification of pathogens using the droplet digital Bio-Rad platform QX100. We present as an example the quantification of the quarantine plant pathogenic bacterium, Erwinia amylovora.

  9. Establishing ion ratio thresholds based on absolute peak area for absolute protein quantification using protein cleavage isotope dilution mass spectrometry.

    PubMed

    Loziuk, Philip L; Sederoff, Ronald R; Chiang, Vincent L; Muddiman, David C

    2014-11-07

    Quantitative mass spectrometry has become central to the field of proteomics and metabolomics. Selected reaction monitoring is a widely used method for the absolute quantification of proteins and metabolites. This method renders high specificity using several product ions measured simultaneously. With growing interest in quantification of molecular species in complex biological samples, confident identification and quantitation has been of particular concern. A method to confirm purity or contamination of product ion spectra has become necessary for achieving accurate and precise quantification. Ion abundance ratio assessments were introduced to alleviate some of these issues. Ion abundance ratios are based on the consistent relative abundance (RA) of specific product ions with respect to the total abundance of all product ions. To date, no standardized method of implementing ion abundance ratios has been established. Thresholds by which product ion contamination is confirmed vary widely and are often arbitrary. This study sought to establish criteria by which the relative abundance of product ions can be evaluated in an absolute quantification experiment. These findings suggest that evaluation of the absolute ion abundance for any given transition is necessary in order to effectively implement RA thresholds. Overall, the variation of the RA value was observed to be relatively constant beyond an absolute threshold ion abundance. Finally, these RA values were observed to fluctuate significantly over a 3 year period, suggesting that these values should be assessed as close as possible to the time at which data is collected for quantification.

  10. LC-MS/MS methods for absolute quantification and identification of proteins associated with chimeric plant oil bodies.

    PubMed

    Capuano, Floriana; Bond, Nicholas J; Gatto, Laurent; Beaudoin, Frédéric; Napier, Johnathan A; Benvenuto, Eugenio; Lilley, Kathryn S; Baschieri, Selene

    2011-12-15

    Oil bodies (OBs) are plant cell organelles that consist of a lipid core surrounded by a phospholipid monolayer embedded with specialized proteins such as oleosins. Recombinant proteins expressed in plants can be targeted to OBs as fusions with oleosin. This expression strategy is attractive because OBs are easily enriched and purified from other cellular components, based on their unique physicochemical properties. For recombinant OBs to be a potential therapeutic agent in biomedical applications, it is necessary to comprehensively analyze and quantify both endogenous and heterologously expressed OB proteins. In this study, a mass spectrometry (MS)-based method was developed to accurately quantify an OB-targeted heterologously expressed fusion protein that has potential as a therapeutic agent. The effect of the chimeric oleosin expression upon the OB proteome in transgenic plants was also investigated, and the identification of new potential OB residents was pursued through a variety of liquid chromatography (LC)-MS/MS approaches. The results showed that the accumulation of the fusion protein on OBs was low. Moreover, no significant differences in the accumulation of OB proteins were revealed between transgenic and wild-type seeds. The identification of five new putative components of OB proteome was also reported.

  11. Absolute Quantification of Endogenous Ras Isoform Abundance

    PubMed Central

    Mageean, Craig J.; Griffiths, John R.; Smith, Duncan L.; Clague, Michael J.; Prior, Ian A.

    2015-01-01

    Ras proteins are important signalling hubs situated near the top of networks controlling cell proliferation, differentiation and survival. Three almost identical isoforms, HRAS, KRAS and NRAS, are ubiquitously expressed yet have differing biological and oncogenic properties. In order to help understand the relative biological contributions of each isoform we have optimised a quantitative proteomics method for accurately measuring Ras isoform protein copy number per cell. The use of isotopic protein standards together with selected reaction monitoring for diagnostic peptides is sensitive, robust and suitable for application to sub-milligram quantities of lysates. We find that in a panel of isogenic SW48 colorectal cancer cells, endogenous Ras proteins are highly abundant with ≥260,000 total Ras protein copies per cell and the rank order of isoform abundance is KRAS>NRAS≥HRAS. A subset of oncogenic KRAS mutants exhibit increased total cellular Ras abundance and altered the ratio of mutant versus wild type KRAS protein. These data and methodology are significant because Ras protein copy number is required to parameterise models of signalling networks and informs interpretation of isoform-specific Ras functional data. PMID:26560143

  12. Improved Strategies and Optimization of Calibration Models for Real-time PCR Absolute Quantification

    EPA Science Inventory

    Real-time PCR absolute quantification applications rely on the use of standard curves to make estimates of DNA target concentrations in unknown samples. Traditional absolute quantification approaches dictate that a standard curve must accompany each experimental run. However, t...

  13. "Absolute" quantification in magnetic resonance spectroscopy: validation of a clinical protocol in multiple sclerosis.

    PubMed

    Bagory, Matthieu; Durand-Dubief, Françoise; Ibarrola, Danielle; Confavreux, Christian; Sappey-Marinier, Dominique

    2007-01-01

    MRS allows to measure cerebral metabolites, thus helping to characterize brain disease diagnosis and followup. Metabolite concentration quantification is usually based on metabolite ratio referring to creatine. If this metabolite concentration is supposed to be constant, it may vary in pathological processes. Therefore, "absolute" concentration methodology is needed. The aim of this study is to validate a clinical "absolute" quantification protocol through the development of an external metabolic phantom, calibration and correction, and the investigation of reproducibility issues. When phantom stability was investigated by a short-term and a long-term reproducibility study, both Standard Deviations (SD) were in agreement with literature values. This "absolute" quantification method was applied to patients with Multiple Sclerosis (MS). The results show a significant decrease in both N-Acetyl Aspartate (NAA) and choline concentrations.

  14. Development of absolute quantification method for genotype-specific Babesia microti using real-time PCR and practical experimental tips of real-time PCR.

    PubMed

    Ohmori, Shiho; Nagano-Fujii, Motoko; Saito-Ito, Atsuko

    2016-10-01

    Babesia microti, a rodent babesia, is known as a pathogen of zoonosis, human babesiosis, is composed of several genotypes of small subunit ribosomal RNA gene (SSUrDNA) and different genotypes have been suggested to have different infectivity and pathogenicity to humans. We established a real-time PCR assay using SYBR Green I, which allows specific detection and absolute quantification for each SSUrDNA-type-B. microti of four SSUrDNA-types found in Japanese rodents even in mixed infection. In this assay, four genotype-specific primer pairs targeted on internal transcribed spacer 1 or 2 sequences were used. Primer pairs have the characteristics for a high specificity for homologous genotype DNA. The calibration curves of cycle threshold (Ct) values versus log concentrations of DNA for all four genotypes were linear over 10(7) fold range of DNA concentrations with correlation coefficient from 0.95 to 1 and sufficient amplification efficiency from 90% to 110%. The standard curves for all four genotypes were not changed even in the presence of heterologous DNA. In this paper, we introduce how to establish and perform the genotype-specific real-time PCR and our practical experimental tips to be recommended.

  15. Absolute quantification of the alleles in somatic point mutations by bioluminometric methods based on competitive polymerase chain reaction in the presence of a locked nucleic acid blocker or an allele-specific primer.

    PubMed

    Iliadi, Alexandra; Petropoulou, Margarita; Ioannou, Penelope C; Christopoulos, Theodore K; Anagnostopoulos, Nikolaos I; Kanavakis, Emmanuel; Traeger-Synodinos, Jan

    2011-09-01

    In somatic (acquired) point mutations, the challenge is to quantify minute amounts of the mutant allele in the presence of a large excess of the normal allele that differs only in a single base pair. We report two bioluminometric methods that enable absolute quantification of the alleles. The first method exploits the ability of a locked nucleic acid (LNA) oligonucleotide to bind to and inhibit effectively the polymerase chain reaction (PCR) amplification of the normal allele while the amplification of the mutant allele remains unaffected. The second method employs allele-specific PCR primers, thereby allowing the amplification of the corresponding allele only. DNA internal standards (competitors) are added to the PCR mixture to compensate for any sample-to-sample variation in the amplification efficiency. The amplification products from the two alleles and the internal standards are quantified by a microtiter well-based bioluminometric hybridization assay using the photoprotein aequorin as a reporter. The methods allow absolute quantification of less than 300 copies of the mutant allele even in samples containing less than 1% of the mutant allele.

  16. Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification.

    PubMed

    Manes, Nathan P; Mann, Jessica M; Nita-Lazar, Aleksandra

    2015-08-17

    Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This protocol provides step-by-step instructions for the development and application of quantitative assays using selected reaction monitoring (SRM) mass spectrometry (MS). First, likely quantotypic target peptides are identified based on numerous criteria. This includes identifying proteotypic peptides, avoiding sites of posttranslational modification, and analyzing the uniqueness of the target peptide to the target protein. Next, crude external peptide standards are synthesized and used to develop SRM assays, and the resulting assays are used to perform qualitative analyses of the biological samples. Finally, purified, quantified, heavy isotope labeled internal peptide standards are prepared and used to perform isotope dilution series SRM assays. Analysis of all of the resulting MS data is presented. This protocol was used to accurately assay the absolute abundance of proteins of the chemotaxis signaling pathway within RAW 264.7 cells (a mouse monocyte/macrophage cell line). The quantification of Gi2 (a heterotrimeric G-protein α-subunit) is described in detail.

  17. Sulfur-based absolute quantification of proteins using isotope dilution inductively coupled plasma mass spectrometry

    NASA Astrophysics Data System (ADS)

    Lee, Hyun-Seok; Heun Kim, Sook; Jeong, Ji-Seon; Lee, Yong-Moon; Yim, Yong-Hyeon

    2015-10-01

    An element-based reductive approach provides an effective means of realizing International System of Units (SI) traceability for high-purity biological standards. Here, we develop an absolute protein quantification method using double isotope dilution (ID) inductively coupled plasma mass spectrometry (ICP-MS) combined with microwave-assisted acid digestion for the first time. We validated the method and applied it to certify the candidate protein certified reference material (CRM) of human growth hormone (hGH). The concentration of hGH was determined by analysing the total amount of sulfur in hGH. Next, the size-exclusion chromatography method was used with ICP-MS to characterize and quantify sulfur-containing impurities. By subtracting the contribution of sulfur-containing impurities from the total sulfur content in the hGH CRM, we obtained a SI-traceable certification value. The quantification result obtained with the present method based on sulfur analysis was in excellent agreement with the result determined via a well-established protein quantification method based on amino acid analysis using conventional acid hydrolysis combined with an ID liquid chromatography-tandem mass spectrometry. The element-based protein quantification method developed here can be generally used for SI-traceable absolute quantification of proteins, especially pure-protein standards.

  18. Absolute peptide quantification by lutetium labeling and nanoHPLC-ICPMS with isotope dilution analysis.

    PubMed

    Rappel, Christina; Schaumlöffel, Dirk

    2009-01-01

    The need of analytical methods for absolute quantitative protein analysis spurred research on new developments in recent years. In this work, a novel approach was developed for accurate absolute peptide quantification based on metal labeling with lutetium diethylenetriamine pentaacetic acid (Lu-DTPA) and nanoflow high-performance liquid chromatography-inductively coupled plasma isotope dilution mass spectrometry (nanoHPLC-ICP-IDMS). In a two-step procedure peptides were derivatized at amino groups with diethylenetriamine pentaacetic anhydride (DTPAA) followed by chelation of lutetium. Electrospray ionization mass spectrometry (ESI MS) of the reaction product demonstrated highly specific peptide labeling. Under optimized nanoHPLC conditions the labeled peptides were baseline-separated, and the excess labeling reagent did not interfere. A 176Lu-labeled spike was continuously added to the column effluent for quantification by ICP-IDMS. The recovery of a Lu-DTPA-labeled standard peptide was close to 100% indicating high labeling efficiency and accurate absolute quantification. The precision of the entire method was 4.9%. The detection limit for Lu-DTPA-tagged peptides was 179 amol demonstrating that lutetium-specific peptide quantification was by 4 orders of magnitude more sensitive than detection by natural sulfur atoms present in cysteine or methionine residues. Furthermore, the application to peptides in insulin tryptic digest allowed the identification of interfering reagents decreasing the labeling efficiency. An additional advantage of this novel approach is the analysis of peptides, which do not naturally feature ICPMS-detectable elements.

  19. Self-digitization microfluidic chip for absolute quantification of mRNA in single cells.

    PubMed

    Thompson, Alison M; Gansen, Alexander; Paguirigan, Amy L; Kreutz, Jason E; Radich, Jerald P; Chiu, Daniel T

    2014-12-16

    Quantification of mRNA in single cells provides direct insight into how intercellular heterogeneity plays a role in disease progression and outcomes. Quantitative polymerase chain reaction (qPCR), the current gold standard for evaluating gene expression, is insufficient for providing absolute measurement of single-cell mRNA transcript abundance. Challenges include difficulties in handling small sample volumes and the high variability in measurements. Microfluidic digital PCR provides far better sensitivity for minute quantities of genetic material, but the typical format of this assay does not allow for counting of the absolute number of mRNA transcripts samples taken from single cells. Furthermore, a large fraction of the sample is often lost during sample handling in microfluidic digital PCR. Here, we report the absolute quantification of single-cell mRNA transcripts by digital, one-step reverse transcription PCR in a simple microfluidic array device called the self-digitization (SD) chip. By performing the reverse transcription step in digitized volumes, we find that the assay exhibits a linear signal across a wide range of total RNA concentrations and agrees well with standard curve qPCR. The SD chip is found to digitize a high percentage (86.7%) of the sample for single-cell experiments. Moreover, quantification of transferrin receptor mRNA in single cells agrees well with single-molecule fluorescence in situ hybridization experiments. The SD platform for absolute quantification of single-cell mRNA can be optimized for other genes and may be useful as an independent control method for the validation of mRNA quantification techniques.

  20. Identification and absolute quantification of enzymes in laundry detergents by liquid chromatography tandem mass spectrometry.

    PubMed

    Gaubert, Alexandra; Jeudy, Jérémy; Rougemont, Blandine; Bordes, Claire; Lemoine, Jérôme; Casabianca, Hervé; Salvador, Arnaud

    2016-07-01

    In a stricter legislative context, greener detergent formulations are developed. In this way, synthetic surfactants are frequently replaced by bio-sourced surfactants and/or used at lower concentrations in combination with enzymes. In this paper, a LC-MS/MS method was developed for the identification and quantification of enzymes in laundry detergents. Prior to the LC-MS/MS analyses, a specific sample preparation protocol was developed due to matrix complexity (high surfactant percentages). Then for each enzyme family mainly used in detergent formulations (protease, amylase, cellulase, and lipase), specific peptides were identified on a high resolution platform. A LC-MS/MS method was then developed in selected reaction monitoring (SRM) MS mode for the light and corresponding heavy peptides. The method was linear on the peptide concentration ranges 25-1000 ng/mL for protease, lipase, and cellulase; 50-1000 ng/mL for amylase; and 5-1000 ng/mL for cellulase in both water and laundry detergent matrices. The application of the developed analytical strategy to real commercial laundry detergents enabled enzyme identification and absolute quantification. For the first time, identification and absolute quantification of enzymes in laundry detergent was realized by LC-MS/MS in a single run. Graphical Abstract Identification and quantification of enzymes by LC-MS/MS.

  1. Absolute quantification of proteins by LCMSE: a virtue of parallel MS acquisition.

    PubMed

    Silva, Jeffrey C; Gorenstein, Marc V; Li, Guo-Zhong; Vissers, Johannes P C; Geromanos, Scott J

    2006-01-01

    Relative quantification methods have dominated the quantitative proteomics field. There is a need, however, to conduct absolute quantification studies to accurately model and understand the complex molecular biology that results in proteome variability among biological samples. A new method of absolute quantification of proteins is described. This method is based on the discovery of an unexpected relationship between MS signal response and protein concentration: the average MS signal response for the three most intense tryptic peptides per mole of protein is constant within a coefficient of variation of less than +/-10%. Given an internal standard, this relationship is used to calculate a universal signal response factor. The universal signal response factor (counts/mol) was shown to be the same for all proteins tested in this study. A controlled set of six exogenous proteins of varying concentrations was studied in the absence and presence of human serum. The absolute quantity of the standard proteins was determined with a relative error of less than +/-15%. The average MS signal responses of the three most intense peptides from each protein were plotted against their calculated protein concentrations, and this plot resulted in a linear relationship with an R(2) value of 0.9939. The analyses were applied to determine the absolute concentration of 11 common serum proteins, and these concentrations were then compared with known values available in the literature. Additionally within an unfractionated Escherichia coli lysate, a subset of identified proteins known to exist as functional complexes was studied. The calculated absolute quantities were used to accurately determine their stoichiometry.

  2. Development and validation of a liquid chromatography/electrospray ionization time-of-flight mass spectrometry method for relative and absolute quantification of steroidal alkaloids in Fritillaria species.

    PubMed

    Zhou, Jian-Liang; Li, Ping; Li, Hui-Jun; Jiang, Yan; Ren, Mei-Ting; Liu, Ying

    2008-01-04

    Steroidal alkaloids are naturally occurring nitrogen-containing compounds in many edible or medicinal plants, such as potato, tomato, Fritillaria and American hellebore, which possess a variety of toxicological and pharmacological effects on humans. The aim of this study is to explore the potential of liquid chromatography/electrospray ionization time-of-flight mass spectrometry (LC/ESI-TOF-MS) method in the determination of these important alkaloids in plant matrices. The application of this method has been proven through 26 naturally occurring steroidal alkaloids in Fritillaria species. Accurate mass measurements within 4 ppm error were obtained for all the alkaloids detected out of various plant matrices, which allowed an unequivocal identification of the target steroidal alkaloids. The bunching factor for mass spectrometer, an important parameter significantly affecting the precision and accuracy of quantitative method, was firstly optimized in this work and satisfactory precision and linearity were achieved by the optimization of that parameter. The ranges of RSD values of intra-day and inter-day variability for all alkaloids were decreased remarkably from 41.8-159% and 13.2-140% to 0.32-7.98% and 2.37-16.1%, respectively, when the value of bunching factor was optimized from 1 to 3. Linearity of response more than two orders of magnitude was also demonstrated (regression coefficient >0.99). The LC/TOF-MS detection method offered improvements to the sensitivity, compared with previously applied LC (or GC) methods, with limits of detection down to 0.0014-0.0335 microg/ml. The results in this paper illustrate the robustness and applicability of LC/TOF-MS for steroidal alkaloids analysis in plant samples. In addition, relative quantitative determination of steroidal alkaloid with one popular analyte verticinone which is commercially available was also investigated in order to break through the choke point of lack of standards in phytochemical analysis. The

  3. Picoliter Well Array Chip-Based Digital Recombinase Polymerase Amplification for Absolute Quantification of Nucleic Acids.

    PubMed

    Li, Zhao; Liu, Yong; Wei, Qingquan; Liu, Yuanjie; Liu, Wenwen; Zhang, Xuelian; Yu, Yude

    2016-01-01

    Absolute, precise quantification methods expand the scope of nucleic acids research and have many practical applications. Digital polymerase chain reaction (dPCR) is a powerful method for nucleic acid detection and absolute quantification. However, it requires thermal cycling and accurate temperature control, which are difficult in resource-limited conditions. Accordingly, isothermal methods, such as recombinase polymerase amplification (RPA), are more attractive. We developed a picoliter well array (PWA) chip with 27,000 consistently sized picoliter reactions (314 pL) for isothermal DNA quantification using digital RPA (dRPA) at 39°C. Sample loading using a scraping liquid blade was simple, fast, and required small reagent volumes (i.e., <20 μL). Passivating the chip surface using a methoxy-PEG-silane agent effectively eliminated cross-contamination during dRPA. Our creative optical design enabled wide-field fluorescence imaging in situ and both end-point and real-time analyses of picoliter wells in a 6-cm(2) area. It was not necessary to use scan shooting and stitch serial small images together. Using this method, we quantified serial dilutions of a Listeria monocytogenes gDNA stock solution from 9 × 10(-1) to 4 × 10(-3) copies per well with an average error of less than 11% (N = 15). Overall dRPA-on-chip processing required less than 30 min, which was a 4-fold decrease compared to dPCR, requiring approximately 2 h. dRPA on the PWA chip provides a simple and highly sensitive method to quantify nucleic acids without thermal cycling or precise micropump/microvalve control. It has applications in fast field analysis and critical clinical diagnostics under resource-limited settings.

  4. Picoliter Well Array Chip-Based Digital Recombinase Polymerase Amplification for Absolute Quantification of Nucleic Acids

    PubMed Central

    Li, Zhao; Liu, Yong; Wei, Qingquan; Liu, Yuanjie; Liu, Wenwen; Zhang, Xuelian; Yu, Yude

    2016-01-01

    Absolute, precise quantification methods expand the scope of nucleic acids research and have many practical applications. Digital polymerase chain reaction (dPCR) is a powerful method for nucleic acid detection and absolute quantification. However, it requires thermal cycling and accurate temperature control, which are difficult in resource-limited conditions. Accordingly, isothermal methods, such as recombinase polymerase amplification (RPA), are more attractive. We developed a picoliter well array (PWA) chip with 27,000 consistently sized picoliter reactions (314 pL) for isothermal DNA quantification using digital RPA (dRPA) at 39°C. Sample loading using a scraping liquid blade was simple, fast, and required small reagent volumes (i.e., <20 μL). Passivating the chip surface using a methoxy-PEG-silane agent effectively eliminated cross-contamination during dRPA. Our creative optical design enabled wide-field fluorescence imaging in situ and both end-point and real-time analyses of picoliter wells in a 6-cm2 area. It was not necessary to use scan shooting and stitch serial small images together. Using this method, we quantified serial dilutions of a Listeria monocytogenes gDNA stock solution from 9 × 10-1 to 4 × 10-3 copies per well with an average error of less than 11% (N = 15). Overall dRPA-on-chip processing required less than 30 min, which was a 4-fold decrease compared to dPCR, requiring approximately 2 h. dRPA on the PWA chip provides a simple and highly sensitive method to quantify nucleic acids without thermal cycling or precise micropump/microvalve control. It has applications in fast field analysis and critical clinical diagnostics under resource-limited settings. PMID:27074005

  5. Absolute protein quantification of the yeast chaperome under conditions of heat shock

    PubMed Central

    Mackenzie, Rebecca J.; Lawless, Craig; Holman, Stephen W.; Lanthaler, Karin; Beynon, Robert J.; Grant, Chris M.; Hubbard, Simon J.

    2016-01-01

    Chaperones are fundamental to regulating the heat shock response, mediating protein recovery from thermal‐induced misfolding and aggregation. Using the QconCAT strategy and selected reaction monitoring (SRM) for absolute protein quantification, we have determined copy per cell values for 49 key chaperones in Saccharomyces cerevisiae under conditions of normal growth and heat shock. This work extends a previous chemostat quantification study by including up to five Q‐peptides per protein to improve confidence in protein quantification. In contrast to the global proteome profile of S. cerevisiae in response to heat shock, which remains largely unchanged as determined by label‐free quantification, many of the chaperones are upregulated with an average two‐fold increase in protein abundance. Interestingly, eight of the significantly upregulated chaperones are direct gene targets of heat shock transcription factor‐1. By performing absolute quantification of chaperones under heat stress for the first time, we were able to evaluate the individual protein‐level response. Furthermore, this SRM data was used to calibrate label‐free quantification values for the proteome in absolute terms, thus improving relative quantification between the two conditions. This study significantly enhances the largely transcriptomic data available in the field and illustrates a more nuanced response at the protein level. PMID:27252046

  6. Recombinant isotope labeled and selenium quantified proteins for absolute protein quantification.

    PubMed

    Zinn, Nico; Winter, Dominic; Lehmann, Wolf D

    2010-03-15

    A novel, widely applicable method for the production of absolutely quantified proteins is described, which can be used as internal standards for quantitative proteomic studies based on mass spectrometry. These standards are recombinant proteins containing an isotope label and selenomethionine. For recombinant protein expression, assembly of expression vectors fitted to cell-free protein synthesis was conducted using the gateway technology which offers fast access to a variety of genes via open reading frame libraries and an easy shuttling of genes between vectors. The proteins are generated by cell-free expression in a medium in which methionine is exchanged against selenomethionine and at least one amino acid is exchanged by a highly stable isotope labeled analogue. After protein synthesis and purification, selenium is used for absolute quantification by element mass spectrometry, while the heavy amino acids in the protein serve as reference in subsequent analyses by LC-ESI-MS or MALDI-MS. Accordingly, these standards are denominated RISQ (for recombinant isotope labeled and selenium quantified) proteins. In this study, a protein was generated containing Lys+6 ([(13)C(6)]-lysine) and Arg+10 ([(13)C(6),(15)N(4)]-arginine) so that each standard tryptic peptide contains a labeled amino acid. Apolipoprotein A1 was synthesized as RISQ protein, and its use as internal standard led to quantification of a reference material within the specified value. Owing to their cell-free expression, RISQ proteins do not contain posttranslational modifications. Thus, correct quantitative data by ESI- or MALDI-MS are restricted to quantifications based on peptides derived from unmodified regions of the analyte protein. Therefore, besides serving as internal standards, RISQ proteins stand out as new tools for quantitative analysis of covalent protein modifications.

  7. Accurate Quantification of Cardiovascular Biomarkers in Serum Using Protein Standard Absolute Quantification (PSAQ™) and Selected Reaction Monitoring*

    PubMed Central

    Huillet, Céline; Adrait, Annie; Lebert, Dorothée; Picard, Guillaume; Trauchessec, Mathieu; Louwagie, Mathilde; Dupuis, Alain; Hittinger, Luc; Ghaleh, Bijan; Le Corvoisier, Philippe; Jaquinod, Michel; Garin, Jérôme; Bruley, Christophe; Brun, Virginie

    2012-01-01

    Development of new biomarkers needs to be significantly accelerated to improve diagnostic, prognostic, and toxicity monitoring as well as therapeutic follow-up. Biomarker evaluation is the main bottleneck in this development process. Selected Reaction Monitoring (SRM) combined with stable isotope dilution has emerged as a promising option to speed this step, particularly because of its multiplexing capacities. However, analytical variabilities because of upstream sample handling or incomplete trypsin digestion still need to be resolved. In 2007, we developed the PSAQ™ method (Protein Standard Absolute Quantification), which uses full-length isotope-labeled protein standards to quantify target proteins. In the present study we used clinically validated cardiovascular biomarkers (LDH-B, CKMB, myoglobin, and troponin I) to demonstrate that the combination of PSAQ and SRM (PSAQ-SRM) allows highly accurate biomarker quantification in serum samples. A multiplex PSAQ-SRM assay was used to quantify these biomarkers in clinical samples from myocardial infarction patients. Good correlation between PSAQ-SRM and ELISA assay results was found and demonstrated the consistency between these analytical approaches. Thus, PSAQ-SRM has the capacity to improve both accuracy and reproducibility in protein analysis. This will be a major contribution to efficient biomarker development strategies. PMID:22080464

  8. Absolute quantification for benzoic acid in processed foods using quantitative proton nuclear magnetic resonance spectroscopy.

    PubMed

    Ohtsuki, Takashi; Sato, Kyoko; Sugimoto, Naoki; Akiyama, Hiroshi; Kawamura, Yoko

    2012-09-15

    The absolute quantification method of benzoic acid (BA) in processed foods using solvent extraction and quantitative proton nuclear magnetic resonance spectroscopy was developed and validated. BA levels were determined using proton signals (δ(H) 7.53 and 7.98) referenced to 2-dimethyl-2-silapentane-5-sulfonate-d(6) sodium salt (DSS-d(6)) after simple solvent extraction from processed foods. All recoveries from several kinds of processed foods, spiked at their specified maximum Japanese usage levels (0.6-2.5 g kg(-1)) and at 0.13 g kg(-1) and 0.063 g kg(-1), were greater than 80%. The limit of quantification was confirmed as 0.063 g kg(-1) in processed foods, which was sufficiently low for the purposes of monitoring BA. The accuracy of the proposed method is equivalent to the conventional method using steam-distillation extraction and high-performance liquid chromatography. The proposed method was both rapid and simple. Moreover, it provided International System of Units traceability without the need for authentic analyte standards. Therefore, the proposed method is a useful and practical tool for determining BA levels in processed foods.

  9. NeuCode labels with parallel reaction monitoring for multiplexed, absolute protein quantification

    PubMed Central

    Potts, Gregory K.; Voigt, Emily A.; Bailey, Derek J.; Westphall, Michael S.; Hebert, Alexander S.; Yin, John; Coon, Joshua J.

    2016-01-01

    We introduce a new method to multiplex the throughput of samples for targeted mass spectrometry analysis. The current paradigm for obtaining absolute quantification from biological samples requires spiking isotopically heavy peptide standards into light biological lysates. Because each lysate must be run individually, this method places limitations on sample throughput and high demands on instrument time. When cell lines are first metabolically labeled with various neutron-encoded (NeuCode) lysine isotopologues possessing mDa mass differences from each other, heavy cell lysates may be mixed and spiked with an additional heavy peptide as an internal standard. We demonstrate that these NeuCode lysate peptides may be co-isolated with their internal standards, fragmented, and analyzed together using high resolving power parallel reaction monitoring (PRM). Instead of running each sample individually, these methods allow samples to be multiplexed to obtain absolute concentrations of target peptides in 5, 15, and even 25 biological samples at a time during single mass spectrometry experiments. PMID:26882330

  10. Refinements to the structure of graphite oxide: absolute quantification of functional groups via selective labelling.

    PubMed

    Eng, Alex Yong Sheng; Chua, Chun Kiang; Pumera, Martin

    2015-12-21

    Chemical modification and functionalization of inherent functional groups within graphite oxide (GO) are essential aspects of graphene-based nano-materials used in wide-ranging applications. Despite extensive research, there remains some discrepancy in its structure, with current knowledge limited primarily to spectroscopic data from XPS, NMR and vibrational spectroscopies. We report herein an innovative electrochemistry-based approach. Four electroactive labels are chosen to selectively functionalize groups in GO, and quantification of each group is achieved by voltammetric analysis. This allows for the first time quantification of absolute amounts of each group, with a further advantage of distinguishing various carbonyl species: namely ortho- and para-quinones from aliphatic ketones. Intrinsic variations in the compositions of permanganate versus chlorate-oxidized GOs were thus observed. Principal differences include permanganate-GO exhibiting substantial quinonyl content, in comparison to chlorate-GO with the vast majority of its carbonyls as isolated ketones. The results confirm that carboxylic groups are rare in actuality, and are in fact entirely absent from chlorate-GO. These observations refine and advance our understanding of GO structure by addressing certain disparities in past models resulting from employment of different oxidation routes, with the vital implication that GO production methods cannot be used interchangeably in the manufacture of graphene-based devices.

  11. Calibration-free absolute quantification of optical absorption coefficients using acoustic spectra in 3D photoacoustic microscopy of biological tissue.

    PubMed

    Guo, Zijian; Hu, Song; Wang, Lihong V

    2010-06-15

    Optical absorption is closely associated with many physiological important parameters, such as the concentration and oxygen saturation of hemoglobin, and it can be used to quantify the concentrations of nonfluorescent molecules. We propose a method to use acoustic spectra of photoacoustic signals to quantify the absolute optical absorption. This method is self-calibrating and thus insensitive to variations in the optical fluence. Factors such as system bandwidth and acoustic attenuation can affect the quantification but can be canceled by dividing the acoustic spectra measured at two optical wavelengths. Using optical-resolution photoacoustic microscopy, we quantified the absolute optical absorption of black ink samples with various concentrations. We also quantified both the concentration and oxygen saturation of hemoglobin in a live mouse in absolute units.

  12. Absolute method of measuring magnetic susceptibility

    USGS Publications Warehouse

    Thorpe, A.; Senftle, F.E.

    1959-01-01

    An absolute method of standardization and measurement of the magnetic susceptibility of small samples is presented which can be applied to most techniques based on the Faraday method. The fact that the susceptibility is a function of the area under the curve of sample displacement versus distance of the magnet from the sample, offers a simple method of measuring the susceptibility without recourse to a standard sample. Typical results on a few substances are compared with reported values, and an error of less than 2% can be achieved. ?? 1959 The American Institute of Physics.

  13. FLEXIQuant: A novel tool for the absolute quantification of proteins, and the simultaneous identification and quantification of potentially modified peptides

    PubMed Central

    Singh, Sasha; Springer, Michael; Steen, Judith; Kirschner, Marc W.; Steen, Hanno

    2010-01-01

    Measurements of protein abundance and quantitative assessment of multiple post-translational modifications (PTMs) within a single protein are increasingly called for to understand the control of protein activity, particularly in metazoan cells. General methods of wide applicability and precision/accuracy for quantitative estimation of protein post-translational regulation are lacking. Protein mass spectrometry has evolved from a high throughput qualitative technique to a potentially general quantitative tool but there are still serious limitations in dynamic range and coverage. To address some of these limitations we introduce a novel MS-based quantitative strategy, FLEXIQuant, (Full-Length Expressed Stable Isotope-labeled Proteins for Quantification), which can track changes in relative peptide abundances as a function of PTM, and determine absolute quantity of a protein from its lysate. We examined two subunits of the anaphase-promoting complex, CDC27 and APC5, as a test of our ability to monitor quantitatively, the PTM status of several peptides over time. We find evidence of differential regulation at different sites, a phenomenon we believe will be very widespread. FLEXIQuant proved itself to be capable of serving as a general quantitative tool. PMID:19344176

  14. Absolute quantification by droplet digital PCR versus analog real-time PCR

    PubMed Central

    Hindson, Christopher M; Chevillet, John R; Briggs, Hilary A; Gallichotte, Emily N; Ruf, Ingrid K; Hindson, Benjamin J; Vessella, Robert L; Tewari, Muneesh

    2014-01-01

    Nanoliter-sized droplet technology paired with digital PCR (ddPCR) holds promise for highly precise, absolute nucleic acid quantification. Our comparison of microRNA quantification by ddPCR and real-time PCR revealed greater precision (coefficients of variation decreased by 37–86%) and improved day-to-day reproducibility (by a factor of seven) of ddPCR but with comparable sensitivity. When we applied ddPCR to serum microRNA biomarker analysis, this translated to superior diagnostic performance for identifying individuals with cancer. PMID:23995387

  15. Refinements to the structure of graphite oxide: absolute quantification of functional groups via selective labelling

    NASA Astrophysics Data System (ADS)

    Eng, Alex Yong Sheng; Chua, Chun Kiang; Pumera, Martin

    2015-11-01

    Chemical modification and functionalization of inherent functional groups within graphite oxide (GO) are essential aspects of graphene-based nano-materials used in wide-ranging applications. Despite extensive research, there remains some discrepancy in its structure, with current knowledge limited primarily to spectroscopic data from XPS, NMR and vibrational spectroscopies. We report herein an innovative electrochemistry-based approach. Four electroactive labels are chosen to selectively functionalize groups in GO, and quantification of each group is achieved by voltammetric analysis. This allows for the first time quantification of absolute amounts of each group, with a further advantage of distinguishing various carbonyl species: namely ortho- and para-quinones from aliphatic ketones. Intrinsic variations in the compositions of permanganate versus chlorate-oxidized GOs were thus observed. Principal differences include permanganate-GO exhibiting substantial quinonyl content, in comparison to chlorate-GO with the vast majority of its carbonyls as isolated ketones. The results confirm that carboxylic groups are rare in actuality, and are in fact entirely absent from chlorate-GO. These observations refine and advance our understanding of GO structure by addressing certain disparities in past models resulting from employment of different oxidation routes, with the vital implication that GO production methods cannot be used interchangeably in the manufacture of graphene-based devices.Chemical modification and functionalization of inherent functional groups within graphite oxide (GO) are essential aspects of graphene-based nano-materials used in wide-ranging applications. Despite extensive research, there remains some discrepancy in its structure, with current knowledge limited primarily to spectroscopic data from XPS, NMR and vibrational spectroscopies. We report herein an innovative electrochemistry-based approach. Four electroactive labels are chosen to selectively

  16. Metal Stable Isotope Tagging: Renaissance of Radioimmunoassay for Multiplex and Absolute Quantification of Biomolecules.

    PubMed

    Liu, Rui; Zhang, Shixi; Wei, Chao; Xing, Zhi; Zhang, Sichun; Zhang, Xinrong

    2016-05-17

    The unambiguous quantification of biomolecules is of great significance in fundamental biological research as well as practical clinical diagnosis. Due to the lack of a detectable moiety, the direct and highly sensitive quantification of biomolecules is often a "mission impossible". Consequently, tagging strategies to introduce detectable moieties for labeling target biomolecules were invented, which had a long and significant impact on studies of biomolecules in the past decades. For instance, immunoassays have been developed with radioisotope tagging by Yalow and Berson in the late 1950s. The later languishment of this technology can be almost exclusively ascribed to the use of radioactive isotopes, which led to the development of nonradioactive tagging strategy-based assays such as enzyme-linked immunosorbent assay, fluorescent immunoassay, and chemiluminescent and electrochemiluminescent immunoassay. Despite great success, these strategies suffered from drawbacks such as limited spectral window capacity for multiplex detection and inability to provide absolute quantification of biomolecules. After recalling the sequences of tagging strategies, an apparent question is why not use stable isotopes from the start? A reasonable explanation is the lack of reliable means for accurate and precise quantification of stable isotopes at that time. The situation has changed greatly at present, since several atomic mass spectrometric measures for metal stable isotopes have been developed. Among the newly developed techniques, inductively coupled plasma mass spectrometry is an ideal technique to determine metal stable isotope-tagged biomolecules, for its high sensitivity, wide dynamic linear range, and more importantly multiplex and absolute quantification ability. Since the first published report by our group, metal stable isotope tagging has become a revolutionary technique and gained great success in biomolecule quantification. An exciting research highlight in this area

  17. Absolute quantification of carnosine in human calf muscle by proton magnetic resonance spectroscopy

    NASA Astrophysics Data System (ADS)

    Özdemir, Mahir S.; Reyngoudt, Harmen; DeDeene, Yves; Sazak, Hakan S.; Fieremans, Els; Delputte, Steven; D'Asseler, Yves; Derave, Wim; Lemahieu, Ignace; Achten, Eric

    2007-12-01

    Carnosine has been shown to be present in the skeletal muscle and in the brain of a variety of animals and humans. Despite the various physiological functions assigned to this metabolite, its exact role remains unclear. It has been suggested that carnosine plays a role in buffering in the intracellular physiological pHi range in skeletal muscle as a result of accepting hydrogen ions released in the development of fatigue during intensive exercise. It is thus postulated that the concentration of carnosine is an indicator for the extent of the buffering capacity. However, the determination of the concentration of this metabolite has only been performed by means of muscle biopsy, which is an invasive procedure. In this paper, we utilized proton magnetic resonance spectroscopy (1H MRS) in order to perform absolute quantification of carnosine in vivo non-invasively. The method was verified by phantom experiments and in vivo measurements in the calf muscles of athletes and untrained volunteers. The measured mean concentrations in the soleus and the gastrocnemius muscles were found to be 2.81 ± 0.57/4.8 ± 1.59 mM (mean ± SD) for athletes and 2.58 ± 0.65/3.3 ± 0.32 mM for untrained volunteers, respectively. These values are in agreement with previously reported biopsy-based results. Our results suggest that 1H MRS can provide an alternative method for non-invasively determining carnosine concentration in human calf muscle in vivo.

  18. Usage of human reliability quantification methods.

    PubMed

    Grozdanovic, Miroljub

    2005-01-01

    Human reliability quantification (HRQ) methods are becoming increasingly important in risk and accident assessment in systems these terms are usually related to (hi-tech industrial systems, including nuclear and chemical plants). These methods began to intensively develop after numerous accidents caused by human error or inadequate activity of people who controlled and managed complex technological processes. For already existing systems, but also for new ones, it is important to assess the possibility of an accident. Determination of possible preventive activities, which include the influence of human error on the safety of a system, is also required. These are the main goals of the HRQ method. Using Absolute Probability Judgment (APJ) and Success Likelihood Index Methods (SLIM) HRQ techniques in control and management centers in electro-power systems in Belgrade and railway traffic in Nis (both in Serbia and Montenegro) are shown in this paper.

  19. Absolute quantification of Bovine Viral Diarrhea Virus (BVDV) RNA by the digital PCR technique

    NASA Astrophysics Data System (ADS)

    Flatschart, R. B.; Almeida, D. O.; Heinemann, M. B.; Medeiros, M. N.; Granjeiro, J. M.; Folgueras-Flatschart, A. V.

    2015-01-01

    The quality control of cell lines used in research and industry is critical to ensure confidence in experimental results and to guarantee the safety of biopharmaceuticals to consumers. The BVDV is a common adventitious agent in many cell lines. We preliminarly evaluate the use of Digital Droplet PCR (ddPCR) for the detection and enumeration of genome copies of BVDV in cell culture and on FBS. The application of a commercial Real-Time PCR kit with the ddPCR technique was successful on different matrices. The technique allowed the absolute quantification of the genome without the use of calibration standards, suggesting its promising application on the development of reference materials for quantification of nucleic acids.

  20. Absolute quantification of genetically modified MON810 maize (Zea mays L.) by digital polymerase chain reaction.

    PubMed

    Corbisier, Philippe; Bhat, Somanath; Partis, Lina; Xie, Vicki Rui Dan; Emslie, Kerry R

    2010-03-01

    Quantitative analysis of genetically modified (GM) foods requires estimation of the amount of the transgenic event relative to an endogenous gene. Regulatory authorities in the European Union (EU) have defined the labelling threshold for GM food on the copy number ratio between the transgenic event and an endogenous gene. Real-time polymerase chain reaction (PCR) is currently being used for quantification of GM organisms (GMOs). Limitations in real-time PCR applications to detect very low number of DNA targets has led to new developments such as the digital PCR (dPCR) which allows accurate measurement of DNA copies without the need for a reference calibrator. In this paper, the amount of maize MON810 and hmg copies present in a DNA extract from seed powders certified for their mass content and for their copy number ratio was measured by dPCR. The ratio of these absolute copy numbers determined by dPCR was found to be identical to the ratios measured by real-time quantitative PCR (qPCR) using a plasmid DNA calibrator. These results indicate that both methods could be applied to determine the copy number ratio in MON810. The reported values were in agreement with estimations from a model elaborated to convert mass fractions into copy number fractions in MON810 varieties. This model was challenged on two MON810 varieties used for the production of MON810 certified reference materials (CRMs) which differ in the parental origin of the introduced GM trait. We conclude that dPCR has a high metrological quality and can be used for certifying GM CRMs in terms of DNA copy number ratio.

  1. Droplet Digital Enzyme-Linked Oligonucleotide Hybridization Assay for Absolute RNA Quantification

    NASA Astrophysics Data System (ADS)

    Guan, Weihua; Chen, Liben; Rane, Tushar D.; Wang, Tza-Huei

    2015-09-01

    We present a continuous-flow droplet-based digital Enzyme-Linked Oligonucleotide Hybridization Assay (droplet digital ELOHA) for sensitive detection and absolute quantification of RNA molecules. Droplet digital ELOHA incorporates direct hybridization and single enzyme reaction via the formation of single probe-RNA-probe (enzyme) complex on magnetic beads. It enables RNA detection without reverse transcription and PCR amplification processes. The magnetic beads are subsequently encapsulated into a large number of picoliter-sized droplets with enzyme substrates in a continuous-flow device. This device is capable of generating droplets at high-throughput. It also integrates in-line enzymatic incubation and detection of fluorescent products. Our droplet digital ELOHA is able to accurately quantify (differentiate 40% difference) as few as ~600 RNA molecules in a 1 mL sample (equivalent to 1 aM or lower) without molecular replication. The absolute quantification ability of droplet digital ELOHA is demonstrated with the analysis of clinical Neisseria gonorrhoeae 16S rRNA to show its potential value in real complex samples.

  2. Novel isotopic N, N-Dimethyl Leucine (iDiLeu) Reagents Enable Absolute Quantification of Peptides and Proteins Using a Standard Curve Approach

    NASA Astrophysics Data System (ADS)

    Greer, Tyler; Lietz, Christopher B.; Xiang, Feng; Li, Lingjun

    2015-01-01

    Absolute quantification of protein targets using liquid chromatography-mass spectrometry (LC-MS) is a key component of candidate biomarker validation. One popular method combines multiple reaction monitoring (MRM) using a triple quadrupole instrument with stable isotope-labeled standards (SIS) for absolute quantification (AQUA). LC-MRM AQUA assays are sensitive and specific, but they are also expensive because of the cost of synthesizing stable isotope peptide standards. While the chemical modification approach using mass differential tags for relative and absolute quantification (mTRAQ) represents a more economical approach when quantifying large numbers of peptides, these reagents are costly and still suffer from lower throughput because only two concentration values per peptide can be obtained in a single LC-MS run. Here, we have developed and applied a set of five novel mass difference reagents, isotopic N, N-dimethyl leucine (iDiLeu). These labels contain an amine reactive group, triazine ester, are cost effective because of their synthetic simplicity, and have increased throughput compared with previous LC-MS quantification methods by allowing construction of a four-point standard curve in one run. iDiLeu-labeled peptides show remarkably similar retention time shifts, slightly lower energy thresholds for higher-energy collisional dissociation (HCD) fragmentation, and high quantification accuracy for trypsin-digested protein samples (median errors <15%). By spiking in an iDiLeu-labeled neuropeptide, allatostatin, into mouse urine matrix, two quantification methods are validated. The first uses one labeled peptide as an internal standard to normalize labeled peptide peak areas across runs (<19% error), whereas the second enables standard curve creation and analyte quantification in one run (<8% error).

  3. Clarity™ digital PCR system: a novel platform for absolute quantification of nucleic acids.

    PubMed

    Low, Huiyu; Chan, Shun-Jie; Soo, Guo-Hao; Ling, Belinda; Tan, Eng-Lee

    2017-03-01

    In recent years, digital polymerase chain reaction (dPCR) has gained recognition in biomedical research as it provides a platform for precise and accurate quantification of nucleic acids without the need for a standard curve. However, this technology has not yet been widely adopted as compared to real-time quantitative PCR due to its more cumbersome workflow arising from the need to sub-divide a PCR sample into a large number of smaller partitions prior to thermal cycling to achieve zero or at least one copy of the target RNA/DNA per partition. A recently launched platform, the Clarity™ system from JN Medsys, simplifies dPCR workflow through the use of a novel chip-in-a-tube technology for sample partitioning. In this study, the performance of Clarity™ was evaluated through quantification of the single-copy human RNase P gene. The system demonstrated high precision and accuracy and also excellent linearity across a range of over 4 orders of magnitude for the absolute quantification of the target gene. Moreover, consistent DNA copy measurements were also attained using a panel of different probe- and dye-based master mixes, demonstrating the system's compatibility with commercial master mixes. The Clarity™ was then compared to the QX100™ droplet dPCR system from Bio-Rad using a set of DNA reference materials, and the copy number concentrations derived from both systems were found to be closely associated. Collectively, the results showed that Clarity™ is a reliable, robust and flexible platform for next-generation genetic analysis.

  4. Global absolute quantification reveals tight regulation of protein expression in single Xenopus eggs

    PubMed Central

    Smits, Arne H.; Lindeboom, Rik G.H.; Perino, Matteo; van Heeringen, Simon J.; Veenstra, Gert Jan C.; Vermeulen, Michiel

    2014-01-01

    While recent developments in genomic sequencing technology have enabled comprehensive transcriptome analyses of single cells, single cell proteomics has thus far been restricted to targeted studies. Here, we perform global absolute protein quantification of fertilized Xenopus laevis eggs using mass spectrometry-based proteomics, quantifying over 5800 proteins in the largest single cell proteome characterized to date. Absolute protein amounts in single eggs are highly consistent, thus indicating a tight regulation of global protein abundance. Protein copy numbers in single eggs range from tens of thousands to ten trillion copies per cell. Comparison between the single-cell proteome and transcriptome reveal poor expression correlation. Finally, we identify 439 proteins that significantly change in abundance during early embryogenesis. Downregulated proteins include ribosomal proteins and upregulated proteins include basal transcription factors, among others. Many of these proteins do not show regulation at the transcript level. Altogether, our data reveal that the transcriptome is a poor indicator of the proteome and that protein levels are tightly controlled in X. laevis eggs. PMID:25056316

  5. Global absolute quantification reveals tight regulation of protein expression in single Xenopus eggs.

    PubMed

    Smits, Arne H; Lindeboom, Rik G H; Perino, Matteo; van Heeringen, Simon J; Veenstra, Gert Jan C; Vermeulen, Michiel

    2014-09-01

    While recent developments in genomic sequencing technology have enabled comprehensive transcriptome analyses of single cells, single cell proteomics has thus far been restricted to targeted studies. Here, we perform global absolute protein quantification of fertilized Xenopus laevis eggs using mass spectrometry-based proteomics, quantifying over 5800 proteins in the largest single cell proteome characterized to date. Absolute protein amounts in single eggs are highly consistent, thus indicating a tight regulation of global protein abundance. Protein copy numbers in single eggs range from tens of thousands to ten trillion copies per cell. Comparison between the single-cell proteome and transcriptome reveal poor expression correlation. Finally, we identify 439 proteins that significantly change in abundance during early embryogenesis. Downregulated proteins include ribosomal proteins and upregulated proteins include basal transcription factors, among others. Many of these proteins do not show regulation at the transcript level. Altogether, our data reveal that the transcriptome is a poor indicator of the proteome and that protein levels are tightly controlled in X. laevis eggs.

  6. Absolute quantification of olive oil DNA by droplet digital-PCR (ddPCR): Comparison of isolation and amplification methodologies.

    PubMed

    Scollo, Francesco; Egea, Leticia A; Gentile, Alessandra; La Malfa, Stefano; Dorado, Gabriel; Hernandez, Pilar

    2016-12-15

    Olive oil is considered a premium product for its nutritional value and health benefits, and the ability to define its origin and varietal composition is a key step towards ensuring the traceability of the product. However, isolating the DNA from such a matrix is a difficult task. In this study, the quality and quantity of olive oil DNA, isolated using four different DNA isolation protocols, was evaluated using the qRT-PCR and ddPCR techniques. The results indicate that CTAB-based extraction methods were the best for unfiltered oil, while Nucleo Spin-based extraction protocols showed greater overall reproducibility. The use of both qRT-PCR and ddPCR led to the absolute quantification of the DNA copy number. The results clearly demonstrate the importance of the choice of DNA-isolation protocol, which should take into consideration the qualitative aspects of DNA and the evaluation of the amplified DNA copy number.

  7. Toward greener analytical techniques for the absolute quantification of peptides in pharmaceutical and biological samples.

    PubMed

    Van Eeckhaut, Ann; Mangelings, Debby

    2015-09-10

    Peptide-based biopharmaceuticals represent one of the fastest growing classes of new drug molecules. New reaction types included in the synthesis strategies to reduce the rapid metabolism of peptides, along with the availability of new formulation and delivery technologies, resulted in an increased marketing of peptide drug products. In this regard, the development of analytical methods for quantification of peptides in pharmaceutical and biological samples is of utmost importance. From the sample preparation step to their analysis by means of chromatographic or electrophoretic methods, many difficulties should be tackled to analyze them. Recent developments in analytical techniques emphasize more and more on the use of green analytical techniques. This review will discuss the progresses in and challenges observed during green analytical method development for the quantification of peptides in pharmaceutical and biological samples.

  8. Centrifugal step emulsification applied for absolute quantification of nucleic acids by digital droplet RPA.

    PubMed

    Schuler, Friedrich; Schwemmer, Frank; Trotter, Martin; Wadle, Simon; Zengerle, Roland; von Stetten, Felix; Paust, Nils

    2015-07-07

    Aqueous microdroplets provide miniaturized reaction compartments for numerous chemical, biochemical or pharmaceutical applications. We introduce centrifugal step emulsification for the fast and easy production of monodisperse droplets. Homogenous droplets with pre-selectable diameters in a range from 120 μm to 170 μm were generated with coefficients of variation of 2-4% and zero run-in time or dead volume. The droplet diameter depends on the nozzle geometry (depth, width, and step size) and interfacial tensions only. Droplet size is demonstrated to be independent of the dispersed phase flow rate between 0.01 and 1 μl s(-1), proving the robustness of the centrifugal approach. Centrifugal step emulsification can easily be combined with existing centrifugal microfluidic unit operations, is compatible to scalable manufacturing technologies such as thermoforming or injection moulding and enables fast emulsification (>500 droplets per second and nozzle) with minimal handling effort (2-3 pipetting steps). The centrifugal microfluidic droplet generation was used to perform the first digital droplet recombinase polymerase amplification (ddRPA). It was used for absolute quantification of Listeria monocytogenes DNA concentration standards with a total analysis time below 30 min. Compared to digital droplet polymerase chain reaction (ddPCR), with processing times of about 2 hours, the overall processing time of digital analysis was reduced by more than a factor of 4.

  9. The Multispecimen Method for Absolute Paleointensity Determination

    NASA Astrophysics Data System (ADS)

    Dekkers, M. J.; de Groot, L. V.; Monster, M.

    2015-12-01

    Paleointensity methods have seen a large improvement in the 21th century. This included optimizing classic Thellier-style protocols along with establishing stringent sets of quality criteria, developing microwave excitation as an alternative to thermal treatment, selecting sample material that contains the most suitable remanence carriers (i.e. single domain magnetic particles), calibrating non-heating paleointensity methods, and the introduction of the multispecimen paleointensity (MSP) protocol. An MSP experiment is carried out at one specific temperature selected to avoid thermochemical alteration; a series of specimens is heated and cooled in various applied furnace fields oriented parallel to the specimen's NRM. The furnace field value at which no change in NRM occurs is the paleofield. While the rationale of the MSP approach is surprisingly straightforward, some of the original claims (Dekkers and Böhnel, 2006) are by now shown to be untenable. This pertains to the claimed domain state independence in the original MSP method, although the Fabian and Leonhardt (2010) extended protocol largely corrects for domain state effects. Here we describe the optimal workflow for MSP experiments derived from our collection of historic flows from four volcanic edifices: Mt. Etna, Hawaii, the Canary Islands, and the Azores. By comparing the experimental outcome derived from historic flows with known paleointensities we found that technically acceptable experiments may yield overestimates, correct determinations, as well as underestimates of the paleofield. The so-called "ARM test" (de Groot et al., 2012) can distinguish between those three options. Based on TRM and ARM being analogues, this test compares ARM acquisition curves of sister samples before and after heating to the MSP experiment temperature. Simulated paleointensity experiments following this workflow consistently deliver the correct answer (Monster et al., submitted).

  10. Absolute gain measurement by the image method under mismatched condition

    NASA Technical Reports Server (NTRS)

    Lee, Richard Q.; Baddour, Maurice F.

    1987-01-01

    Purcell's image method for measuring the absolute gain of an antenna is particularly attractive for small test antennas. The method is simple to use and utilizes only one antenna with a reflecting plane to provide an image for the receiving antenna. However, the method provides accurate results only if the antenna is matched to its waveguide. In this paper, a waveguide junction analysis is developed to determine the gain of an antenna under mismatched condition. Absolute gain measurements for two standard gain horn antennas have been carried out. Experimental results agree closely with published data.

  11. BACOM2.0 facilitates absolute normalization and quantification of somatic copy number alterations in heterogeneous tumor

    NASA Astrophysics Data System (ADS)

    Fu, Yi; Yu, Guoqiang; Levine, Douglas A.; Wang, Niya; Shih, Ie-Ming; Zhang, Zhen; Clarke, Robert; Wang, Yue

    2015-09-01

    Most published copy number datasets on solid tumors were obtained from specimens comprised of mixed cell populations, for which the varying tumor-stroma proportions are unknown or unreported. The inability to correct for signal mixing represents a major limitation on the use of these datasets for subsequent analyses, such as discerning deletion types or detecting driver aberrations. We describe the BACOM2.0 method with enhanced accuracy and functionality to normalize copy number signals, detect deletion types, estimate tumor purity, quantify true copy numbers, and calculate average-ploidy value. While BACOM has been validated and used with promising results, subsequent BACOM analysis of the TCGA ovarian cancer dataset found that the estimated average tumor purity was lower than expected. In this report, we first show that this lowered estimate of tumor purity is the combined result of imprecise signal normalization and parameter estimation. Then, we describe effective allele-specific absolute normalization and quantification methods that can enhance BACOM applications in many biological contexts while in the presence of various confounders. Finally, we discuss the advantages of BACOM in relation to alternative approaches. Here we detail this revised computational approach, BACOM2.0, and validate its performance in real and simulated datasets.

  12. Non-Invasive Method of Determining Absolute Intracranial Pressure

    NASA Technical Reports Server (NTRS)

    Yost, William T. (Inventor); Cantrell, John H., Jr. (Inventor); Hargens, Alan E. (Inventor)

    2004-01-01

    A method is presented for determining absolute intracranial pressure (ICP) in a patient. Skull expansion is monitored while changes in ICP are induced. The patient's blood pressure is measured when skull expansion is approximately zero. The measured blood pressure is indicative of a reference ICP value. Subsequently, the method causes a known change in ICP and measured the change in skull expansion associated therewith. The absolute ICP is a function of the reference ICP value, the known change in ICP and its associated change in skull expansion; and a measured change in skull expansion.

  13. Phantom Validation of Tc-99m Absolute Quantification in a SPECT/CT Commercial Device

    PubMed Central

    Leite Ferreira, Paulo; Malterre, Jerome; Laub, Priscille; Prior, John O.; Verdun, Francis R.

    2016-01-01

    Aim. Similar to PET, absolute quantitative imaging is becoming available in commercial SPECT/CT devices. This study's goal was to assess quantitative accuracy of activity recovery as a function of image reconstruction parameters and count statistics in a variety of phantoms. Materials and Methods. We performed quantitative 99mTc-SPECT/CT acquisitions (Siemens Symbia Intevo, Erlangen, Germany) of a uniform cylindrical, NEMA/IEC, and an anthropomorphic abdominal phantom. Background activity concentrations tested ranged: 2–80 kBq/mL. SPECT acquisitions used 120 projections (20 s/projection). Reconstructions were performed with the proprietary iterative conjugate gradient algorithm. NEMA phantom reconstructions were obtained as a function of the iteration number (range: 4–48). Recovery coefficients, hot contrast, relative lung error (NEMA phantom), and image noise were assessed. Results. In all cases, absolute activity and activity concentration were measured within 10% of the expected value. Recovery coefficients and hot contrast in hot inserts did not vary appreciably with count statistics. RC converged at 16 iterations for insert size > 22 mm. Relative lung errors were comparable to PET levels indicating the efficient integration of attenuation and scatter corrections with adequate detector modeling. Conclusions. The tested device provided accurate activity recovery within 10% of correct values; these performances are comparable to current generation PET/CT systems. PMID:28096891

  14. Monte Carlo Modeling-Based Digital Loop-Mediated Isothermal Amplification on a Spiral Chip for Absolute Quantification of Nucleic Acids.

    PubMed

    Xia, Yun; Yan, Shuangqian; Zhang, Xian; Ma, Peng; Du, Wei; Feng, Xiaojun; Liu, Bi-Feng

    2017-03-21

    Digital loop-mediated isothermal amplification (dLAMP) is an attractive approach for absolute quantification of nucleic acids with high sensitivity and selectivity. Theoretical and numerical analysis of dLAMP provides necessary guidance for the design and analysis of dLAMP devices. In this work, a mathematical model was proposed on the basis of the Monte Carlo method and the theories of Poisson statistics and chemometrics. To examine the established model, we fabricated a spiral chip with 1200 uniform and discrete reaction chambers (9.6 nL) for absolute quantification of pathogenic DNA samples by dLAMP. Under the optimized conditions, dLAMP analysis on the spiral chip realized quantification of nucleic acids spanning over 4 orders of magnitude in concentration with sensitivity as low as 8.7 × 10(-2) copies/μL in 40 min. The experimental results were consistent with the proposed mathematical model, which could provide useful guideline for future development of dLAMP devices.

  15. System and method for calibrating a rotary absolute position sensor

    NASA Technical Reports Server (NTRS)

    Davis, Donald R. (Inventor); Permenter, Frank Noble (Inventor); Radford, Nicolaus A (Inventor)

    2012-01-01

    A system includes a rotary device, a rotary absolute position (RAP) sensor generating encoded pairs of voltage signals describing positional data of the rotary device, a host machine, and an algorithm. The algorithm calculates calibration parameters usable to determine an absolute position of the rotary device using the encoded pairs, and is adapted for linearly-mapping an ellipse defined by the encoded pairs to thereby calculate the calibration parameters. A method of calibrating the RAP sensor includes measuring the rotary position as encoded pairs of voltage signals, linearly-mapping an ellipse defined by the encoded pairs to thereby calculate the calibration parameters, and calculating an absolute position of the rotary device using the calibration parameters. The calibration parameters include a positive definite matrix (A) and a center point (q) of the ellipse. The voltage signals may include an encoded sine and cosine of a rotary angle of the rotary device.

  16. Method and apparatus for two-dimensional absolute optical encoding

    NASA Technical Reports Server (NTRS)

    Leviton, Douglas B. (Inventor)

    2004-01-01

    This invention presents a two-dimensional absolute optical encoder and a method for determining position of an object in accordance with information from the encoder. The encoder of the present invention comprises a scale having a pattern being predetermined to indicate an absolute location on the scale, means for illuminating the scale, means for forming an image of the pattern; and detector means for outputting signals derived from the portion of the image of the pattern which lies within a field of view of the detector means, the field of view defining an image reference coordinate system, and analyzing means, receiving the signals from the detector means, for determining the absolute location of the object. There are two types of scale patterns presented in this invention: grid type and starfield type.

  17. Neutron activation analysis of certified samples by the absolute method

    NASA Astrophysics Data System (ADS)

    Kadem, F.; Belouadah, N.; Idiri, Z.

    2015-07-01

    The nuclear reactions analysis technique is mainly based on the relative method or the use of activation cross sections. In order to validate nuclear data for the calculated cross section evaluated from systematic studies, we used the neutron activation analysis technique (NAA) to determine the various constituent concentrations of certified samples for animal blood, milk and hay. In this analysis, the absolute method is used. The neutron activation technique involves irradiating the sample and subsequently performing a measurement of the activity of the sample. The fundamental equation of the activation connects several physical parameters including the cross section that is essential for the quantitative determination of the different elements composing the sample without resorting to the use of standard sample. Called the absolute method, it allows a measurement as accurate as the relative method. The results obtained by the absolute method showed that the values are as precise as the relative method requiring the use of standard sample for each element to be quantified.

  18. Progress Report of CNES Activities Regarding the Absolute Calibration Method

    DTIC Science & Technology

    2010-11-01

    several receivers (Ashtech Z12-T, Septentrio PolaRx2, and Dicom GTR50) and a GNSS signal simulator (Spirent 4760) according to the temperature and...laboratories, Ashtech Z12- T, Septentrio PolaRx2, and Dicom GTR50, can be calibrated with the absolute method [6,8]. The last works concerned the...Ashtech, Septentrio, and Dicom receiver calibrations. Table 2. Uncertainty of the different receiver calibrations. Uncertainty Source

  19. Chemo-Enzymatic Synthesis of (13)C Labeled Complex N-Glycans As Internal Standards for the Absolute Glycan Quantification by Mass Spectrometry.

    PubMed

    Echeverria, Begoña; Etxebarria, Juan; Ruiz, Nerea; Hernandez, Álvaro; Calvo, Javier; Haberger, Markus; Reusch, Dietmar; Reichardt, Niels-Christian

    2015-11-17

    Methods for the absolute quantification of glycans are needed in glycoproteomics, during development and production of biopharmaceuticals and for the clinical analysis of glycan disease markers. Here we present a strategy for the chemo-enzymatic synthesis of (13)C labeled N-glycan libraries and provide an example for their use as internal standards in the profiling and absolute quantification of mAb glycans by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. A synthetic biantennary glycan precursor was (13)C-labeled on all four amino sugar residues and enzymatically derivatized to produce a library of 15 glycan isotopologues with a mass increment of 8 Da over the natural products. Asymmetrically elongated glycans were accessible by performing enzymatic reactions on partially protected UV-absorbing intermediates, subsequent fractionation by preparative HPLC, and final hydrogenation. Using a preformulated mixture of eight internal standards, we quantified the glycans in a monoclonal therapeutic antibody with excellent precision and speed.

  20. Absolute Quantification of Rifampicin by MALDI Imaging Mass Spectrometry Using Multiple TOF/TOF Events in a Single Laser Shot.

    PubMed

    Prentice, Boone M; Chumbley, Chad W; Caprioli, Richard M

    2017-01-01

    Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) allows for the visualization of molecular distributions within tissue sections. While providing excellent molecular specificity and spatial information, absolute quantification by MALDI IMS remains challenging. Especially in the low molecular weight region of the spectrum, analysis is complicated by matrix interferences and ionization suppression. Though tandem mass spectrometry (MS/MS) can be used to ensure chemical specificity and improve sensitivity by eliminating chemical noise, typical MALDI MS/MS modalities only scan for a single MS/MS event per laser shot. Herein, we describe TOF/TOF instrumentation that enables multiple fragmentation events to be performed in a single laser shot, allowing the intensity of the analyte to be referenced to the intensity of the internal standard in each laser shot while maintaining the benefits of MS/MS. This approach is illustrated by the quantitative analyses of rifampicin (RIF), an antibiotic used to treat tuberculosis, in pooled human plasma using rifapentine (RPT) as an internal standard. The results show greater than 4-fold improvements in relative standard deviation as well as improved coefficients of determination (R(2)) and accuracy (>93% quality controls, <9% relative errors). This technology is used as an imaging modality to measure absolute RIF concentrations in liver tissue from an animal dosed in vivo. Each microspot in the quantitative image measures the local RIF concentration in the tissue section, providing absolute pixel-to-pixel quantification from different tissue microenvironments. The average concentration determined by IMS is in agreement with the concentration determined by HPLC-MS/MS, showing a percent difference of 10.6%. Graphical Abstract ᅟ.

  1. Absolute Quantification of Rifampicin by MALDI Imaging Mass Spectrometry Using Multiple TOF/TOF Events in a Single Laser Shot

    NASA Astrophysics Data System (ADS)

    Prentice, Boone M.; Chumbley, Chad W.; Caprioli, Richard M.

    2017-01-01

    Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) allows for the visualization of molecular distributions within tissue sections. While providing excellent molecular specificity and spatial information, absolute quantification by MALDI IMS remains challenging. Especially in the low molecular weight region of the spectrum, analysis is complicated by matrix interferences and ionization suppression. Though tandem mass spectrometry (MS/MS) can be used to ensure chemical specificity and improve sensitivity by eliminating chemical noise, typical MALDI MS/MS modalities only scan for a single MS/MS event per laser shot. Herein, we describe TOF/TOF instrumentation that enables multiple fragmentation events to be performed in a single laser shot, allowing the intensity of the analyte to be referenced to the intensity of the internal standard in each laser shot while maintaining the benefits of MS/MS. This approach is illustrated by the quantitative analyses of rifampicin (RIF), an antibiotic used to treat tuberculosis, in pooled human plasma using rifapentine (RPT) as an internal standard. The results show greater than 4-fold improvements in relative standard deviation as well as improved coefficients of determination (R2) and accuracy (>93% quality controls, <9% relative errors). This technology is used as an imaging modality to measure absolute RIF concentrations in liver tissue from an animal dosed in vivo. Each microspot in the quantitative image measures the local RIF concentration in the tissue section, providing absolute pixel-to-pixel quantification from different tissue microenvironments. The average concentration determined by IMS is in agreement with the concentration determined by HPLC-MS/MS, showing a percent difference of 10.6%.

  2. Method of differential-phase/absolute-amplitude QAM

    DOEpatents

    Dimsdle, Jeffrey William

    2008-10-21

    A method of quadrature amplitude modulation involving encoding phase differentially and amplitude absolutely, allowing for a high data rate and spectral efficiency in data transmission and other communication applications, and allowing for amplitude scaling to facilitate data recovery; amplitude scale tracking to track-out rapid and severe scale variations and facilitate successful demodulation and data retrieval; 2.sup.N power carrier recovery; incoherent demodulation where coherent carrier recovery is not possible or practical due to signal degradation; coherent demodulation; multipath equalization to equalize frequency dependent multipath; and demodulation filtering.

  3. Method of differential-phase/absolute-amplitude QAM

    DOEpatents

    Dimsdle, Jeffrey William

    2007-07-03

    A method of quadrature amplitude modulation involving encoding phase differentially and amplitude absolutely, allowing for a high data rate and spectral efficiency in data transmission and other communication applications, and allowing for amplitude scaling to facilitate data recovery; amplitude scale tracking to track-out rapid and severe scale variations and facilitate successful demodulation and data retrieval; 2.sup.N power carrier recovery; incoherent demodulation where coherent carrier recovery is not possible or practical due to signal degradation; coherent demodulation; multipath equalization to equalize frequency dependent multipath; and demodulation filtering.

  4. Method of differential-phase/absolute-amplitude QAM

    DOEpatents

    Dimsdle, Jeffrey William

    2007-10-02

    A method of quadrature amplitude modulation involving encoding phase differentially and amplitude absolutely, allowing for a high data rate and spectral efficiency in data transmission and other communication applications, and allowing for amplitude scaling to facilitate data recovery; amplitude scale tracking to track-out rapid and severe scale variations and facilitate successful demodulation and data retrieval; 2.sup.N power carrier recovery; incoherent demodulation where coherent carrier recovery is not possible or practical due to signal degradation; coherent demodulation; multipath equalization to equalize frequency dependent multipath; and demodulation filtering.

  5. Method of differential-phase/absolute-amplitude QAM

    SciTech Connect

    Dimsdle, Jeffrey William

    2009-09-01

    A method of quadrature amplitude modulation involving encoding phase differentially and amplitude absolutely, allowing for a high data rate and spectral efficiency in data transmission and other communication applications, and allowing for amplitude scaling to facilitate data recovery; amplitude scale tracking to track-out rapid and severe scale variations and facilitate successful demodulation and data retrieval; 2.sup.N power carrier recovery; incoherent demodulation where coherent carrier recovery is not possible or practical due to signal degradation; coherent demodulation; multipath equalization to equalize frequency dependent multipath; and demodulation filtering.

  6. Method of differential-phase/absolute-amplitude QAM

    DOEpatents

    Dimsdle, Jeffrey William

    2007-07-17

    A method of quadrature amplitude modulation involving encoding phase differentially and amplitude absolutely, allowing for a high data rate and spectral efficiency in data transmission and other communication applications, and allowing for amplitude scaling to facilitate data recovery; amplitude scale tracking to track-out rapid and severe scale variations and facilitate successful demodulation and data retrieval; 2.sup.N power carrier recovery; incoherent demodulation where coherent carrier recovery is not possible or practical due to signal degradation; coherent demodulation; multipath equalization to equalize frequency dependent multipath; and demodulation filtering.

  7. Tutorial examples for uncertainty quantification methods.

    SciTech Connect

    De Bord, Sarah

    2015-08-01

    This report details the work accomplished during my 2015 SULI summer internship at Sandia National Laboratories in Livermore, CA. During this internship, I worked on multiple tasks with the common goal of making uncertainty quantification (UQ) methods more accessible to the general scientific community. As part of my work, I created a comprehensive numerical integration example to incorporate into the user manual of a UQ software package. Further, I developed examples involving heat transfer through a window to incorporate into tutorial lectures that serve as an introduction to UQ methods.

  8. Absolute quantification of myosin heavy chain isoforms by selected reaction monitoring can underscore skeletal muscle changes in a mouse model of amyotrophic lateral sclerosis.

    PubMed

    Peggion, Caterina; Massimino, Maria Lina; Biancotto, Giancarlo; Angeletti, Roberto; Reggiani, Carlo; Sorgato, Maria Catia; Bertoli, Alessandro; Stella, Roberto

    2017-03-01

    Skeletal muscle fibers contain different isoforms of myosin heavy chain (MyHC) that define distinctive contractile properties. In light of the muscle capacity to adapt MyHC expression to pathophysiological conditions, a rapid and quantitative assessment of MyHC isoforms in small muscle tissue quantities would represent a valuable diagnostic tool for (neuro)muscular diseases. As past protocols did not meet these requirements, in the present study we applied a targeted proteomic approach based on selected reaction monitoring that allowed the absolute quantification of slow and fast MyHC isoforms in different mouse skeletal muscles with high reproducibility. This mass-spectrometry-based method was validated also in a pathological specimen, by comparison of the MyHC expression profiles in different muscles from healthy mice and a genetic mouse model of amyotrophic lateral sclerosis (ALS) expressing the SOD1(G93A) mutant. This analysis showed that terminally ill ALS mice have a fast-to-slow shift in the fiber type composition of the tibialis anterior and gastrocnemius muscles, as previously reported. These results will likely open the way to accurate and rapid diagnoses of human (neuro)muscular diseases by the proposed method. Graphical Abstract Methods for myosin heavy chain (MyHC) quantification: a comparison of classical methods and selected reaction monitoring (SRM)-based mass spectrometry approaches.

  9. Method and apparatus for making absolute range measurements

    DOEpatents

    Earl, Dennis D [Knoxville, TN; Allison, Stephen W [Knoxville, TN; Cates, Michael R [Oak Ridge, TN; Sanders, Alvin J [Knoxville, TN

    2002-09-24

    This invention relates to a method and apparatus for making absolute distance or ranging measurements using Fresnel diffraction. The invention employs a source of electromagnetic radiation having a known wavelength or wavelength distribution, which sends a beam of electromagnetic radiation through a screen at least partially opaque at the wavelength. The screen has an aperture sized so as to produce a Fresnel diffraction pattern. A portion of the beam travels through the aperture to a detector spaced some distance from the screen. The detector detects the central intensity of the beam as well as a set of intensities displaced from a center of the aperture. The distance from the source to the target can then be calculated based upon the known wavelength, aperture radius, and beam intensity.

  10. Music Proficiency and Quantification of Absolute Pitch: A Large-Scale Study among Brazilian Musicians.

    PubMed

    Leite, Raphael B C; Mota-Rolim, Sergio A; Queiroz, Claudio M T

    2016-01-01

    Absolute pitch (AP) is the ability to identify and name the pitch of a sound without external reference. Often, accuracy and speed at naming isolated musical pitches are correlated with demographic, biological, and acoustical parameters to gain insight into the genesis and evolution of this ability in specific cohorts. However, the majority of those studies were conducted in North America, Europe, or Asia. To fill this gap, here we investigated the pitch-naming performance in a large population of Brazilian conservatory musicians (N = 200). As previously shown, we found that the population performance was rather a continuum than an "all-or-none" ability. By comparing the observed distribution of correct responses to a theoretical binomial distribution, we estimated the prevalence of AP as being 18% amongst regular music students. High accuracy thresholds (e.g., 85% of correct responses) yielded a prevalence of 4%, suggesting that AP might have been underestimated in previous reports. Irrespective of the threshold used, AP prevalence was higher in musicians who started their musical practice and formal musical education early in life. Finally, we compared the performance of those music students (average proficiency group) with another group of students selected to take part in the conservatory orchestra (high proficiency group, N = 30). Interestingly, the prevalence of AP was higher in the latter in comparison to the former group. In addition, even when the response was incorrect, the mean absolute deviation from the correct response was smaller in the high proficiency group compared to the average proficiency group (Glass's Δ: 0.5). Taken together, our results show that the prevalence of AP in Brazilian students is similar to other non-tonal language populations, although this measure is highly dependent on the scoring threshold used. Despite corroborating that early involvement with musical practice and formal education can foster AP ability, the present data

  11. Music Proficiency and Quantification of Absolute Pitch: A Large-Scale Study among Brazilian Musicians

    PubMed Central

    Leite, Raphael B. C.; Mota-Rolim, Sergio A.; Queiroz, Claudio M. T.

    2016-01-01

    Absolute pitch (AP) is the ability to identify and name the pitch of a sound without external reference. Often, accuracy and speed at naming isolated musical pitches are correlated with demographic, biological, and acoustical parameters to gain insight into the genesis and evolution of this ability in specific cohorts. However, the majority of those studies were conducted in North America, Europe, or Asia. To fill this gap, here we investigated the pitch-naming performance in a large population of Brazilian conservatory musicians (N = 200). As previously shown, we found that the population performance was rather a continuum than an “all-or-none” ability. By comparing the observed distribution of correct responses to a theoretical binomial distribution, we estimated the prevalence of AP as being 18% amongst regular music students. High accuracy thresholds (e.g., 85% of correct responses) yielded a prevalence of 4%, suggesting that AP might have been underestimated in previous reports. Irrespective of the threshold used, AP prevalence was higher in musicians who started their musical practice and formal musical education early in life. Finally, we compared the performance of those music students (average proficiency group) with another group of students selected to take part in the conservatory orchestra (high proficiency group, N = 30). Interestingly, the prevalence of AP was higher in the latter in comparison to the former group. In addition, even when the response was incorrect, the mean absolute deviation from the correct response was smaller in the high proficiency group compared to the average proficiency group (Glass's Δ: 0.5). Taken together, our results show that the prevalence of AP in Brazilian students is similar to other non-tonal language populations, although this measure is highly dependent on the scoring threshold used. Despite corroborating that early involvement with musical practice and formal education can foster AP ability, the present data

  12. Comprehensive Absolute Quantification of the Cytosolic Proteome of Bacillus subtilis by Data Independent, Parallel Fragmentation in Liquid Chromatography/Mass Spectrometry (LC/MSE)*

    PubMed Central

    Muntel, Jan; Fromion, Vincent; Goelzer, Anne; Maaβ, Sandra; Mäder, Ulrike; Büttner, Knut; Hecker, Michael; Becher, Dörte

    2014-01-01

    In the growing field of systems biology, the knowledge of protein concentrations is highly required to truly understand metabolic and adaptational networks within the cells. Therefore we established a workflow relying on long chromatographic separation and mass spectrometric analysis by data independent, parallel fragmentation of all precursor ions at the same time (LC/MSE). By prevention of discrimination of co-eluting low and high abundant peptides a high average sequence coverage of 40% could be achieved, resulting in identification of almost half of the predicted cytosolic proteome of the Gram-positive model organism Bacillus subtilis (>1,050 proteins). Absolute quantification was achieved by correlation of average MS signal intensities of the three most intense peptides of a protein to the signal intensity of a spiked standard protein digest. Comparative analysis with heavily labeled peptides (AQUA approach) showed the use of only one standard digest is sufficient for global quantification. The quantification results covered almost four orders of magnitude, ranging roughly from 10 to 150,000 copies per cell. To prove this method for its biological relevance selected physiological aspects of B. subtilis cells grown under conditions requiring either amino acid synthesis or alternatively amino acid degradation were analyzed. This allowed both in particular the validation of the adjustment of protein levels by known regulatory events and in general a perspective of new insights into bacterial physiology. Within new findings the analysis of “protein costs” of cellular processes is extremely important. Such a comprehensive and detailed characterization of cellular protein concentrations based on data independent, parallel fragmentation in liquid chromatography/mass spectrometry (LC/MSE) data has been performed for the first time and should pave the way for future comprehensive quantitative characterization of microorganisms as physiological entities. PMID

  13. A regularized method for peptide quantification.

    PubMed

    Yang, Chao; Yang, Can; Yu, Weichuan

    2010-05-07

    Peptide abundance estimation is generally the first step in protein quantification. In peptide abundance estimation, peptide overlapping and peak intensity variation are two challenges. The main objective of this paper is to estimate peptide abundance by taking advantage of peptide isotopic distribution and smoothness of peptide elution profile. Our method proposes to solve the peptide overlapping problem and provides a way to control the variance of estimation. We compare our method with a commonly used method on simulated data sets and two real data sets of standard protein mixtures. The results show that our method achieves more accurate estimation of peptide abundance on different samples. In our method, there is a variance-related parameter. Considering the well-known trade-off between the variance and the bias of estimation, we should not only focus on reducing the variance in real applications. A suggestion about parameter selection is given based on the discussion of variance and bias. Matlab source codes and detailed experimental results are available at http://bioinformatics.ust.hk/PeptideQuant/peptidequant.htm.

  14. Comparison of analysis methods for airway quantification

    NASA Astrophysics Data System (ADS)

    Odry, Benjamin L.; Kiraly, Atilla P.; Novak, Carol L.; Naidich, David P.

    2012-03-01

    Diseased airways have been known for several years as a possible contributing factor to airflow limitation in Chronic Obstructive Pulmonary Diseases (COPD). Quantification of disease severity through the evaluation of airway dimensions - wall thickness and lumen diameter - has gained increased attention, thanks to the availability of multi-slice computed tomography (CT). Novel approaches have focused on automated methods of measurement as a faster and more objective means that the visual assessment routinely employed in the clinic. Since the Full-Width Half-Maximum (FWHM) method of airway measurement was introduced two decades ago [1], several new techniques for quantifying airways have been detailed in the literature, but no approach has truly become a standard for such analysis. Our own research group has presented two alternative approaches for determining airway dimensions, one involving a minimum path and the other active contours [2, 3]. With an increasing number of techniques dedicated to the same goal, we decided to take a step back and analyze the differences of these methods. We consequently put to the test our two methods of analysis and the FWHM approach. We first measured a set of 5 airways from a phantom of known dimensions. Then we compared measurements from the three methods to those of two independent readers, performed on 35 airways in 5 patients. We elaborate on the differences of each approach and suggest conclusions on which could be defined as the best one.

  15. Weighted Wilcoxon-type Smoothly Clipped Absolute Deviation Method

    PubMed Central

    Wang, Lan; Li, Runze

    2009-01-01

    Summary Shrinkage-type variable selection procedures have recently seen increasing applications in biomedical research. However, their performance can be adversely influenced by outliers in either the response or the covariate space. This paper proposes a weighted Wilcoxon-type smoothly clipped absolute deviation (WW-SCAD) method, which deals with robust variable selection and robust estimation simultaneously. The new procedure can be conveniently implemented with the statistical software R. We establish that the WW-SCAD correctly identifies the set of zero coefficients with probability approaching one and estimates the nonzero coefficients with the rate n−1/2. Moreover, with appropriately chosen weights the WW-SCAD is robust with respect to outliers in both the x and y directions. The important special case with constant weights yields an oracle-type estimator with high efficiency at the presence of heavier-tailed random errors. The robustness of the WW-SCAD is partly justified by its asymptotic performance under local shrinking contamination. We propose a BIC-type tuning parameter selector for the WW-SCAD. The performance of the WW-SCAD is demonstrated via simulations and by an application to a study that investigates the effects of personal characteristics and dietary factors on plasma beta-carotene level. PMID:18647294

  16. Calibration method of absolute orientation of camera optical axis

    NASA Astrophysics Data System (ADS)

    Xu, Yong; Guo, Pengyu; Zhang, Xiaohu; Ding, Shaowen; Su, Ang; Li, Lichun

    2013-08-01

    Camera calibration is one of the most basic and important processes in optical measuring field. Generally, the objective of camera calibration is to estimate the internal and external parameters of object cameras, while the orientation error of optical axis is not included yet. Orientation error of optical axis is a important factor, which seriously affects measuring precision in high-precision measurement field, especially for those distant aerospace measurement in which object distance is much longer than focal length, that lead to magnifying the orientation errors to thousands times. In order to eliminate the influence of orientation error of camera optical axis, the imaging model of camera is analysed and established in this paper, and the calibration method is also introduced: Firstly, we analyse the reasons that cause optical axis error and its influence. Then, we find the model of optical axis orientation error and imaging model of camera basing on it's practical physical meaning. Furthermore, we derive the bundle adjustment algorithm which could compute the internal and external camera parameters and absolute orientation of camera optical axis simultaneously at high precision. In numeric simulation, we solve the camera parameters by using bundle adjustment optimization algorithm, then we correct the image points by calibration results according to the model of optical axis error, and the simulation result shows that our calibration model is reliable, effective and precise.

  17. Absolute Quantification of Human Liver Phosphorus-Containing Metabolites In Vivo Using an Inhomogeneous Spoiling Magnetic Field Gradient

    PubMed Central

    Bashir, Adil; Gropler, Robert; Ackerman, Joseph

    2015-01-01

    Purpose Absolute concentrations of high-energy phosphorus (31P) metabolites in liver provide more important insight into physiologic status of liver disease compared to resonance integral ratios. A simple method for measuring absolute concentrations of 31P metabolites in human liver is described. The approach uses surface spoiling inhomogeneous magnetic field gradient to select signal from liver tissue. The technique avoids issues caused by respiratory motion, chemical shift dispersion associated with linear magnetic field gradients, and increased tissue heat deposition due to radiofrequency absorption, especially at high field strength. Methods A method to localize signal from liver was demonstrated using superficial and highly non-uniform magnetic field gradients, which eliminate signal(s) from surface tissue(s) located between the liver and RF coil. A double standard method was implemented to determine absolute 31P metabolite concentrations in vivo. 8 healthy individuals were examined in a 3 T MR scanner. Results Concentrations of metabolites measured in eight healthy individuals are: γ-adenosine triphosphate (ATP) = 2.44 ± 0.21 (mean ± sd) mmol/l of wet tissue volume, α-ATP = 3.2 ± 0.63 mmol/l, β-ATP = 2.98 ± 0.45 mmol/l, inorganic phosphates (Pi) = 1.87 ± 0.25 mmol/l, phosphodiesters (PDE) = 10.62 ± 2.20 mmol/l and phosphomonoesters (PME) = 2.12 ± 0.51 mmol/l. All are in good agreement with literature values. Conclusions The technique offers robust and fast means to localize signal from liver tissue, allows absolute metabolite concentration determination, and avoids problems associated with constant field gradient (linear field variation) localization methods. PMID:26633549

  18. Elucidation of Xylem-Specific Transcription Factors and Absolute Quantification of Enzymes Regulating Cellulose Biosynthesis in Populus trichocarpa.

    PubMed

    Loziuk, Philip L; Parker, Jennifer; Li, Wei; Lin, Chien-Yuan; Wang, Jack P; Li, Quanzi; Sederoff, Ronald R; Chiang, Vincent L; Muddiman, David C

    2015-10-02

    Cellulose, the main chemical polymer of wood, is the most abundant polysaccharide in nature.1 The ability to perturb the abundance and structure of cellulose microfibrils is of critical importance to the pulp and paper industry as well as for the textile, wood products, and liquid biofuels industries. Although much has been learned at the transcript level about the biosynthesis of cellulose, a quantitative understanding at the proteome level has yet to be established. The study described herein sought to identify the proteins directly involved in cellulose biosynthesis during wood formation in Populus trichocarpa along with known xylem-specific transcription factors involved in regulating these key proteins. Development of an effective discovery proteomic strategy through a combination of subcellular fractionation of stem differentiating xylem tissue (SDX) with recently optimized FASP digestion protocols, StageTip fractionation, as well as optimized instrument parameters for global proteomic analysis using the quadrupole-orbitrap mass spectrometer resulted in the deepest proteomic coverage of SDX protein from P. trichocarpa with 9,146 protein groups being identified (1% FDR). Of these, 20 cellulosic/hemicellulosic enzymes and 43 xylem-specific transcription factor groups were identified. Finally, selection of surrogate peptides led to an assay for absolute quantification of 14 cellulosic proteins in SDX of P. trichocarpa.

  19. Two methods for absolute calibration of dynamic pressure transducers

    NASA Astrophysics Data System (ADS)

    Swift, G. W.; Migliori, A.; Garrett, S. L.; Wheatley, J. C.

    1982-12-01

    Two techniques are described for absolute calibration of a dynamic pressure transducer from 0 to 400 Hz in 1-MPa helium gas. One technique is based on a comparison to a mercury manometer; the other is based on the principle of reciprocity. The two techniques agree within the instrumental uncertainties of 1%.

  20. Single-track absolute position encoding method based on spatial frequency of stripes

    NASA Astrophysics Data System (ADS)

    Xiang, Xiansong; Lu, Yancong; Wei, Chunlong; Zhou, Changhe

    2014-11-01

    A new method of single-track absolute position encoding based on spatial frequency of stripes is proposed. Instead of using pseudorandom-sequence arranged stripes as in conventional situations, this kind of encoding method stores the location information in the frequency space of the stripes, which means the spatial frequency of stripes varies with position and indicates position. This encoding method has a strong fault-tolerant capability with single-stripe detecting errors. The method can be applied to absolute linear encoders, absolute photoelectric angle encoders or two-dimensional absolute linear encoders. The measuring apparatus includes a CCD image sensor and a microscope system, and the method of decoding this frequency code is based on FFT algorithm. This method should be highly interesting for practical applications as an absolute position encoding method.

  1. Noninvasive optical quantification of absolute blood flow, blood oxygenation, and oxygen consumption rate in exercising skeletal muscle

    PubMed Central

    Gurley, Katelyn; Shang, Yu

    2012-01-01

    Abstract. This study investigates a method using novel hybrid diffuse optical spectroscopies [near-infrared spectroscopy (NIRS) and diffuse correlation spectroscopy (DCS)] to obtain continuous, noninvasive measurement of absolute blood flow (BF), blood oxygenation, and oxygen consumption rate (V˙O2) in exercising skeletal muscle. Healthy subjects (n=9) performed a handgrip exercise to increase BF and V˙O2 in forearm flexor muscles, while a hybrid optical probe on the skin surface directly monitored oxy-, deoxy-, and total hemoglobin concentrations ([HbO2], [Hb], and THC), tissue oxygen saturation (StO2), relative BF (rBF), and relative oxygen consumption rate (rV˙O2). The rBF and rV˙O2 signals were calibrated with absolute baseline BF and V˙O2 obtained through venous and arterial occlusions, respectively. Known problems with muscle-fiber motion artifacts in optical measurements during exercise were mitigated using a novel gating algorithm that determined muscle contraction status based on control signals from a dynamometer. Results were consistent with previous findings in the literature. This study supports the application of NIRS/DCS technology to quantitatively evaluate hemodynamic and metabolic parameters in exercising skeletal muscle and holds promise for improving diagnosis and treatment evaluation for patients suffering from diseases affecting skeletal muscle and advancing fundamental understanding of muscle and exercise physiology. PMID:22894482

  2. Noninvasive optical quantification of absolute blood flow, blood oxygenation, and oxygen consumption rate in exercising skeletal muscle

    NASA Astrophysics Data System (ADS)

    Gurley, Katelyn; Shang, Yu; Yu, Guoqiang

    2012-07-01

    This study investigates a method using novel hybrid diffuse optical spectroscopies [near-infrared spectroscopy (NIRS) and diffuse correlation spectroscopy (DCS)] to obtain continuous, noninvasive measurement of absolute blood flow (BF), blood oxygenation, and oxygen consumption rate (\\Vdot O2) in exercising skeletal muscle. Healthy subjects (n=9) performed a handgrip exercise to increase BF and \\Vdot O2 in forearm flexor muscles, while a hybrid optical probe on the skin surface directly monitored oxy-, deoxy-, and total hemoglobin concentrations ([HbO2], [Hb], and THC), tissue oxygen saturation (StO2), relative BF (rBF), and relative oxygen consumption rate (r\\Vdot O2). The rBF and r\\Vdot O2 signals were calibrated with absolute baseline BF and \\Vdot O2 obtained through venous and arterial occlusions, respectively. Known problems with muscle-fiber motion artifacts in optical measurements during exercise were mitigated using a novel gating algorithm that determined muscle contraction status based on control signals from a dynamometer. Results were consistent with previous findings in the literature. This study supports the application of NIRS/DCS technology to quantitatively evaluate hemodynamic and metabolic parameters in exercising skeletal muscle and holds promise for improving diagnosis and treatment evaluation for patients suffering from diseases affecting skeletal muscle and advancing fundamental understanding of muscle and exercise physiology.

  3. Absolute quantification of Pru av 2 in sweet cherry fruit by liquid chromatography/tandem mass spectrometry with the use of a stable isotope-labelled peptide.

    PubMed

    Ippoushi, Katsunari; Sasanuma, Motoe; Oike, Hideaki; Kobori, Masuko; Maeda-Yamamoto, Mari

    2016-08-01

    Pru av 2, a pathogenesis-related (PR) protein present in the sweet cherry (Prunus avium L.) fruit, is the principal allergen of cherry and one of the chief causes of pollen food syndrome (oral allergy syndrome). In this study, a quantitative assay for this protein was developed with the use of the protein absolute quantification (AQUA) method, which consists of liquid chromatography/tandem mass spectrometry (LC/MS/MS) employing TGC[CAM]STDASGK[(13)C6,(15)N2], a stable isotope-labelled internal standard (SIIS) peptide. This assay gave a linear relationship (r(2)>0.99) in a concentration range (2.3-600fmol/μL), and the overall coefficient of variation (CV) for multiple tests was 14.6%. Thus, the contents of this allergenic protein in sweet cherry products could be determined using this assay. This assay should be valuable for allergological investigations of Pru av 2 in sweet cherry and detection of protein contamination in foods.

  4. A quick colorimetric method for total lipid quantification in microalgae.

    PubMed

    Byreddy, Avinesh R; Gupta, Adarsha; Barrow, Colin J; Puri, Munish

    2016-06-01

    Discovering microalgae with high lipid productivity are among the key milestones for achieving sustainable biodiesel production. Current methods of lipid quantification are time intensive and costly. A rapid colorimetric method based on sulfo-phospho-vanillin (SPV) reaction was developed for the quantification of microbial lipids to facilitate screening for lipid producing microalgae. This method was successfully tested on marine thraustochytrid strains and vegetable oils. The colorimetric method results correlated well with gravimetric method estimates. The new method was less time consuming than gravimetric analysis and is quantitative for lipid determination, even in the presence of carbohydrates, proteins and glycerol.

  5. A detailed analysis of next generation sequencing reads of microRNA expression in Barrett’s Esophagus: absolute versus relative quantification

    PubMed Central

    2014-01-01

    Background Next generation sequencing (NGS) is a state of the art technology for microRNA (miRNA) analysis. The quantitative interpretation of the primary output of NGS i.e. the read counts for a miRNA sequence that can vary by several orders of magnitude (1 to 107) remains incompletely understood. Findings NGS (SOLiD 3 technology) was performed on biopsies from 6 Barrett’s esophagus (BE) and 5 Gastroesophageal Reflux Disease (GERD) patients. Read sequences were aligned to miRBase 18.0. Differential expression analysis was adjusted for false discovery rate of 5%. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed for 36 miRNA in a validation cohort of 47 patients (27 BE and 20 GERD). Correlation coefficients, accuracy, precision and recall of NGS compared to qRT-PCR were calculated. Increase in NGS reads was associated with progressively lower Cq values, p < 0.05. Although absolute quantification between NGS reads and Cq values correlated modestly: -0.38, p = 0.01 for BE and -0.32, p = 0.05 for GERD, relative quantification (fold changes) of miRNA expression between BE &GERD by NGS correlated highly with qRT-PCR 0.86, p = 2.45E-11. Fold change correlations were unaffected when different thresholds of NGS read counts were compared (>1000 vs. <1000, >500 vs. <500 and >100 vs. <100). The accuracy, precision and recall of NGS to label a miRNA as differentially expressed were 0.71, 0.88 and 0.74 respectively. Conclusion Absolute NGS reads correlated modestly with qRT-PCR but fold changes correlated highly. NGS is robust at relative but not absolute quantification of miRNA levels and accurate for high-throughput identification of differentially expressed miRNA. PMID:24708854

  6. Absolute Quantification of Prion Protein (90-231) Using Stable Isotope-Labeled Chymotryptic Peptide Standards in a LC-MRM AQUA Workflow

    NASA Astrophysics Data System (ADS)

    Sturm, Robert; Sheynkman, Gloria; Booth, Clarissa; Smith, Lloyd M.; Pedersen, Joel A.; Li, Lingjun

    2012-09-01

    Substantial evidence indicates that the disease-associated conformer of the prion protein (PrPTSE) constitutes the etiologic agent in prion diseases. These diseases affect multiple mammalian species. PrPTSE has the ability to convert the conformation of the normal prion protein (PrPC) into a β-sheet rich form resistant to proteinase K digestion. Common immunological techniques lack the sensitivity to detect PrPTSE at subfemtomole levels, whereas animal bioassays, cell culture, and in vitro conversion assays offer higher sensitivity but lack the high-throughput the immunological assays offer. Mass spectrometry is an attractive alternative to the above assays as it offers high-throughput, direct measurement of a protein's signature peptide, often with subfemtomole sensitivities. Although a liquid chromatography-multiple reaction monitoring (LC-MRM) method has been reported for PrPTSE, the chemical composition and lack of amino acid sequence conservation of the signature peptide may compromise its accuracy and make it difficult to apply to multiple species. Here, we demonstrate that an alternative protease (chymotrypsin) can produce signature peptides suitable for a LC-MRM absolute quantification (AQUA) experiment. The new method offers several advantages, including: (1) a chymotryptic signature peptide lacking chemically active residues (Cys, Met) that can confound assay accuracy; (2) low attomole limits of detection and quantitation (LOD and LOQ); and (3) a signature peptide retaining the same amino acid sequence across most mammals naturally susceptible to prion infection as well as important laboratory models. To the authors' knowledge, this is the first report on the use of a non-tryptic peptide in a LC-MRM AQUA workflow.

  7. Methods to calibrate the absolute receive sensitivity of single-element, focused transducers.

    PubMed

    Rich, Kyle T; Mast, T Douglas

    2015-09-01

    Absolute pressure measurements of acoustic emissions by single-element, focused passive cavitation detectors would be facilitated by improved wideband receive calibration techniques. Here, calibration methods were developed to characterize the absolute, frequency-dependent receive sensitivity of a spherically focused, single-element transducer using pulse-echo and pitch-catch techniques. Validation of these calibration methods on a focused receiver were made by generating a pulse from a small diameter source at the focus of the transducer and comparing the absolute pressure measured by a calibrated hydrophone to that of the focused transducer using the receive sensitivities determined here.

  8. Lignin-degrading peroxidases in white-rot fungus Trametes hirsuta 072. Absolute expression quantification of full multigene family

    PubMed Central

    Vasina, Daria V.; Moiseenko, Konstantin V.; Fedorova, Tatiana V.; Tyazhelova, Tatiana V.

    2017-01-01

    Ligninolytic heme peroxidases comprise an extensive family of enzymes, which production is characteristic for white-rot Basidiomycota. The majority of fungal heme peroxidases are encoded by multigene families that differentially express closely related proteins. Currently, there were very few attempts to characterize the complete multigene family of heme peroxidases in a single fungus. Here we are focusing on identification and characterization of peroxidase genes, which are transcribed and secreted by basidiomycete Trametes hirsuta 072, an efficient lignin degrader. The T. hirsuta genome contains 18 ligninolytic peroxidase genes encoding 9 putative lignin peroxidases (LiP), 7 putative short manganese peroxidases (MnP) and 2 putative versatile peroxidases (VP). Using ddPCR method we have quantified the absolute expression of the 18 peroxidase genes under different culture conditions and on different growth stages of basidiomycete. It was shown that only two genes (one MnP and one VP) were prevalently expressed as well as secreted into cultural broth under all conditions investigated. However their transcriptome and protein profiles differed in time depending on the effector used. The expression of other peroxidase genes revealed a significant variability, so one can propose the specific roles of these enzymes in fungal development and lifestyle. PMID:28301519

  9. Absolute quantification of dengue virus serotype 4 chimera vaccine candidate in Vero cell culture by targeted mass spectrometry.

    PubMed

    Rougemont, Blandine; Simon, Romain; Carrière, Romain; Biarc, Jordane; Fonbonne, Catherine; Salvador, Arnaud; Huillet, Céline; Berard, Yves; Adam, Olivier; Manin, Catherine; Lemoine, Jérôme

    2015-10-01

    Infection by dengue flavivirus is transmitted by mosquitoes and affects tens to hundreds of millions people around the world each year. Four serotypes have been described, all of which cause similar disease. Currently, there no approved vaccines or specific therapeutics for dengue, although several vaccine prototypes are in different stages of clinical development. Among them, a chimeric vaccine, built from the replication machinery of the yellow fever 17D virus, has shown promising results in phase III trials. Accurate quantitation of expressed viral particles in alive attenuated viral antigen vaccine is essential and determination of infectious titer is usually the method of choice. The current paper describes an alternative or orthogonal strategy, namely, a multiplexed and absolute assay of four proteins of the chimera yellow fever/dengue serotype 4 virus using targeted MS in SRM mode. Over 1 month, variability of the assay using a partially purified Vero cell extract was between 8 and 17%, and accuracy was between 80 and 120%. In addition, the assay was linear between 6.25 and 200 nmol/L and could therefore be used in the near future to quantify dengue virus type 4 during production and purification from Vero cells.

  10. Tissue-specific Calibration of Real-time PCR Facilitates Absolute Quantification of Plasmid DNA in Biodistribution Studies

    PubMed Central

    Ho, Joan K; White, Paul J; Pouton, Colin W

    2016-01-01

    Analysis of the tissue distribution of plasmid DNA after administration of nonviral gene delivery systems is best accomplished using quantitative real-time polymerase chain reaction (qPCR), although published strategies do not allow determination of the absolute mass of plasmid delivered to different tissues. Generally, data is expressed as the mass of plasmid relative to the mass of genomic DNA (gDNA) in the sample. This strategy is adequate for comparisons of efficiency of delivery to a single site but it does not allow direct comparison of delivery to multiple tissues, as the mass of gDNA extracted per unit mass of each tissue is different. We show here that by constructing qPCR standard curves for each tissue it is possible to determine the dose of intact plasmid remaining in each tissue, which is a more useful parameter when comparing the fates of different formulations of DNA. We exemplify the use of this tissue-specific qPCR method by comparing the delivery of naked DNA, cationic DNA complexes, and neutral PEGylated DNA complexes after intramuscular injection. Generally, larger masses of intact plasmid were present 24 hours after injection of DNA complexes, and neutral complexes resulted in delivery of a larger mass of intact plasmid to the spleen. PMID:27701400

  11. Proteomics of Microparticles with SILAC Quantification (PROMIS-Quan): A Novel Proteomic Method for Plasma Biomarker Quantification*

    PubMed Central

    Harel, Michal; Oren-Giladi, Pazit; Kaidar-Person, Orit; Shaked, Yuval; Geiger, Tamar

    2015-01-01

    Unbiased proteomic analysis of plasma samples holds the promise to reveal clinically invaluable disease biomarkers. However, the tremendous dynamic range of the plasma proteome has so far hampered the identification of such low abundant markers. To overcome this challenge we analyzed the plasma microparticle proteome, and reached an unprecedented depth of over 3000 plasma proteins in single runs. To add a quantitative dimension, we developed PROMIS-Quan—PROteomics of MIcroparticles with Super-Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) Quantification, a novel mass spectrometry-based technology for plasma microparticle proteome quantification. PROMIS-Quan enables a two-step relative and absolute SILAC quantification. First, plasma microparticle proteomes are quantified relative to a super-SILAC mix composed of cell lines from distinct origins. Next, the absolute amounts of selected proteins of interest are quantified relative to the super-SILAC mix. We applied PROMIS-Quan to prostate cancer and compared plasma microparticle samples of healthy individuals and prostate cancer patients. We identified in total 5374 plasma-microparticle proteins, and revealed a predictive signature of three proteins that were elevated in the patient-derived plasma microparticles. Finally, PROMIS-Quan enabled determination of the absolute quantitative changes in prostate specific antigen (PSA) upon treatment. We propose PROMIS-Quan as an innovative platform for biomarker discovery, validation, and quantification in both the biomedical research and in the clinical worlds. PMID:25624350

  12. A method to estimate the absolute ultrasonic nonlinearity parameter from relative measurements.

    PubMed

    Kim, Jongbeom; Song, Dong-Gi; Jhang, Kyung-Young

    2017-02-17

    The ultrasonic nonlinearity parameter, β, is determined from the displacement amplitude of the second-order harmonic frequency component generated during the propagation of ultrasonic waves through a material. This parameter is generally referred to as the absolute parameter. Meanwhile, it is difficult to measure the small displacement amplitude of the second-order harmonic component; therefore, most studies measure the relative parameter determined from the detected signal amplitude. However, for quantitative assessment of material degradation, the absolute parameter is still required. This study proposes a method to estimate the absolute parameter for damaged material by measuring the relative parameter. This method is based on the fact that the fractional ratio of the relative parameters between different materials is identical to that of the absolute parameters after compensation for material dependent differences such as the wavenumber and detection-sensitivity. In order to experimentally verify the method, the relative parameters of heat-treated Al6061-T6 alloy specimens with different aging times were measured to compare with absolute parameters directly measured by piezo-electric detection. The results show that the fluctuations of both parameters with respect to aging time were very similar to each other, and that the absolute parameters estimated by the proposed method were in good agreement with those measured directly.

  13. Macroscopic inspection of ape feces: what's in a quantification method?

    PubMed

    Phillips, Caroline A; McGrew, William C

    2014-06-01

    Macroscopic inspection of feces has been used to investigate primate diet. The limitations of this method to identify food-items to species level have long been recognized, but ascertaining aspects of diet (e.g., folivory) are achievable by quantifying food-items in feces. Quantification methods applied include rating food-items using a scale of abundance, estimating their percentage volume, and weighing food-items. However, verification as to whether or not composition data differ, depending on which quantification method is used during macroscopic inspection, has not been done. We analyzed feces collected from ten adult chimpanzees (Pan troglodytes schweinfurthii) of the Kanyawara community in Kibale National Park, Uganda. We compare dietary composition totals obtained from using different quantification methods and ascertain if sieve mesh size influences totals calculated. Finally, this study validates findings from direct observation of feeding by the same individuals from whom the fecal samples had been collected. Contrasting diet composition totals obtained by using different quantification methods and sieve mesh sizes can influence folivory and frugivory estimates. However, our findings were based on the assumption that fibrous matter contained pith and leaf fragments only, which remains to be verified. We advocate macroscopic inspection of feces can be a valuable tool to provide a generalized overview of dietary composition for primate populations. As most populations remain unhabituated, scrutinizing and validating indirect measures are important if they are to be applied to further understand inter- and intra-species dietary variation.

  14. Comparison of Two Methods for Anthocyanin Quantification

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The pH differential method (AOAC method 2005.02) by spectrophotometer and high performance liquid chromatography (HPLC) are methods commonly used by researchers and the food industry for quantifying anthocyanins of samples or products. This study was carried out to establish a relationship between t...

  15. Comparison of Two Methods for Anthocyanin Quantification

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The pH differential method (AOAC method 2005.02) by spectrophotometer, and high performance liquid chromatography (HPLC) are methods commonly used by researchers and the food industry for quantifying anthocyanins of samples or products. This study was carried out to establish a relationship between...

  16. Comparison of Two Methods for Anthocyanin Quantification

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The pH differential method (AOAC method 2005.02) by spectrophotometer, and high performance liquid chromatography (HPLC) are methods commonly used by researchers and the food industry for quantifying anthocyanins of samples or products. This study was carried out to establish a relationship between ...

  17. Digital Droplet PCR for the Absolute Quantification of Exon Skipping Induced by Antisense Oligonucleotides in (Pre-)Clinical Development for Duchenne Muscular Dystrophy

    PubMed Central

    Verheul, Ruurd C.; van Deutekom, Judith C. T.; Datson, Nicole A.

    2016-01-01

    Antisense oligonucleotides (AONs) in clinical development for Duchenne muscular dystrophy (DMD) aim to induce skipping of a specific exon of the dystrophin transcript during pre-mRNA splicing. This results in restoration of the open reading frame and consequently synthesis of a dystrophin protein with a shorter yet functional central rod domain. To monitor the molecular therapeutic effect of exon skip-inducing AONs in clinical studies, accurate quantification of pre- and post-treatment exon skip levels is required. With the recent introduction of 3rd generation digital droplet PCR (ddPCR), a state-of-the-art technology became available which allows absolute quantification of transcript copy numbers with and without specific exon skip with high precision, sensitivity and reproducibility. Using Taqman assays with probes targeting specific exon-exon junctions, we here demonstrate that ddPCR reproducibly quantified cDNA fragments with and without exon 51 of the DMD gene over a 4-log dynamic range. In a comparison of conventional nested PCR, qPCR and ddPCR using cDNA constructs with and without exon 51 mixed in different molar ratios using, ddPCR quantification came closest to the expected outcome over the full range of ratios (0–100%), while qPCR and in particular nested PCR overestimated the relative percentage of the construct lacking exon 51. Highest accuracy was similarly obtained with ddPCR in DMD patient-derived muscle cells treated with an AON inducing exon 51 skipping. We therefore recommend implementation of ddPCR for quantification of exon skip efficiencies of AONs in (pre)clinical development for DMD. PMID:27612288

  18. Error quantification in strain mapping methods.

    PubMed

    Guerrero, Elisa; Galindo, Pedro; Yáñez, Andrés; Ben, Teresa; Molina, Sergio I

    2007-10-01

    In this article a method for determining errors of the strain values when applying strain mapping techniques has been devised. This methodology starts with the generation of a thickness/defocus series of simulated high-resolution transmission electron microscopy images of InAsxP1-x/InP heterostructures and the application of geometric phase. To obtain optimal defocusing conditions, a comparison of different defocus values is carried out by the calculation of the strain profile standard deviations among different specimen thicknesses. Finally, based on the analogy of real state strain to a step response, a characterization of strain mapping error near an interface is proposed.

  19. Analytical Methods for the Quantification of Histamine and Histamine Metabolites.

    PubMed

    Bähre, Heike; Kaever, Volkhard

    2017-03-21

    The endogenous metabolite histamine (HA) is synthesized in various mammalian cells but can also be ingested from exogenous sources. It is involved in a plethora of physiological and pathophysiological processes. So far, four different HA receptors (H1R-H4R) have been described and numerous HAR antagonists have been developed. Contemporary investigations regarding the various roles of HA and its main metabolites have been hampered by the lack of highly specific and sensitive analytic methods for all of these analytes. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) is the method of choice for identification and sensitive quantification of many low-molecular weight endogenous metabolites. In this chapter, different methodological aspects of HA quantification as well as recommendations for LC-MS/MS methods suitable for analysis of HA and its main metabolites are summarized.

  20. Validation of a digital PCR method for quantification of DNA copy number concentrations by using a certified reference material.

    PubMed

    Deprez, Liesbet; Corbisier, Philippe; Kortekaas, Anne-Marie; Mazoua, Stéphane; Beaz Hidalgo, Roxana; Trapmann, Stefanie; Emons, Hendrik

    2016-09-01

    Digital PCR has become the emerging technique for the sequence-specific detection and quantification of nucleic acids for various applications. During the past years, numerous reports on the development of new digital PCR methods have been published. Maturation of these developments into reliable analytical methods suitable for diagnostic or other routine testing purposes requires their validation for the intended use. Here, the results of an in-house validation of a droplet digital PCR method are presented. This method is intended for the quantification of the absolute copy number concentration of a purified linearized plasmid in solution with a nucleic acid background. It has been investigated which factors within the measurement process have a significant effect on the measurement results, and the contribution to the overall measurement uncertainty has been estimated. A comprehensive overview is provided on all the aspects that should be investigated when performing an in-house method validation of a digital PCR method.

  1. Absolute Quantification of Lipophilic Shellfish Toxins by Quantitative Nuclear Magnetic Resonance Using Removable Internal Reference Substance with SI Traceability.

    PubMed

    Kato, Tsuyoshi; Saito, Maki; Nagae, Mika; Fujita, Kazuhiro; Watai, Masatoshi; Igarashi, Tomoji; Yasumoto, Takeshi; Inagaki, Minoru

    2016-01-01

    Okadaic acid (OA), a lipophilic shellfish toxin, was accurately quantified using quantitative nuclear magnetic resonance with internal standards for the development of an authentic reference standard. Pyridine and the residual proton in methanol-d4 were used as removable internal standards to limit any contamination. They were calibrated based on a maleic acid certified reference material. Thus, the concentration of OA was traceable to the SI units through accurate quantitative NMR with an internal reference substance. Signals from the protons on the oxygenated and unsaturated carbons of OA were used for quantification. A reasonable accuracy was obtained by integrating between the lower and upper (13)C satellite signal range when more than 4 mg of OA was used. The best-determined purity was 97.4% (0.16% RSD) when 20 mg of OA was used. Dinophysistoxin-1, a methylated analog of OA having an almost identical spectrum, was also quantified by using the same methodology.

  2. Re-creating Gauss's method for non-electrical absolute measurements of magnetic fields and moments

    NASA Astrophysics Data System (ADS)

    Van Baak, D. A.

    2013-10-01

    In 1832, Gauss made the first absolute measurements of magnetic fields and of magnetic moments in experiments that are straightforward and instructive to replicate. We show, using rare-earth permanent magnets and a variation of Gauss's technique, that the horizontal component of the ambient geomagnetic field, as well as the size of the magnetic moments of such magnets, can be found. The method shows the connection between the SI and cgs emu unit systems for these quantities and permits an absolute realization of the Ampere with considerable precision.

  3. Relative and Absolute Error Control in a Finite-Difference Method Solution of Poisson's Equation

    ERIC Educational Resources Information Center

    Prentice, J. S. C.

    2012-01-01

    An algorithm for error control (absolute and relative) in the five-point finite-difference method applied to Poisson's equation is described. The algorithm is based on discretization of the domain of the problem by means of three rectilinear grids, each of different resolution. We discuss some hardware limitations associated with the algorithm,…

  4. Optically based quantification of absolute cerebral metabolic rate of oxygen (CMRO2) with high spatial resolution in rodents

    NASA Astrophysics Data System (ADS)

    Yaseen, Mohammad A.; Srinivasan, Vivek J.; Sakadžić, Sava; Vinogradov, Sergei A.; Boas, David A.

    2010-02-01

    Measuring oxygen delivery in brain tissue is important for identifying the pathophysiological changes associated with brain injury and various diseases such as cancer, stroke, and Alzheimer's disease. We have developed a multi-modal imaging system for minimally invasive measurement of cerebral oxygenation and blood flow in small animals with high spatial resolution. The system allows for simultaneous measurement of blood flow using Fourier-domain optical coherence tomography, and oxygen partial pressure (pO2) using either confocal or multiphoton phosphorescence lifetime imaging with exogenous porphyrin-based dyes sensitive to dissolved oxygen. Here we present the changes in pO2 and blood flow in superficial cortical vessels of Sprague Dawley rats in response to conditions such as hypoxia, hyperoxia, and functional stimulation. pO2 measurements display considerable heterogeneity over distances that cannot be resolved with more widely used oxygen-monitoring techniques such as BOLD-fMRI. Large increases in blood flow are observed in response to functional stimulation and hypoxia. Our system allows for quantification of cerebral metabolic rate of oxygen (CMRO2) with high spatial resolution, providing a better understanding of metabolic dynamics during functional stimulation and under various neuropathologies. Ultimately, better insight into the underlying mechanisms of neuropathologies will facilitate the development of improved therapeutic strategies to minimize damage to brain tissue.

  5. [Study on the absolute spectral irradiation calibration method for far ultraviolet spectrometer in remote sensing].

    PubMed

    Yu, Lei; Lin, Guan-Yu; Chen, Bin

    2013-01-01

    The present paper studied spectral irradiation responsivities calibration method which can be applied to the far ultraviolet spectrometer for upper atmosphere remote sensing. It is difficult to realize the calibration for far ultraviolet spectrometer for many reasons. Standard instruments for far ultraviolet waveband calibration are few, the degree of the vacuum experiment system is required to be high, the stabilities of the experiment are hardly maintained, and the limitation of the far ultraviolet waveband makes traditional diffuser and the integrating sphere radiance calibration method difficult to be used. To solve these problems, a new absolute spectral irradiance calibration method was studied, which can be applied to the far ultraviolet calibration. We build a corresponding special vacuum experiment system to verify the calibration method. The light source system consists of a calibrated deuterium lamp, a vacuum ultraviolet monochromater and a collimating system. We used the calibrated detector to obtain the irradiance responsivities of it. The three instruments compose the calibration irradiance source. We used the "calibration irradiance source" to illuminate the spectrometer prototype and obtained the spectral irradiance responsivities. It realized the absolute spectral irradiance calibration for the far ultraviolet spectrometer utilizing the calibrated detector. The absolute uncertainty of the calibration is 7.7%. The method is significant for the ground irradiation calibration of the far ultraviolet spectrometer in upper atmosphere remote sensing.

  6. Effect of the absolute statistic on gene-sampling gene-set analysis methods.

    PubMed

    Nam, Dougu

    2015-03-02

    Gene-set enrichment analysis and its modified versions have commonly been used for identifying altered functions or pathways in disease from microarray data. In particular, the simple gene-sampling gene-set analysis methods have been heavily used for datasets with only a few sample replicates. The biggest problem with this approach is the highly inflated false-positive rate. In this paper, the effect of absolute gene statistic on gene-sampling gene-set analysis methods is systematically investigated. Thus far, the absolute gene statistic has merely been regarded as a supplementary method for capturing the bidirectional changes in each gene set. Here, it is shown that incorporating the absolute gene statistic in gene-sampling gene-set analysis substantially reduces the false-positive rate and improves the overall discriminatory ability. Its effect was investigated by power, false-positive rate, and receiver operating curve for a number of simulated and real datasets. The performances of gene-set analysis methods in one-tailed (genome-wide association study) and two-tailed (gene expression data) tests were also compared and discussed.

  7. Verification of 235U mass content in nuclear fuel plates by an absolute method

    NASA Astrophysics Data System (ADS)

    El-Gammal, W.

    2007-01-01

    Nuclear Safeguards is referred to a verification System by which a State can control all nuclear materials (NM) and nuclear activities under its authority. An effective and efficient Safeguards System must include a system of measurements with capabilities sufficient to verify such NM. Measurements of NM using absolute methods could eliminate the dependency on NM Standards, which are necessary for other relative or semi-absolute methods. In this work, an absolute method has been investigated to verify the 235U mass content in nuclear fuel plates of Material Testing Reactor (MTR) type. The most intense gamma-ray signature at 185.7 keV emitted after α-decay of the 235U nuclei was employed in the method. The measuring system (an HPGe-spectrometer) was mathematically calibrated for efficiency using the general Monte Carlo transport code MCNP-4B. The calibration results and the measured net count rate were used to estimate the 235U mass content in fuel plates at different detector-to-fuel plate distances. Two sets of fuel plates, containing natural and low enriched uranium, were measured at the Fuel Fabrication Facility. Average accuracies for the estimated 235U masses of about 2.62% and 0.3% are obtained for the fuel plates containing natural and low enriched uranium; respectively, with a precision of about 3%.

  8. Measurements of absolute concentrations of NADH in cells using the phasor FLIM method.

    PubMed

    Ma, Ning; Digman, Michelle A; Malacrida, Leonel; Gratton, Enrico

    2016-07-01

    We propose a graphical method using the phasor representation of the fluorescence decay to derive the absolute concentration of NADH in cells. The method requires the measurement of a solution of NADH at a known concentration. The phasor representation of the fluorescence decay accounts for the differences in quantum yield of the free and bound form of NADH, pixel by pixel of an image. The concentration of NADH in every pixel in a cell is obtained after adding to each pixel in the phasor plot a given amount of unmodulated light which causes a shift of the phasor towards the origin by an amount that depends on the intensity at the pixel and the fluorescence lifetime at the pixel. The absolute concentration of NADH is obtained by comparison of the shift obtained at each pixel of an image with the shift of the calibrated solution.

  9. Measurements of absolute concentrations of NADH in cells using the phasor FLIM method

    PubMed Central

    Ma, Ning; Digman, Michelle A.; Malacrida, Leonel; Gratton, Enrico

    2016-01-01

    We propose a graphical method using the phasor representation of the fluorescence decay to derive the absolute concentration of NADH in cells. The method requires the measurement of a solution of NADH at a known concentration. The phasor representation of the fluorescence decay accounts for the differences in quantum yield of the free and bound form of NADH, pixel by pixel of an image. The concentration of NADH in every pixel in a cell is obtained after adding to each pixel in the phasor plot a given amount of unmodulated light which causes a shift of the phasor towards the origin by an amount that depends on the intensity at the pixel and the fluorescence lifetime at the pixel. The absolute concentration of NADH is obtained by comparison of the shift obtained at each pixel of an image with the shift of the calibrated solution. PMID:27446681

  10. A new method to calibrate the absolute sensitivity of a soft X-ray streak camera

    NASA Astrophysics Data System (ADS)

    Yu, Jian; Liu, Shenye; Li, Jin; Yang, Zhiwen; Chen, Ming; Guo, Luting; Yao, Li; Xiao, Shali

    2016-12-01

    In this paper, we introduce a new method to calibrate the absolute sensitivity of a soft X-ray streak camera (SXRSC). The calibrations are done in the static mode by using a small laser-produced X-ray source. A calibrated X-ray CCD is used as a secondary standard detector to monitor the X-ray source intensity. In addition, two sets of holographic flat-field grating spectrometers are chosen as the spectral discrimination systems of the SXRSC and the X-ray CCD. The absolute sensitivity of the SXRSC is obtained by comparing the signal counts of the SXRSC to the output counts of the X-ray CCD. Results show that the calibrated spectrum covers the range from 200 eV to 1040 eV. The change of the absolute sensitivity in the vicinity of the K-edge of the carbon can also be clearly seen. The experimental values agree with the calculated values to within 29% error. Compared with previous calibration methods, the proposed method has several advantages: a wide spectral range, high accuracy, and simple data processing. Our calibration results can be used to make quantitative X-ray flux measurements in laser fusion research.

  11. An absolute method for determination of misalignment of an immersion ultrasonic transducer.

    PubMed

    Narayanan, M M; Singh, Narender; Kumar, Anish; Babu Rao, C; Jayakumar, T

    2014-12-01

    An absolute methodology has been developed for quantification of misalignment of an ultrasonic transducer using a corner-cube retroreflector. The amplitude based and the time of flight (TOF) based C-scans of the reflector are obtained for various misalignments of the transducer. At zero degree orientation of the transducer, the vertical positions of the maximum amplitude and the minimum TOF in the C-scan coincide. At any other orientation of the transducer with the horizontal plane, there is a vertical shift in the position of the maximum amplitude with respect to the minimum TOF. The position of the minimum (TOF) remains the same irrespective of the orientation of the transducer and hence is used as a reference for any misalignment of the transducer. With the measurement of the vertical shift and the horizontal distance between the transducer and the vertex of the reflector, the misalignment of the transducer is quantified. Based on the methodology developed in the present study, retroreflectors are placed in the Indian 500MWe Prototype Fast Breeder Reactor for assessment of the orientation of the ultrasonic transducer prior to the under-sodium ultrasonic scanning for detection of any protrusion of the subassemblies.

  12. Position error correction in absolute surface measurement based on a multi-angle averaging method

    NASA Astrophysics Data System (ADS)

    Wang, Weibo; Wu, Biwei; Liu, Pengfei; Liu, Jian; Tan, Jiubin

    2017-04-01

    We present a method for position error correction in absolute surface measurement based on a multi-angle averaging method. Differences in shear rotation measurements at overlapping areas can be used to estimate the unknown relative position errors of the measurements. The model and the solving of the estimation algorithm have been discussed in detail. The estimation algorithm adopts a least-squares technique to eliminate azimuthal errors caused by rotation inaccuracy. The cost functions can be minimized to determine the true values of the unknowns of Zernike polynomial coefficients and rotation angle. Experimental results show the validity of the method proposed.

  13. Unexpected patterns of Epstein-Barr virus transcription revealed by a high throughput PCR array for absolute quantification of viral mRNA.

    PubMed

    Tierney, Rosemary J; Shannon-Lowe, Claire D; Fitzsimmons, Leah; Bell, Andrew I; Rowe, Martin

    2015-01-01

    We have validated a flexible, high-throughput and relatively inexpensive RT-QPCR array platform for absolute quantification of Epstein-Barr virus transcripts in different latent and lytic infection states. Several novel observations are reported. First, during infection of normal B cells, Wp-initiated latent gene transcripts remain far more abundant following activation of the Cp promoter than was hitherto suspected. Second, EBNA1 transcript levels are remarkably low in all forms of latency, typically ranging from 1 to 10 transcripts per cell. EBNA3A, -3B and -3C transcripts are likewise very low in Latency III, typically at levels similar to or less than EBNA1 transcripts. Thirdly, a subset of lytic gene transcripts is detectable in Burkitt lymphoma lines at low levels, including: BILF1, which has oncogenic properties, and the poorly characterized LF1, LF2 and LF3 genes. Analysis of seven African BL biopsies confirmed this transcription profile but additionally revealed significant expression of LMP2 transcripts.

  14. Comparison of three digital fringe signal processing methods in a ballistic free-fall absolute gravimeter

    NASA Astrophysics Data System (ADS)

    Svitlov, S.; Masłyk, P.; Rothleitner, Ch; Hu, H.; Wang, L. J.

    2010-12-01

    This paper reports results of comparison of three digital fringe signal processing methods implemented in the same free-fall absolute gravimeter. A two-sample zero-crossing method, a windowed second-difference method and a method of non-linear least-squares adjustment on the undersampled fringe signal are compared in numerical simulations, hardware tests and actual measurements with the MPG-2 absolute gravimeter, developed at the Max Planck Institute for the Science of Light, Germany. The two-sample zero-crossing method realizes data location schemes that are both equally spaced in distance and equally spaced in time (EST) along the free-fall trajectory. The windowed second-difference method and the method of non-linear least-squares adjustment with complex heterodyne demodulation operate with the EST data. Results of the comparison verify an agreement of the three methods within one part in 109 of the measured gravity value, provided a common data location scheme is considered.

  15. Reliability and discriminatory power of methods for dental plaque quantification

    PubMed Central

    RAGGIO, Daniela Prócida; BRAGA, Mariana Minatel; RODRIGUES, Jonas Almeida; FREITAS, Patrícia Moreira; IMPARATO, José Carlos Pettorossi; MENDES, Fausto Medeiros

    2010-01-01

    Objective This in situ study evaluated the discriminatory power and reliability of methods of dental plaque quantification and the relationship between visual indices (VI) and fluorescence camera (FC) to detect plaque. Material and Methods Six volunteers used palatal appliances with six bovine enamel blocks presenting different stages of plaque accumulation. The presence of plaque with and without disclosing was assessed using VI. Images were obtained with FC and digital camera in both conditions. The area covered by plaque was assessed. Examinations were done by two independent examiners. Data were analyzed by Kruskal-Wallis and Kappa tests to compare different conditions of samples and to assess the inter-examiner reproducibility. Results Some methods presented adequate reproducibility. The Turesky index and the assessment of area covered by disclosed plaque in the FC images presented the highest discriminatory powers. Conclusions The Turesky index and images with FC with disclosing present good reliability and discriminatory power in quantifying dental plaque. PMID:20485931

  16. Stochastic methods for uncertainty quantification in radiation transport

    SciTech Connect

    Fichtl, Erin D; Prinja, Anil K; Warsa, James S

    2009-01-01

    The use of generalized polynomial chaos (gPC) expansions is investigated for uncertainty quantification in radiation transport. The gPC represents second-order random processes in terms of an expansion of orthogonal polynomials of random variables and is used to represent the uncertain input(s) and unknown(s). We assume a single uncertain input-the total macroscopic cross section-although this does not represent a limitation of the approaches considered here. Two solution methods are examined: The Stochastic Finite Element Method (SFEM) and the Stochastic Collocation Method (SCM). The SFEM entails taking Galerkin projections onto the orthogonal basis, which, for fixed source problems, yields a linear system of fully -coupled equations for the PC coefficients of the unknown. For k-eigenvalue calculations, the SFEM system is non-linear and a Newton-Krylov method is employed to solve it. The SCM utilizes a suitable quadrature rule to compute the moments or PC coefficients of the unknown(s), thus the SCM solution involves a series of independent deterministic transport solutions. The accuracy and efficiency of the two methods are compared and contrasted. The PC coefficients are used to compute the moments and probability density functions of the unknown(s), which are shown to be accurate by comparing with Monte Carlo results. Our work demonstrates that stochastic spectral expansions are a viable alternative to sampling-based uncertainty quantification techniques since both provide a complete characterization of the distribution of the flux and the k-eigenvalue. Furthermore, it is demonstrated that, unlike perturbation methods, SFEM and SCM can handle large parameter uncertainty.

  17. Rapid quantification method for Legionella pneumophila in surface water.

    PubMed

    Wunderlich, Anika; Torggler, Carmen; Elsässer, Dennis; Lück, Christian; Niessner, Reinhard; Seidel, Michael

    2016-03-01

    World-wide legionellosis outbreaks caused by evaporative cooling systems have shown that there is a need for rapid screening methods for Legionella pneumophila in water. Antibody-based methods for the quantification of L. pneumophila are rapid, non-laborious, and relatively cheap but not sensitive enough for establishment as a screening method for surface and drinking water. Therefore, preconcentration methods have to be applied in advance to reach the needed sensitivity. In a basic test, monolithic adsorption filtration (MAF) was used as primary preconcentration method that adsorbs L. pneumophila with high efficiency. Ten-liter water samples were concentrated in 10 min and further reduced to 1 mL by centrifugal ultrafiltration (CeUF). The quantification of L. pneumophila strains belonging to the monoclonal subtype Bellingham was performed via flow-based chemiluminescence sandwich microarray immunoassays (CL-SMIA) in 36 min. The whole analysis process takes 90 min. A polyclonal antibody (pAb) against L. pneumophila serogroup 1-12 and a monoclonal antibody (mAb) against L. pneumophila SG 1 strain Bellingham were immobilized on a microarray chip. Without preconcentration, the detection limit was 4.0 × 10(3) and 2.8 × 10(3) CFU/mL determined by pAb and mAb 10/6, respectively. For samples processed by MAF-CeUF prior to SMIA detection, the limit of detection (LOD) could be decreased to 8.7 CFU/mL and 0.39 CFU/mL, respectively. A recovery of 99.8 ± 15.9% was achieved for concentrations between 1-1000 CFU/mL. The established combined analytical method is sensitive for rapid screening of surface and drinking water to allow fast hygiene control of L. pneumophila.

  18. Absolute quantification of E1, ubiquitin-like proteins and Nedd8-MLN4924 adduct by mass spectrometry.

    PubMed

    Yang, Xiaofeng; Brownell, James E; Xu, Qing; Zhu, Fengying; Ma, Jingya; Loke, Huay-Keng; Rollins, Neil; Soucy, Teresa A; Minissale, James J; Thomas, Michael P; Mallender, William D; Dick, Lawrence R; Li, Ping; Liao, Hua

    2013-09-01

    Ubiquitin (Ub) and ubiquitin-like (Ubl) proteins regulate a variety of important cellular processes by forming covalent conjugates with target proteins or lipids. Ubl conjugation is catalyzed by a cascade of proteins including activating enzymes (E1), conjugating enzymes (E2), and in many cases ligation enzymes (E3). The discovery of MLN4924 (Brownell et al., Mol Cell 37: 102-111, 1), an investigational small molecule that is a mechanism-based inhibitor of NEDD8-activating enzyme (NAE), reveals a promising strategy of targeting E1/Ubl pathway for therapeutic purposes. In order to better understand, the biochemical dynamics of Ubl conjugation in cells and tissues, we have developed a mass spectrometry-based method to quantify E1 and Ubls using isotope-labeled proteins as internal standards. Furthermore, we have used the described method to quantify levels of the covalent Nedd8-inhibitor adduct formed in MLN4924 treated cells and tissues. The Nedd8-MLN4924 adduct is a tight-binding inhibitor of NAE, and its cellular concentration represents an indirect pharmacodynamic readout of NAE/Nedd8 pathway inhibition.

  19. Correlation of Two Anthocyanin Quantification Methods: HPLC and Spectrophotometric Methods

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The pH differential method and HPLC are methods that are commonly used by researchers and the food industry for quantifying anthocyanins in a sample. This study was conducted to establish a relationship between the two analytical methods. Seven juice samples containing an array of different individu...

  20. Absolute quantification of the total and antidrug antibody-bound concentrations of recombinant human α-glucosidase in human plasma using protein G extraction and LC-MS/MS.

    PubMed

    Bronsema, Kees J; Bischoff, Rainer; Pijnappel, W W M Pim; van der Ploeg, Ans T; van de Merbel, Nico C

    2015-04-21

    The administration of protein-based pharmaceuticals can cause the in vivo formation of antidrug antibodies (ADAs), which may reduce the efficacy of the therapy by binding to the protein drug. An accurate determination of the total and ADA-bound concentrations of the drug gives information on the extent of this immune response and its consequences and may help develop improved therapeutic regimens. We present an absolute quantitative method to differentiate between total, free, and ADA-bound drug for recombinant human alpha acid glucosidase (rhGAA) in plasma from patients suffering from Pompe's disease. LC-MS/MS quantification of a signature peptide after trypsin digestion of plasma samples before and after an extraction of the total IgG content of plasma with protein G coated beads was used to determine the total and the ADA-bound fractions of rhGAA in samples from Pompe patients after enzyme infusion. The methods for total and ADA-bound rhGAA allow quantitation of the drug in the range of 0.5 to 500 μg/mL using 20 μL of plasma and met the regular bioanalytical validation requirements, both in the absence and presence of high levels of anti-rhGAA antibodies. This demonstrates that the ADA-bound rhGAA fraction can be accurately and precisely determined and is not influenced by sample dilution, repeated freezing and thawing, or extended benchtop or frozen storage. In samples from a patient with a reduced response to therapy due to ADAs, high ADA-bound concentrations of rhGAA were found, while in the samples from a patient lacking ADAs, no significant ADA-bound concentrations were found. Since protein G captures the complete IgG content of plasma, including all antidrug antibodies, the described extraction approach is universally applicable for the quantification of ADA-bound concentrations of all non-IgG-based biopharmaceuticals.

  1. A Simple Optical Coherence Tomography Quantification Method for Choroidal Neovascularization

    PubMed Central

    Sulaiman, Rania S.; Quigley, Judith; Qi, Xiaoping; O'Hare, Michael N.; Grant, Maria B.; Boulton, Michael E.

    2015-01-01

    Abstract Purpose: Therapeutic efficacy is routinely assessed by measurement of lesion size using flatmounted choroids and confocal microscopy in the laser-induced choroidal neovascularization (L-CNV) rodent model. We investigated whether optical coherence tomography (OCT) quantification, using an ellipsoid volume measurement, was comparable to standard ex vivo evaluation methods for this model and whether this approach could be used to monitor treatment-related lesion changes. Methods: Bruch's membrane was ruptured by argon laser in the dilated eyes of C57BL/6J mice, followed by intravitreal injections of anti-VEGF164 or vehicle, or no injection. In vivo OCT images were acquired using Micron III or InVivoVue systems at 7, 10, and/or 14 days post-laser and neovascular lesion volume was calculated as an ellipsoid. Subsequently, lesion volume was compared to that calculated from confocal Z-stack images of agglutinin-stained choroidal flatmounts. Results: Ellipsoid volume measurement of orthogonal 2-dimensional OCT images obtained from different imaging systems correlated with ex vivo lesion volumes for L-CNV (Spearman's ρ=0.82, 0.75, and 0.82 at days 7, 10, and 14, respectively). Ellipsoid volume calculation allowed temporal monitoring and evaluation of CNV lesions in response to antivascular endothelial growth factor treatment. Conclusions: Ellipsoid volume measurements allow rapid, quantitative use of OCT for the assessment of CNV lesions in vivo. This novel method can be used with different OCT imaging systems with sensitivity to distinguish between treatment conditions. It may serve as a useful adjunct to the standard ex vivo confocal quantification, to assess therapeutic efficacy in preclinical models of CNV, and in models of other ocular diseases. PMID:26060878

  2. Dirhodium complexes: determination of absolute configuration by the exciton chirality method using VCD spectroscopy.

    PubMed

    Szilvágyi, Gábor; Brém, Balázs; Báti, Gábor; Tölgyesi, László; Hollósi, Miklós; Vass, Elemér

    2013-09-28

    Inherently chiral dinuclear rhodium complexes have been synthesized from the well-known dirhodium(II)-acetate and chiral/achiral amino acids. These complexes have a twisted paddlewheel structure due to axial chirality. Chiral induction could be observed when the ligands were chiral, opposite to the case of achiral ligands, where a racemic mixture was formed. The racemic mixture was separated by chiral HPLC-ECD. The stereochemical properties of these complexes were determined by VCD spectroscopy supported by theoretical calculations at the DFT level. We present a simple route to determine the absolute configuration by an exciton chirality method using VCD spectroscopy.

  3. Easy Absolute Values? Absolutely

    ERIC Educational Resources Information Center

    Taylor, Sharon E.; Mittag, Kathleen Cage

    2015-01-01

    The authors teach a problem-solving course for preservice middle-grades education majors that includes concepts dealing with absolute-value computations, equations, and inequalities. Many of these students like mathematics and plan to teach it, so they are adept at symbolic manipulations. Getting them to think differently about a concept that they…

  4. Improved HPLC method for total plasma homocysteine detection and quantification.

    PubMed

    Sawuła, Wojciech; Banecka-Majkutewicz, Zyta; Kadziński, Leszek; Jakóbkiewicz-Banecka, Joanna; Wegrzyn, Grzegorz; Nyka, Walenty; Banecki, Bogdan

    2008-01-01

    Recent clinical research has pointed at hyperhomocysteinemia as an independent risk factor in a number of cardiovascular and neurological diseases. We have improved a chromatographic method of total plasma homocysteine measurements in order to obtain higher sensitivity, reliability and reproducibility. The method demonstrates excellent linearity (R=0.999), range (<2-100 microM), precision (instrumental RSD 0.06 and method RSD 1.17), accuracy (recovery of 99.92 and RSD 1.27), reproducibility, quantification limit and ruggedness (e.g. pH from 2.0 to 2.5). Because even a small increase in homocysteine level can be a significant risk factor of cardiovascular diseases, such a precise method is required. The constructed method allows the measurement of plasma pyridoxal phosphate, PLP, the co-enzyme form of vitamin B(6), on the same column and similar reagents. The developed method has been successfully applied to measure both total plasma and serum homocysteine in a group of acute stroke patients.

  5. Numerical Continuation Methods for Intrusive Uncertainty Quantification Studies

    SciTech Connect

    Safta, Cosmin; Najm, Habib N.; Phipps, Eric Todd

    2014-09-01

    Rigorous modeling of engineering systems relies on efficient propagation of uncertainty from input parameters to model outputs. In recent years, there has been substantial development of probabilistic polynomial chaos (PC) Uncertainty Quantification (UQ) methods, enabling studies in expensive computational models. One approach, termed ”intrusive”, involving reformulation of the governing equations, has been found to have superior computational performance compared to non-intrusive sampling-based methods in relevant large-scale problems, particularly in the context of emerging architectures. However, the utility of intrusive methods has been severely limited due to detrimental numerical instabilities associated with strong nonlinear physics. Previous methods for stabilizing these constructions tend to add unacceptably high computational costs, particularly in problems with many uncertain parameters. In order to address these challenges, we propose to adapt and improve numerical continuation methods for the robust time integration of intrusive PC system dynamics. We propose adaptive methods, starting with a small uncertainty for which the model has stable behavior and gradually moving to larger uncertainty where the instabilities are rampant, in a manner that provides a suitable solution.

  6. Teaching Absolute Value Meaningfully

    ERIC Educational Resources Information Center

    Wade, Angela

    2012-01-01

    What is the meaning of absolute value? And why do teachers teach students how to solve absolute value equations? Absolute value is a concept introduced in first-year algebra and then reinforced in later courses. Various authors have suggested instructional methods for teaching absolute value to high school students (Wei 2005; Stallings-Roberts…

  7. Novel Parachlamydia acanthamoebae quantification method based on coculture with amoebae.

    PubMed

    Matsuo, Junji; Hayashi, Yasuhiro; Nakamura, Shinji; Sato, Marie; Mizutani, Yoshihiko; Asaka, Masahiro; Yamaguchi, Hiroyuki

    2008-10-01

    Parachlamydia acanthamoebae, belonging to the order Chlamydiales, is an obligately intracellular bacterium that infects free-living amoebae and is a potential human pathogen. However, no method exists to accurately quantify viable bacterial numbers. We present a novel quantification method for P. acanthamoebae based on coculture with amoebae. P. acanthamoebae was cultured either with Acanthamoeba spp. or with mammalian epithelial HEp-2 or Vero cells. The infection rate of P. acanthamoebae (amoeba-infectious dose [AID]) was determined by DAPI (4',6-diamidino-2-phenylindole) staining and was confirmed by fluorescent in situ hybridization. AIDs were plotted as logistic sigmoid dilution curves, and P. acanthamoebae numbers, defined as amoeba-infectious units (AIU), were calculated. During culture, amoeba numbers and viabilities did not change, and amoebae did not change from trophozoites to cysts. Eight amoeba strains showed similar levels of P. acanthamoebae growth, and bacterial numbers reached ca. 1,000-fold (10(9) AIU preculture) after 4 days. In contrast, no increase was observed for P. acanthamoebae in either mammalian cell line. However, aberrant structures in epithelial cells, implying possible persistent infection, were seen by transmission electron microscopy. Thus, our method could monitor numbers of P. acanthamoebae bacteria in host cells and may be useful for understanding chlamydiae present in the natural environment as human pathogens.

  8. Intrusive Method for Uncertainty Quantification in a Multiphase Flow Solver

    NASA Astrophysics Data System (ADS)

    Turnquist, Brian; Owkes, Mark

    2016-11-01

    Uncertainty quantification (UQ) is a necessary, interesting, and often neglected aspect of fluid flow simulations. To determine the significance of uncertain initial and boundary conditions, a multiphase flow solver is being created which extends a single phase, intrusive, polynomial chaos scheme into multiphase flows. Reliably estimating the impact of input uncertainty on design criteria can help identify and minimize unwanted variability in critical areas, and has the potential to help advance knowledge in atomizing jets, jet engines, pharmaceuticals, and food processing. Use of an intrusive polynomial chaos method has been shown to significantly reduce computational cost over non-intrusive collocation methods such as Monte-Carlo. This method requires transforming the model equations into a weak form through substitution of stochastic (random) variables. Ultimately, the model deploys a stochastic Navier Stokes equation, a stochastic conservative level set approach including reinitialization, as well as stochastic normals and curvature. By implementing these approaches together in one framework, basic problems may be investigated which shed light on model expansion, uncertainty theory, and fluid flow in general. NSF Grant Number 1511325.

  9. Absolute Geostrophic Velocity Inverted from World Ocean Atlas 2013 (WOAV13) with the P-Vector Method

    DTIC Science & Technology

    2015-11-01

    1 WOAV13: world ocean absolute geostrophic velocity Absolute Geostrophic Velocity Inverted from... World Ocean Atlas 2013 (WOAV13) with the P-Vector Method P. C. Chu* and C. W. Fan Naval Ocean Analysis and Prediction (NOAP) Laboratory...from World Ocean Atlas-2013 (WOA13) temperature and salinity fields using the P-vector method. It provides a climatological velocity field that is

  10. Sulfathiazole: analytical methods for quantification in seawater and macroalgae.

    PubMed

    Leston, Sara; Nebot, Carolina; Nunes, Margarida; Cepeda, Alberto; Pardal, Miguel Ângelo; Ramos, Fernando

    2015-01-01

    The awareness of the interconnection between pharmaceutical residues, human health, and aquaculture has highlighted the concern with the potential harmful effects it can induce. Furthermore, to better understand the consequences more research is needed and to achieve that new methodologies on the detection and quantification of pharmaceuticals are necessary. Antibiotics are a major class of drugs included in the designation of emerging contaminants, representing a high risk to natural ecosystems. Among the most prescribed are sulfonamides, with sulfathiazole being the selected compound to be investigated in this study. In the environment, macroalgae are an important group of producers, continuously exposed to contaminants, with a significant role in the trophic web. Due to these characteristics are already under scope for the possibility of being used as bioindicators. The present study describes two new methodologies based on liquid chromatography for the determination of sulfathiazole in seawater and in the green macroalgae Ulva lactuca. Results show both methods were validated according to international standards, with MS/MS detection showing more sensitivity as expected with LODs of 2.79ng/g and 1.40ng/mL for algae and seawater, respectively. As for UV detection the values presented were respectively 2.83μg/g and 2.88μg/mL, making it more suitable for samples originated in more contaminated sites. The methods were also applied to experimental data with success with results showing macroalgae have potential use as indicators of contamination.

  11. Real-time RT-PCR for detection, identification and absolute quantification of viral haemorrhagic septicaemia virus using different types of standards.

    PubMed

    Lopez-Vazquez, C; Bandín, I; Dopazo, C P

    2015-05-21

    In the present study, 2 systems of real-time RT-PCR-one based on SYBR Green and the other on TaqMan-were designed to detect strains from any genotype of viral haemorrhagic septicaemia virus (VHSV), with high sensitivity and repeatability/reproducibility. In addition, the method was optimized for quantitative purposes (qRT-PCR), and standard curves with different types of reference templates were constructed and compared. Specificity was tested against 26 isolates from 4 genotypes. The sensitivity of the procedures was first tested against cell culture isolation, obtaining a limit of detection (LD) of 100 TCID50 ml-1 (100-fold below the LD using cell culture), at a threshold cycle value (Ct) of 36. Sensitivity was also evaluated using RNA from crude (LD = 1 fg; 160 genome copies) and purified virus (100 ag; 16 copies), plasmid DNA (2 copies) and RNA transcript (15 copies). No differences between both chemistries were observed in sensitivity and dynamic range. To evaluate repeatability and reproducibility, all experiments were performed in triplicate and on 3 different days, by workers with different levels of experience, obtaining Ct values with coefficients of variation always <5. This fact, together with the high efficiency and R2 values of the standard curves, encouraged us to analyse the reliability of the method for viral quantification. The results not only demonstrated that the procedure can be used for detection, identification and quantification of this virus, but also demonstrated a clear correlation between the regression lines obtained with different standards, which will help scientists to compare sensitivity results between different studies.

  12. Stochastic methods for uncertainty quantification in radiation transport

    NASA Astrophysics Data System (ADS)

    Fichtl, Erin D.

    The use of stochastic spectral expansions, specifically generalized polynomial chaos (gPC) and Karhunen-Loeve (KL) expansions, is investigated for uncertainty quantification in radiation transport. The gPC represents second-order random processes in terms of an expansion of orthogonal polynomials of random variables. The KL expansion is a Fourier-type expansion that represents a second-order random process with known covariance function in terms of a set of uncorrelated random variables and the eigenmodes of the covariance function. The flux and, in multiplying materials, the k-eigenvalue, which are the problem unknowns, are always expanded in a gPC expansion since their covariance functions are also unknown. This work assumes a single uncertain input---the total macroscopic cross section---although this does not represent a limitation of the approaches considered here. Two particular types of input parameter uncertainty are investigated: The cross section as a univariate Gaussian, log-normal, gamma or beta random variable, and the cross section as a spatially varying Gaussian or log-normal random process. In the first case, a gPC expansion in terms of a univariate random variable suffices, while in the second, a truncated KL expansion is first necessary followed by a gPC expansion in terms of multivariate random variables. Two solution methods are examined: The Stochastic Finite Element Method (SFEM) and the Stochastic Collocation Method (SCM). The SFEM entails taking Galerkin projections onto the orthogonal basis, which yields a system of fully-coupled equations for the PC coefficients of the flux and the k-eigenvalue. This system is linear when there is no multiplication and can be solved using Richardson iteration, employing a standard operator splitting such as block Gauss-Seidel or block Jacobi, or a Krylov iterative method, which can be preconditioned using these splittings. When multiplication is present, the SFEM system is non-linear and a Newton

  13. In vivo cell tracking and quantification method in adult zebrafish

    NASA Astrophysics Data System (ADS)

    Zhang, Li; Alt, Clemens; Li, Pulin; White, Richard M.; Zon, Leonard I.; Wei, Xunbin; Lin, Charles P.

    2012-03-01

    Zebrafish have become a powerful vertebrate model organism for drug discovery, cancer and stem cell research. A recently developed transparent adult zebrafish using double pigmentation mutant, called casper, provide unparalleled imaging power in in vivo longitudinal analysis of biological processes at an anatomic resolution not readily achievable in murine or other systems. In this paper we introduce an optical method for simultaneous visualization and cell quantification, which combines the laser scanning confocal microscopy (LSCM) and the in vivo flow cytometry (IVFC). The system is designed specifically for non-invasive tracking of both stationary and circulating cells in adult zebrafish casper, under physiological conditions in the same fish over time. The confocal imaging part in this system serves the dual purposes of imaging fish tissue microstructure and a 3D navigation tool to locate a suitable vessel for circulating cell counting. The multi-color, multi-channel instrument allows the detection of multiple cell populations or different tissues or organs simultaneously. We demonstrate initial testing of this novel instrument by imaging vasculature and tracking circulating cells in CD41: GFP/Gata1: DsRed transgenic casper fish whose thrombocytes/erythrocytes express the green and red fluorescent proteins. Circulating fluorescent cell incidents were recorded and counted repeatedly over time and in different types of vessels. Great application opportunities in cancer and stem cell researches are discussed.

  14. Free volume hypothetical scanning molecular dynamics method for the absolute free energy of liquids

    PubMed Central

    White, Ronald P.; Meirovitch, Hagai

    2006-01-01

    The hypothetical scanning (HS) method is a general approach for calculating the absolute entropy, S, and free energy, F, by analyzing Boltzmann samples obtained by Monte Carlo (MC) or molecular dynamics (MD) techniques. With HS applied to a fluid, each configuration i of the sample is reconstructed by gradually placing the molecules in their positions at i using transition probabilities (TPs). With our recent version of HS, called HSMC-EV, each TP is calculated from MC simulations, where the simulated particles are excluded from the volume reconstructed in previous steps. In this paper we remove the excluded volume (EV) restriction, replacing it by a “free volume” (FV) approach. For liquid argon, HSMC-FV leads to an improvement in efficiency over HSMC-EV by a factor of 2–3. Importantly, the FV treatment greatly simplifies the HS implementation for liquids, allowing a much more natural application of the method for MD simulations. Given the success and popularity of MD, the present development of the HSMD method for liquids is an important advancement for HS methodology. Results for the HSMD-FV approach presented here agree well with our HSMC and thermodynamic integration results. The efficiency of HSMD-FV is equivalent to HSMC-EV. The potential use of HSMC(MD)-FV in protein systems with explicit water is discussed. PMID:16774320

  15. The high-precision videometrics methods to determining absolute vertical benchmark

    NASA Astrophysics Data System (ADS)

    Liu, Jinbo; Zhu, Zhaokun

    2013-01-01

    The mobile measurement equipment plays an important role in engineering measurement tasks and its measuring device is fixed with the vehicle platform. Therefore, how to correct the measured error in time that caused by swayed platform is a basic problem. Videometrics has its inherent advantages in solving this problem. First of all, videometrics technology is non-contact measurement, which has no effect on the target's structural characteristics and motion characteristics. Secondly, videometrics technology has high precision especially for surface targets and linear targets in the field of view. Thirdly, videometrics technology has the advantages of automatic, real-time and dynamic. This paper is mainly for mobile theodolite.etc that works under the environment of absolute vertical benchmark and proposed two high-precision methods to determine vertical benchmark: Direct-Extracting, which is based on the intersection of plats under the help of two cameras; Benchmark-Transformation, which gets the vertical benchmark by reconstructing the level-plat. Two methods both have the precision of under 10 seconds by digital simulation and physical experiments. The methods proposed by this paper have significance both on the theory and application.

  16. Multimode Surface Functional Group Determination: Combining Steady-State and Time-Resolved Fluorescence with X-ray Photoelectron Spectroscopy and Absorption Measurements for Absolute Quantification.

    PubMed

    Fischer, Tobias; Dietrich, Paul M; Unger, Wolfgang E S; Rurack, Knut

    2016-01-19

    The quantitative determination of surface functional groups is approached in a straightforward laboratory-based method with high reliability. The application of a multimode BODIPY-type fluorescence, photometry, and X-ray photoelectron spectroscopy (XPS) label allows estimation of the labeling ratio, i.e., the ratio of functional groups carrying a label after reaction, from the elemental ratios of nitrogen and fluorine. The amount of label on the surface is quantified with UV/vis spectrophotometry based on the molar absorption coefficient as molecular property. The investigated surfaces with varying density are prepared by codeposition of 3-(aminopropyl)triethoxysilane (APTES) and cyanoethyltriethoxysilane (CETES) from vapor. These surfaces show high functional group densities that result in significant fluorescence quenching of surface-bound labels. Since alternative quantification of the label on the surface is available through XPS and photometry, a novel method to quantitatively account for fluorescence quenching based on fluorescence lifetime (τ) measurements is shown. Due to the complex distribution of τ on high-density surfaces, the stretched exponential (or Kohlrausch) function is required to determine representative mean lifetimes. The approach is extended to a commercial Rhodamine B isothiocyanate (RITC) label, clearly revealing the problems that arise from such charged labels used in conjunction with silane surfaces.

  17. Tsunakawa-Shaw method - an absolute paleointensity technique using alternating field demagnetization

    NASA Astrophysics Data System (ADS)

    Yamamoto, Y.; Mochizuki, N.; Shibuya, H.; Tsunakawa, H.

    2015-12-01

    Among geologic materials volcanic rocks have been typically used to deduce an absolute paleointensity. In the last decade, however, there seems a becoming consensus that volcanic rocks are not so ideal materials due to such as magnetic grains other than non-interacting single domain particles. One approach to obtain a good paleointensity estimate from the rocks is to reduce and correct the non-ideality, suppress alterations in laboratory and screen out suspicious results. We have been working on a development and an application of the Tsunakawa-Shaw method, which has been previously called the LTD-DHT Shaw method. This method is an AF(alternating field)-based technique and thus a paleointensity is estimated using coercivity spectra. To reduce the non-ideality, all remanences undergo low-temperature demagnetization (LTD) before any AF demagnetizations to remove multi-domain like component. To correct the non-ideality, anhysteretic remanent magnetizations (ARMs) are imparted with their directions parallel to natural remanent magnetizations and laboratory-imparted thermoremanent magnetizations (TRMs) and measured before and after laboratory heating. These ARMs are used to correct remanence anisotropies, possible interaction effects originated from the non-ideal grains and TRM changes caused by laboratory alterations. TRMs are imparted by heating specimens above their Curie temperatures and then cooling to room temperature at once to simulate nature conditions. These cycles are done in vacuum to suppress alterations in laboratory. Obtained results are judged by selection criteria, including a check for validity of the ARM corrections.It has been demonstrated that successful paleointensities are obtained from historical lavas in Japan and Hawaii, and from baked clay samples from a reconstructed ancient kiln, with the flow-mean precision of 5-10%. In case of old volcanic rocks, however, the method does not necessarily seem to be perfect. We will summarize these points in

  18. A Method for Selecting between Fisher's Linear Classification Functions and Least Absolute Deviation in Predictive Discriminant Analysis.

    ERIC Educational Resources Information Center

    Meshbane, Alice; Morris, John D.

    A method for comparing the cross-validated classification accuracy of Fisher's linear classification functions (FLCFs) and the least absolute deviation is presented under varying data conditions for the two-group classification problem. With this method, separate-group as well as total-sample proportions of current classifications can be compared…

  19. Optimizing methods to recover absolute FRET efficiency from immobilized single molecules.

    PubMed

    McCann, James J; Choi, Ucheor B; Zheng, Liqiang; Weninger, Keith; Bowen, Mark E

    2010-08-04

    Microscopy-based fluorescence resonance energy transfer (FRET) experiments measure donor and acceptor intensities by isolating these signals with a series of optical elements. Because this filtering discards portions of the spectrum, the observed FRET efficiency is dependent on the set of filters in use. Similarly, observed FRET efficiency is also affected by differences in fluorophore quantum yield. Recovering the absolute FRET efficiency requires normalization for these effects to account for differences between the donor and acceptor fluorophores in their quantum yield and detection efficiency. Without this correction, FRET is consistent across multiple experiments only if the photophysical and instrument properties remain unchanged. Here we present what is, to our knowledge, the first systematic study of methods to recover the true FRET efficiency using DNA rulers with known fluorophore separations. We varied optical elements to purposefully alter observed FRET and examined protein samples to achieve quantum yields distinct from those in the DNA samples. Correction for calculated instrument transmission reduced FRET deviations, which can facilitate comparison of results from different instruments. Empirical normalization was more effective but required significant effort. Normalization based on single-molecule photobleaching was the most effective depending on how it is applied. Surprisingly, per-molecule gamma-normalization reduced the peak width in the DNA FRET distribution because anomalous gamma-values correspond to FRET outliers. Thus, molecule-to-molecule variation in gamma has an unrecognized effect on the FRET distribution that must be considered to extract information on sample dynamics from the distribution width.

  20. ID-SERS Based Reference Method for Quantification of Large Biomolecules on a Single Chip

    NASA Astrophysics Data System (ADS)

    Yaghobian, Fatemeh; Stosch, Rainer; Henrion, André; Güttler, Bernd

    2010-08-01

    Accuracy and precision of quantitative SERS results have been significantly increased by applying a method based on the so-called isotope-dilution (ID) principle. In this ID-SERS approach, an isotopically labeled analogue of the target molecule (isotopologue) is spiked to the sample at a known concentration. Due to the slight difference in their molar masses, some Raman bands of the heavier isotopologue are red-shifted with respect to the same signals resulting from the unlabelled compound. As a result, spectra evaluation is reduced to the determination of intensity ratios rather than absolute intensities, and the unknown quantity of the analyte can be calculated from the known quantity of the standard. This procedure is of particular interest in the development of highly accurate reference procedures for metrology in chemistry. Because the sample is spiked prior to any further treatment, potential loss of material or matrix effects would equally affect both isotopologues, without influencing the final result. The method has been successfully applied for quantifying small diagnostic marker molecules like creatinine at their relevant serum concentration levels using silver colloids as SERS substrates. Now, the ID-SERS approach has been realized as a "one-chip" approach using "Bio-chips" made of intrinsically grown spherical silver nanoparticles with gaps less than 10 nm in between (Fig. 1). In addition, the scope of the method has been extended to larger biomolecules like peptides which will be shown using the example of the human growth-hormone (hGH) peptide T12 at physiologically relevant serum concentration levels (Fig. 2). Further developments towards the quantification of full proteins will also be reported.

  1. ID-SERS Based Reference Method for Quantification of Large Biomolecules on a Single Chip

    SciTech Connect

    Yaghobian, Fatemeh; Stosch, Rainer; Henrion, Andre; Guettler, Bernd

    2010-08-06

    Accuracy and precision of quantitative SERS results have been significantly increased by applying a method based on the so-called isotope-dilution (ID) principle. In this ID-SERS approach, an isotopically labeled analogue of the target molecule (isotopologue) is spiked to the sample at a known concentration. Due to the slight difference in their molar masses, some Raman bands of the heavier isotopologue are red-shifted with respect to the same signals resulting from the unlabelled compound. As a result, spectra evaluation is reduced to the determination of intensity ratios rather than absolute intensities, and the unknown quantity of the analyte can be calculated from the known quantity of the standard. This procedure is of particular interest in the development of highly accurate reference procedures for metrology in chemistry. Because the sample is spiked prior to any further treatment, potential loss of material or matrix effects would equally affect both isotopologues, without influencing the final result. The method has been successfully applied for quantifying small diagnostic marker molecules like creatinine at their relevant serum concentration levels using silver colloids as SERS substrates. Now, the ID-SERS approach has been realized as a 'one-chip' approach using 'Bio-chips' made of intrinsically grown spherical silver nanoparticles with gaps less than 10 nm in between (Fig. 1). In addition, the scope of the method has been extended to larger biomolecules like peptides which will be shown using the example of the human growth-hormone (hGH) peptide T12 at physiologically relevant serum concentration levels (Fig. 2). Further developments towards the quantification of full proteins will also be reported.

  2. Absolute calibration method for nanosecond-resolved, time-streaked, fiber optic light collection, spectroscopy systems

    NASA Astrophysics Data System (ADS)

    Johnston, Mark D.; Oliver, Bryan V.; Droemer, Darryl W.; Frogget, Brent; Crain, Marlon D.; Maron, Yitzhak

    2012-08-01

    This paper describes a convenient and accurate method to calibrate fast (<1 ns resolution) streaked, fiber optic light collection, spectroscopy systems. Such systems are inherently difficult to calibrate due to the lack of sufficiently intense, calibrated light sources. Such a system is used to collect spectral data on plasmas generated in electron beam diodes fielded on the RITS-6 accelerator (8-12MV, 140-200kA) at Sandia National Laboratories. On RITS, plasma light is collected through a small diameter (200 μm) optical fiber and recorded on a fast streak camera at the output of a 1 meter Czerny-Turner monochromator. For this paper, a 300 W xenon short arc lamp (Oriel Model 6258) was used as the calibration source. Since the radiance of the xenon arc varies from cathode to anode, just the area around the tip of the cathode ("hotspot") was imaged onto the fiber, to produce the highest intensity output. To compensate for chromatic aberrations, the signal was optimized at each wavelength measured. Output power was measured using 10 nm bandpass interference filters and a calibrated photodetector. These measurements give power at discrete wavelengths across the spectrum, and when linearly interpolated, provide a calibration curve for the lamp. The shape of the spectrum is determined by the collective response of the optics, monochromator, and streak tube across the spectral region of interest. The ratio of the spectral curve to the measured bandpass filter curve at each wavelength produces a correction factor (Q) curve. This curve is then applied to the experimental data and the resultant spectra are given in absolute intensity units (photons/sec/cm2/steradian/nm). Error analysis shows this method to be accurate to within +/- 20%, which represents a high level of accuracy for this type of measurement.

  3. Absolute calibration method for nanosecond-resolved, time-streaked, fiber optic light collection, spectroscopy systems.

    PubMed

    Johnston, Mark D; Oliver, Bryan V; Droemer, Darryl W; Frogget, Brent; Crain, Marlon D; Maron, Yitzhak

    2012-08-01

    This paper describes a convenient and accurate method to calibrate fast (<1 ns resolution) streaked, fiber optic light collection, spectroscopy systems. Such systems are inherently difficult to calibrate due to the lack of sufficiently intense, calibrated light sources. Such a system is used to collect spectral data on plasmas generated in electron beam diodes fielded on the RITS-6 accelerator (8-12MV, 140-200kA) at Sandia National Laboratories. On RITS, plasma light is collected through a small diameter (200 μm) optical fiber and recorded on a fast streak camera at the output of a 1 meter Czerny-Turner monochromator. For this paper, a 300 W xenon short arc lamp (Oriel Model 6258) was used as the calibration source. Since the radiance of the xenon arc varies from cathode to anode, just the area around the tip of the cathode ("hotspot") was imaged onto the fiber, to produce the highest intensity output. To compensate for chromatic aberrations, the signal was optimized at each wavelength measured. Output power was measured using 10 nm bandpass interference filters and a calibrated photodetector. These measurements give power at discrete wavelengths across the spectrum, and when linearly interpolated, provide a calibration curve for the lamp. The shape of the spectrum is determined by the collective response of the optics, monochromator, and streak tube across the spectral region of interest. The ratio of the spectral curve to the measured bandpass filter curve at each wavelength produces a correction factor (Q) curve. This curve is then applied to the experimental data and the resultant spectra are given in absolute intensity units (photons/sec/cm(2)/steradian/nm). Error analysis shows this method to be accurate to within +∕- 20%, which represents a high level of accuracy for this type of measurement.

  4. Absolute quantitation of protein posttranslational modification isoform.

    PubMed

    Yang, Zhu; Li, Ning

    2015-01-01

    Mass spectrometry has been widely applied in characterization and quantification of proteins from complex biological samples. Because the numbers of absolute amounts of proteins are needed in construction of mathematical models for molecular systems of various biological phenotypes and phenomena, a number of quantitative proteomic methods have been adopted to measure absolute quantities of proteins using mass spectrometry. The liquid chromatography-tandem mass spectrometry (LC-MS/MS) coupled with internal peptide standards, i.e., the stable isotope-coded peptide dilution series, which was originated from the field of analytical chemistry, becomes a widely applied method in absolute quantitative proteomics research. This approach provides more and more absolute protein quantitation results of high confidence. As quantitative study of posttranslational modification (PTM) that modulates the biological activity of proteins is crucial for biological science and each isoform may contribute a unique biological function, degradation, and/or subcellular location, the absolute quantitation of protein PTM isoforms has become more relevant to its biological significance. In order to obtain the absolute cellular amount of a PTM isoform of a protein accurately, impacts of protein fractionation, protein enrichment, and proteolytic digestion yield should be taken into consideration and those effects before differentially stable isotope-coded PTM peptide standards are spiked into sample peptides have to be corrected. Assisted with stable isotope-labeled peptide standards, the absolute quantitation of isoforms of posttranslationally modified protein (AQUIP) method takes all these factors into account and determines the absolute amount of a protein PTM isoform from the absolute amount of the protein of interest and the PTM occupancy at the site of the protein. The absolute amount of the protein of interest is inferred by quantifying both the absolute amounts of a few PTM

  5. AN ACCURATE NEW METHOD OF CALCULATING ABSOLUTE MAGNITUDES AND K-CORRECTIONS APPLIED TO THE SLOAN FILTER SET

    SciTech Connect

    Beare, Richard; Brown, Michael J. I.; Pimbblet, Kevin

    2014-12-20

    We describe an accurate new method for determining absolute magnitudes, and hence also K-corrections, that is simpler than most previous methods, being based on a quadratic function of just one suitably chosen observed color. The method relies on the extensive and accurate new set of 129 empirical galaxy template spectral energy distributions from Brown et al. A key advantage of our method is that we can reliably estimate random errors in computed absolute magnitudes due to galaxy diversity, photometric error and redshift error. We derive K-corrections for the five Sloan Digital Sky Survey filters and provide parameter tables for use by the astronomical community. Using the New York Value-Added Galaxy Catalog, we compare our K-corrections with those from kcorrect. Our K-corrections produce absolute magnitudes that are generally in good agreement with kcorrect. Absolute griz magnitudes differ by less than 0.02 mag and those in the u band by ∼0.04 mag. The evolution of rest-frame colors as a function of redshift is better behaved using our method, with relatively few galaxies being assigned anomalously red colors and a tight red sequence being observed across the whole 0.0 < z < 0.5 redshift range.

  6. Method for Indirect Quantification of CH4 Production via H2O Production Using Hydrogenotrophic Methanogens

    PubMed Central

    Taubner, Ruth-Sophie; Rittmann, Simon K.-M. R.

    2016-01-01

    Hydrogenotrophic methanogens are an intriguing group of microorganisms from the domain Archaea. Methanogens exhibit extraordinary ecological, biochemical, and physiological characteristics and possess a huge biotechnological potential. Yet, the only possibility to assess the methane (CH4) production potential of hydrogenotrophic methanogens is to apply gas chromatographic quantification of CH4. In order to be able to effectively screen pure cultures of hydrogenotrophic methanogens regarding their CH4 production potential we developed a novel method for indirect quantification of the volumetric CH4 production rate by measuring the volumetric water production rate. This method was established in serum bottles for cultivation of methanogens in closed batch cultivation mode. Water production was estimated by determining the difference in mass increase in a quasi-isobaric setting. This novel CH4 quantification method is an accurate and precise analytical technique, which can be used to rapidly screen pure cultures of methanogens regarding their volumetric CH4 evolution rate. It is a cost effective alternative determining CH4 production of methanogens over CH4 quantification by using gas chromatography, especially if applied as a high throughput quantification method. Eventually, the method can be universally applied for quantification of CH4 production from psychrophilic, thermophilic and hyperthermophilic hydrogenotrophic methanogens. PMID:27199898

  7. A Concurrent Mixed Methods Approach to Examining the Quantitative and Qualitative Meaningfulness of Absolute Magnitude Estimation Scales in Survey Research

    ERIC Educational Resources Information Center

    Koskey, Kristin L. K.; Stewart, Victoria C.

    2014-01-01

    This small "n" observational study used a concurrent mixed methods approach to address a void in the literature with regard to the qualitative meaningfulness of the data yielded by absolute magnitude estimation scaling (MES) used to rate subjective stimuli. We investigated whether respondents' scales progressed from less to more and…

  8. A new objective method for acquisition and quantification of reflex receptive fields.

    PubMed

    Jensen, Michael Brun; Manresa, José Biurrun; Andersen, Ole Kæseler

    2015-03-01

    The nociceptive withdrawal reflex (NWR) is a polysynaptic spinal reflex correlated with pain perception. Assessment of this objective physiological measure constitutes the core of existing methods for quantification of reflex receptive fields (RRFs), which however still suffer from a certain degree of subjective involvement. This article proposes a strictly objective methodology for RRF quantification based on automated identification of NWR thresholds (NWR-Ts). Nociceptive withdrawal reflex thresholds were determined for 10 individual stimulation sites using an interleaved up-down staircase method. Reflexes were detected from electromyography by evaluation of interval peak z scores and application of conduction velocity analysis. Reflex receptive field areas were quantified from interpolated mappings of NWR-Ts and compared with existing RRF quantifications. A total of 3 repeated measures were performed in 2 different sessions to evaluate the test-retest reliability of the various quantifications, using coefficients of repeatability (CRs) and hypothetical sample sizes. The novel quantifications based on identification of NWR-Ts showed a similar level of reliability within and between sessions, whereas existing quantifications all demonstrated worse between-session than within-session reliability. The NWR-T-based quantifications required a smaller sample size than any of the existing RRF measures to detect a clinically relevant effect in a crossover study design involving more than 1 session. Of all measures, quantification from mapping of inversed NWR-Ts demonstrated superior reliability both within (CR, 0.25) and between sessions (CR, 0.28). The study presents a more reliable and robust quantification of the RRF to be used as biomarker of pain hypersensitivity in clinical and experimental research.

  9. The performance of different propensity score methods for estimating absolute effects of treatments on survival outcomes: A simulation study.

    PubMed

    Austin, Peter C; Schuster, Tibor

    2016-10-01

    Observational studies are increasingly being used to estimate the effect of treatments, interventions and exposures on outcomes that can occur over time. Historically, the hazard ratio, which is a relative measure of effect, has been reported. However, medical decision making is best informed when both relative and absolute measures of effect are reported. When outcomes are time-to-event in nature, the effect of treatment can also be quantified as the change in mean or median survival time due to treatment and the absolute reduction in the probability of the occurrence of an event within a specified duration of follow-up. We describe how three different propensity score methods, propensity score matching, stratification on the propensity score and inverse probability of treatment weighting using the propensity score, can be used to estimate absolute measures of treatment effect on survival outcomes. These methods are all based on estimating marginal survival functions under treatment and lack of treatment. We then conducted an extensive series of Monte Carlo simulations to compare the relative performance of these methods for estimating the absolute effects of treatment on survival outcomes. We found that stratification on the propensity score resulted in the greatest bias. Caliper matching on the propensity score and a method based on earlier work by Cole and Hernán tended to have the best performance for estimating absolute effects of treatment on survival outcomes. When the prevalence of treatment was less extreme, then inverse probability of treatment weighting-based methods tended to perform better than matching-based methods.

  10. Selected methods for quantification of community exposure to aircraft noise

    NASA Technical Reports Server (NTRS)

    Edge, P. M., Jr.; Cawthorn, J. M.

    1976-01-01

    A review of the state-of-the-art for the quantification of community exposure to aircraft noise is presented. Physical aspects, people response considerations, and practicalities of useful application of scales of measure are included. Historical background up through the current technology is briefly presented. The developments of both single-event and multiple-event scales are covered. Selective choice is made of scales currently in the forefront of interest and recommended methodology is presented for use in computer programing to translate aircraft noise data into predictions of community noise exposure. Brief consideration is given to future programing developments and to supportive research needs.

  11. The realization of the dipole (γ, γ) method and its application to determine the absolute optical oscillator strengths of helium

    PubMed Central

    Xu, Long-Quan; Liu, Ya-Wei; Kang, Xu; Ni, Dong-Dong; Yang, Ke; Hiraoka, Nozomu; Tsuei, Ku-Ding; Zhu, Lin-Fan

    2015-01-01

    The dipole (γ, γ) method, which is the inelastic x-ray scattering operated at a negligibly small momentum transfer, is proposed and realized to determine the absolute optical oscillator strengths of the vanlence-shell excitations of atoms and molecules. Compared with the conventionally used photoabsorption method, this new method is free from the line saturation effect, which can seriously limit the accuracies of the measured photoabsorption cross sections for discrete transitions with narrow natural linewidths. Furthermore, the Bethe-Born conversion factor of the dipole (γ, γ) method varies much more slowly with the excitation energy than does that of the dipole (e, e) method. Absolute optical oscillator strengths for the excitations of 1s2 → 1 snp(n = 3 − 7) of atomic helium have been determined using the high-resolution dipole (γ, γ) method, and the excellent agreement of the present measurements with both those measured by the dipole (e, e) method and the previous theoretical calculations indicates that the dipole (γ, γ) method is a powerful tool to measure the absolute optical oscillator strengths of the valence-shell excitations of atoms and molecules. PMID:26678298

  12. A new formalism for the quantification of tissue perfusion by the destruction-replenishment method in contrast ultrasound imaging.

    PubMed

    Arditi, Marcel; Frinking, Peter J A; Zhou, Xiang; Rognin, Nicolas G

    2006-06-01

    A new formalism is presented for the destruction-replenishment perfusion quantification approach at low mechanical index. On the basis of physical considerations, best-fit methods should be applied using perfusion functions with S-shape characteristics. These functions are first described for the case of a geometry with a single flow velocity, then extended to the case of vascular beds with blood vessels having multiple flow velocity values and directions. The principles guiding the analysis are, on one hand, a linearization of video echo signals to overcome the log-compression of the imaging instrument, and, on the other hand, the spatial distribution of the transmit-receive ultrasound beam in the elevation direction. An in vitro model also is described; it was used to confirm experimentally the validity of the approach using a commercial contrast agent. The approach was implemented in the form of a computer program, taking as input a sequence of contrast-specific images, as well as parameters related to the ultrasound imaging equipment used. The generated output is either flow-parameter values computed in regions-of-interest, or parametric flow-images (e.g., mean velocity, mean transit time, mean flow, flow variance, or skewness). This approach thus establishes a base for extracting information about the morphology of vascular beds in vivo, and could allow absolute quantification provided that appropriate instrument calibration is implemented.

  13. Determination of absolute configuration using heavy atom based co-crystallization method: Halogen atom effects

    NASA Astrophysics Data System (ADS)

    Wang, Jian-Rong; Fan, Xiaowu; Ding, Qiaoce; Mei, Xuefeng

    2016-09-01

    Heavy atom (chloride, bromide, and iodide) based co-crystals for determination of absolute configuration (AC) for chiral molecules were synthesized and evaluated. Co-crystals of cholestanol and L-ascorbic acid were analysed and the effects and potential benefits of varying the heavy atom are discussed. Changing the halogen atoms (chloride, bromide, or iodide) affects the co-crystal formation, X-ray absorption, and anomalous dispersion, and hence the ability to determine AC.

  14. A Robust Method for Transcript Quantification with RNA-Seq Data

    PubMed Central

    Huang, Yan; Hu, Yin; Jones, Corbin D.; MacLeod, James N.; Chiang, Derek Y.; Liu, Yufeng; Prins, Jan F.

    2013-01-01

    Abstract The advent of high throughput RNA-seq technology allows deep sampling of the transcriptome, making it possible to characterize both the diversity and the abundance of transcript isoforms. Accurate abundance estimation or transcript quantification of isoforms is critical for downstream differential analysis (e.g., healthy vs. diseased cells) but remains a challenging problem for several reasons. First, while various types of algorithms have been developed for abundance estimation, short reads often do not uniquely identify the transcript isoforms from which they were sampled. As a result, the quantification problem may not be identifiable, i.e., lacks a unique transcript solution even if the read maps uniquely to the reference genome. In this article, we develop a general linear model for transcript quantification that leverages reads spanning multiple splice junctions to ameliorate identifiability. Second, RNA-seq reads sampled from the transcriptome exhibit unknown position-specific and sequence-specific biases. We extend our method to simultaneously learn bias parameters during transcript quantification to improve accuracy. Third, transcript quantification is often provided with a candidate set of isoforms, not all of which are likely to be significantly expressed in a given tissue type or condition. By resolving the linear system with LASSO, our approach can infer an accurate set of dominantly expressed transcripts while existing methods tend to assign positive expression to every candidate isoform. Using simulated RNA-seq datasets, our method demonstrated better quantification accuracy and the inference of dominant set of transcripts than existing methods. The application of our method on real data experimentally demonstrated that transcript quantification is effective for differential analysis of transcriptomes. PMID:23461570

  15. Quantification of Training and Competition Loads in Endurance Sports: Methods and Applications.

    PubMed

    Mujika, Iñigo

    2016-12-05

    Training quantification is basic to evaluate an endurance athlete's responses to the training loads, ensure adequate stress/recovery balance and determine the relationship between training and performance. Quantifying both external and internal workload is important, because the external workload does not measure the biological stress imposed by the exercise sessions. Generally used quantification methods include retrospective questionnaires, diaries, direct observation and physiological monitoring, often based on the measurement of oxygen uptake, heart rate and blood lactate concentration. Other methods in use in endurance sports include speed measurement and the measurement of power output, made possible by recent technological advances, such as power meters in cycling and triathlon. Among subjective methods of quantification the RPE stands out because of its wide use. Concurrent assessments of the various quantification methods allow researchers and practitioners to evaluate stress/recovery balance, adjust individual training programmes and determine the relationships between external load, internal load and athletes' performance. This brief review summarizes the most relevant external and internal workload quantification methods in endurance sports, and provides practical examples of their implementation to adjust the training programmes of elite athletes in accordance to their individualized stress/recovery balance.

  16. Diagnosis of human herpes virus 1 and 2 (HHV-1 and HHV-2): use of a synthetic standard curve for absolute quantification by real time polymerase chain reaction

    PubMed Central

    Lima, Lyana Rodrigues Pinto; da Silva, Amanda Perse; Schmidt-Chanasit, Jonas; de Paula, Vanessa Salete

    2017-01-01

    The use of quantitative real time polymerase chain reaction (qPCR) for herpesvirus detection has improved the sensitivity and specificity of diagnosis, as it is able to detect shedding episodes in the absence of clinical lesions and diagnose clinical specimens that have low viral loads. With an aim to improve the detection and quantification of herpesvirus by qPCR, synthetic standard curves for human herpesvirus 1 and 2 (HHV-1 and HHV-2) targeting regions gD and gG, respectively, were designed and evaluated. The results show that synthetic curves can replace DNA standard curves in diagnostic herpes qPCR. PMID:28225902

  17. Diagnosis of human herpes virus 1 and 2 (HHV-1 and HHV-2): use of a synthetic standard curve for absolute quantification by real time polymerase chain reaction.

    PubMed

    Lima, Lyana Rodrigues Pinto; Silva, Amanda Perse da; Schmidt-Chanasit, Jonas; Paula, Vanessa Salete de

    2017-02-16

    The use of quantitative real time polymerase chain reaction (qPCR) for herpesvirus detection has improved the sensitivity and specificity of diagnosis, as it is able to detect shedding episodes in the absence of clinical lesions and diagnose clinical specimens that have low viral loads. With an aim to improve the detection and quantification of herpesvirus by qPCR, synthetic standard curves for human herpesvirus 1 and 2 (HHV-1 and HHV-2) targeting regions gD and gG, respectively, were designed and evaluated. The results show that synthetic curves can replace DNA standard curves in diagnostic herpes qPCR.

  18. Diagnosis of human herpes virus 1 and 2 (HHV-1 and HHV-2): use of a synthetic standard curve for absolute quantification by real time polymerase chain reaction.

    PubMed

    Lima, Lyana Rodrigues Pinto; Silva, Amanda Perse da; Schmidt-Chanasit, Jonas; Paula, Vanessa Salete de

    2017-03-01

    The use of quantitative real time polymerase chain reaction (qPCR) for herpesvirus detection has improved the sensitivity and specificity of diagnosis, as it is able to detect shedding episodes in the absence of clinical lesions and diagnose clinical specimens that have low viral loads. With an aim to improve the detection and quantification of herpesvirus by qPCR, synthetic standard curves for human herpesvirus 1 and 2 (HHV-1 and HHV-2) targeting regions gD and gG, respectively, were designed and evaluated. The results show that synthetic curves can replace DNA standard curves in diagnostic herpes qPCR.

  19. Absolute age Determinations on Diamond by Radioisotopic Methods: NOT the way to Accurately Identify Diamond Provenance

    NASA Astrophysics Data System (ADS)

    Shirey, S. B.

    2002-05-01

    Gem-quality diamond contains such low abundances of parent-daughter radionuclides that dating the diamond lattice directly by isotopic measurements has been and will be impossible. Absolute ages on diamonds typically are obtained through measurements of their syngenetic mineral inclusions: Rb-Sr in garnet; Sm-Nd in garnet and pyroxene; Re-Os and U-Th-Pb in sulfide; K-Ar in pyroxene; and U-Pb in zircon. The application of the first two isotope schemes in the list requires putting together many inclusions from many diamonds whereas the latter isotope schemes permit ages on single diamonds. The key limitations on the application of these decay pairs are the availability and size of the inclusions, the abundance levels of the radionuclides, and instrumental sensitivity. Practical complications of radioisotope dating of inclusions are fatal to the application of the technique for diamond provenance. In all mines, the ratio of gem-quality diamonds to stones with datable inclusions is very high. Thus there is no way to date the valuable, marketable stones that are part of the conflict diamond problem, just their rare, flawed cousins. Each analysis destroys the diamond host plus the inclusion and can only be carried out in research labs by highly trained scientists. Thus, these methods can not be automated or applied to the bulk of diamond production. The geological problems with age dating are equally fatal to its application to diamond provenance. From the geological perspective, for age determination to work as a tool for diamond provenance studies, diamond ages would have to be specific to particular kimberlites or kimberlite fields and different between fields. The southern African Kaapvaal-Zimbabwe Craton and Limpopo Mobile Belt is the only cratonic region where age determinations have been applied on a large enough scale to a number of kimberlites to illustrate the geological problems in age measurements for diamond provenance. However, this southern African example

  20. Label-free relative quantification method for low-abundance glycoproteins in human serum by micrOTOF-Q.

    PubMed

    Hao, Piliang; Ren, Yan; Xie, Yongming

    2009-06-01

    In this study, a label-free relative quantification strategy was developed for quantifying low-abundance glycoproteins in human serum. It included three steps: (1) immunodepletion of 12 high-abundance proteins, (2) enrichment of low-abundance glycoproteins by multi-lectin column, (3) relative quantification of them between different samples by micrOTOF-Q. We also evaluated the specificity and efficiency of immunodepletion, the accuracy of protein quantification and the possible influence of immunodepletion, glycoprotein enrichment, trypsin digestion and peptide ionization on quantification. In conclusion, the relative quantification method can be effectively applied to the screening of low-abundance biomarkers.

  1. Application of Mean of Absolute Deviation Method for the Selection of Best Nonlinear Component Based on Video Encryption

    NASA Astrophysics Data System (ADS)

    Anees, Amir; Khan, Waqar Ahmad; Gondal, Muhammad Asif; Hussain, Iqtadar

    2013-07-01

    The aim of this work is to make use of the mean of absolute deviation (MAD) method for the evaluation process of substitution boxes used in the advanced encryption standard. In this paper, we use the MAD technique to analyze some popular and prevailing substitution boxes used in encryption processes. In particular, MAD is applied to advanced encryption standard (AES), affine power affine (APA), Gray, Lui J., Residue Prime, S8 AES, SKIPJACK, and Xyi substitution boxes.

  2. Absolute nuclear material assay

    DOEpatents

    Prasad, Manoj K [Pleasanton, CA; Snyderman, Neal J [Berkeley, CA; Rowland, Mark S [Alamo, CA

    2012-05-15

    A method of absolute nuclear material assay of an unknown source comprising counting neutrons from the unknown source and providing an absolute nuclear material assay utilizing a model to optimally compare to the measured count distributions. In one embodiment, the step of providing an absolute nuclear material assay comprises utilizing a random sampling of analytically computed fission chain distributions to generate a continuous time-evolving sequence of event-counts by spreading the fission chain distribution in time.

  3. Absolute nuclear material assay

    DOEpatents

    Prasad, Manoj K.; Snyderman, Neal J.; Rowland, Mark S.

    2010-07-13

    A method of absolute nuclear material assay of an unknown source comprising counting neutrons from the unknown source and providing an absolute nuclear material assay utilizing a model to optimally compare to the measured count distributions. In one embodiment, the step of providing an absolute nuclear material assay comprises utilizing a random sampling of analytically computed fission chain distributions to generate a continuous time-evolving sequence of event-counts by spreading the fission chain distribution in time.

  4. Direct liquid chromatography method for the simultaneous quantification of hydroxytyrosol and tyrosol in red wines.

    PubMed

    Piñeiro, Zulema; Cantos-Villar, Emma; Palma, Miguel; Puertas, Belen

    2011-11-09

    A validated HPLC method with fluorescence detection for the simultaneous quantification of hydroxytyrosol and tyrosol in red wines is described. Detection conditions for both compounds were optimized (excitation at 279 and 278 and emission at 631 and 598 nm for hydroxytyrosol and tyrosol, respectively). The validation of the analytical method was based on selectivity, linearity, robustness, detection and quantification limits, repeatability, and recovery. The detection and quantification limits in red wines were set at 0.023 and 0.076 mg L(-1) for hydroxytyrosol and at 0.007 and 0.024 mg L(-1) for tyrosol determination, respectively. Precision values, both within-day and between-day (n = 5), remained below 3% for both compounds. In addition, a fractional factorial experimental design was developed to analyze the influence of six different conditions on analysis. The final optimized HPLC-fluorescence method allowed the analysis of 30 nonpretreated Spanish red wines to evaluate their hydroxytyrosol and tyrosol contents.

  5. Antioxidant Activity and Validation of Quantification Method for Lycopene Extracted from Tomato.

    PubMed

    Cefali, Letícia Caramori; Cazedey, Edith Cristina Laignier; Souza-Moreira, Tatiana Maria; Correa, Marcos Antônio; Salgado, Hérida Regina Nunes; Isaac, Vera Lucia Borges

    2015-01-01

    Lycopene is a carotenoid found in tomatoes with potent antioxidant activity. The aim of the study was to obtain an extract containing lycopene from four types of tomatoes, validate a quantification method for the extracts by HPLC, and assess its antioxidant activity. Results revealed that the tomatoes analyzed contained lycopene and antioxidant activity. Salad tomato presented the highest concentration of this carotenoid and antioxidant activity. The quantification method exhibited linearity with a correlation coefficient of 0.9992. Tests for the assessment of precision, accuracy, and robustness achieved coefficients with variation of less than 5%. The LOD and LOQ were 0.0012 and 0.0039 μg/mL, respectively. Salad tomato can be used as a source of lycopene for the development of topical formulations, and based on performed tests, the chosen method for the identification and quantification of lycopene was considered to be linear, precise, exact, selective, and robust.

  6. Development of defined microbial population standards using fluorescence activated cell sorting for the absolute quantification of S. aureus using real-time PCR.

    PubMed

    Martinon, Alice; Cronin, Ultan P; Wilkinson, Martin G

    2012-01-01

    In this article, four types of standards were assessed in a SYBR Green-based real-time PCR procedure for the quantification of Staphylococcus aureus (S. aureus) in DNA samples. The standards were purified S. aureus genomic DNA (type A), circular plasmid DNA containing a thermonuclease (nuc) gene fragment (type B), DNA extracted from defined populations of S. aureus cells generated by Fluorescence Activated Cell Sorting (FACS) technology with (type C) or without purification of DNA by boiling (type D). The optimal efficiency of 2.016 was obtained on Roche LightCycler(®) 4.1. software for type C standards, whereas the lowest efficiency (1.682) corresponded to type D standards. Type C standards appeared to be more suitable for quantitative real-time PCR because of the use of defined populations for construction of standard curves. Overall, Fieller Confidence Interval algorithm may be improved for replicates having a low standard deviation in Cycle Threshold values such as found for type B and C standards. Stabilities of diluted PCR standards stored at -20°C were compared after 0, 7, 14 and 30 days and were lower for type A or C standards compared with type B standards. However, FACS generated standards may be useful for bacterial quantification in real-time PCR assays once optimal storage and temperature conditions are defined.

  7. Critical review of current and emerging quantification methods for the development of influenza vaccine candidates.

    PubMed

    Manceur, Aziza P; Kamen, Amine A

    2015-11-04

    Significant improvements in production and purification have been achieved since the first approved influenza vaccines were administered 75 years ago. Global surveillance and fast response have limited the impact of the last pandemic in 2009. In case of another pandemic, vaccines can be generated within three weeks with certain platforms. However, our Achilles heel is at the quantification level. Production of reagents for the quantification of new vaccines using the SRID, the main method formally approved by regulatory bodies, requires two to three months. The impact of such delays can be tragic for vulnerable populations. Therefore, efforts have been directed toward developing alternative quantification methods, which are sensitive, accurate, easy to implement and independent of the availability of specific reagents. The use of newly-developed antibodies against a conserved region of hemagglutinin (HA), a surface protein of influenza, holds great promises as they are able to recognize multiple subtypes of influenza; these new antibodies could be used in immunoassays such as ELISA and slot-blot analysis. HA concentration can also be determined using reversed-phase high performance liquid chromatography (RP-HPLC), which obviates the need for antibodies but still requires a reference standard. The number of viral particles can be evaluated using ion-exchange HPLC and techniques based on flow cytometry principles, but non-viral vesicles have to be taken into account with cellular production platforms. As new production systems are optimized, new quantification methods that are adapted to the type of vaccine produced are required. The nature of these new-generation vaccines might dictate which quantification method to use. In all cases, an alternative method will have to be validated against the current SRID assay. A consensus among the scientific community would have to be reached so that the adoption of new quantification methods would be harmonized between

  8. Motion compensation method using dynamic programming for quantification of neovascularization in carotid atherosclerotic plaques with contrast enhanced ultrasound (CEUS)

    NASA Astrophysics Data System (ADS)

    Akkus, Zeynettin; Hoogi, Assaf; Renaud, Guillaume; ten Kate, Gerrit L.; van den Oord, Stijn C. H.; Schinkel, Arend F. L.; de Jong, Nico; van der Steen, Antonius F. W.; Bosch, Johan G.

    2012-03-01

    Intraplaque neovascularization (IPN) has been linked with progressive atherosclerotic disease and plaque instability in several studies. Quantification of IPN may allow early detection of vulnerable plaques. A dedicated motion compensation method with normalized-cross-correlation (NCC) block matching combined with multidimensional (2D+time) dynamic programming (MDP) was developed for quantification of IPN in small plaques (<30% diameter stenosis). The method was compared to NCC block matching without MDP (forward tracking (FT)) and showed to improve motion tracking. Side-by-side CEUS and B-mode ultrasound images of carotid arteries were acquired by a Philips iU22 system with a L9-3 linear array probe. The motion pattern for the plaque region was obtained from the Bmode images with MDP. MDP results were evaluated in-vitro by a phantom and in-vivo by comparing to manual tracking of three experts for multibeat-image-sequences (MIS) of 11 plaques. In the in-vivo images, the absolute error was 72+/-55μm (mean+/-SD) for X (longitudinal) and 34+/-23μm for Y (radial). The method's success rate was visually assessed on 67 MIS. The tracking was considered failed if it deviated >2 pixels (~200μm) from true motion in any frame. Tracking was scored as fully successful in 63 MIS (94%) for MDP vs. 52(78%) for FT. The range of displacement over these 63 was 1045+/-471μm (X) and 395+/-216μm (Y). The tracking sporadically failed in 4 MIS (6%) due to poor image quality, jugular vein proximity and out-of-plane motion. Motion compensation showed improved lumen-plaque contrast separation. In conclusion, the proposed method is sufficiently accurate and successful for in vivo application.

  9. A Clinical Method for the Detection and Quantification of Quick Respiratory Hyperkinesia

    ERIC Educational Resources Information Center

    Hixon, Thomas J.; Hoit, Jeannette D.

    2006-01-01

    Purpose: Quick respiratory hyperkinesia can be difficult to detect with the naked eye. A clinical method is described for the detection and quantification of quick respiratory hyperkinesia. Method: Flow at the airway opening is sensed during spontaneous apnea (rest), voluntary breath holding (postural fixation), and voluntary volume displacement…

  10. A SIMPLE METHOD FOR THE EXTRACTION AND QUANTIFICATION OF PHOTOPIGMENTS FROM SYMBIODINIUM SPP.

    EPA Science Inventory

    John E. Rogers and Dragoslav Marcovich. Submitted. Simple Method for the Extraction and Quantification of Photopigments from Symbiodinium spp.. Limnol. Oceanogr. Methods. 19 p. (ERL,GB 1192).

    We have developed a simple, mild extraction procedure using methanol which, when...

  11. Comparison between titrimetric and spectrophotometric methods for quantification of vitamin C.

    PubMed

    da Silva, T L; Aguiar-Oliveira, E; Mazalli, M R; Kamimura, E S; Maldonado, R R

    2017-06-01

    Vitamin C is usually quantified by titrimetric or chromatographic methods. However, these methods have limitations: food color interferes with the titrimetric method and the chromatographic method is costly. The aim of this study was to compare a spectrophotometric method, based on reduction of cupric ions in the presence of cuproine complex, with a titrimetric method, based on reduction of 2,6-dicholorophenolindophenol. Linearity, precision, accuracy, and limits of detection (LOD) and quantification (LOQ) were evaluated using a standard vitamin C solution. Both methods were also applied for AA quantification in industrialized orange and pineapple juices. The methods were precise and accurate when applied to the standard solution. The spectrophotometric method was more sensitive, with lower values for LOD (0.002mgmL(-1)) and LOQ (0.010mgmL(-1)), and more accurate with error less than 5% while results from the titrimetric method were affected by the juice color, which generated errors in excess of 15%.

  12. Dakota uncertainty quantification methods applied to the NEK-5000 SAHEX model.

    SciTech Connect

    Weirs, V. Gregory

    2014-03-01

    This report summarizes the results of a NEAMS project focused on the use of uncertainty and sensitivity analysis methods within the NEK-5000 and Dakota software framework for assessing failure probabilities as part of probabilistic risk assessment. NEK-5000 is a software tool under development at Argonne National Laboratory to perform computational fluid dynamics calculations for applications such as thermohydraulics of nuclear reactor cores. Dakota is a software tool developed at Sandia National Laboratories containing optimization, sensitivity analysis, and uncertainty quantification algorithms. The goal of this work is to demonstrate the use of uncertainty quantification methods in Dakota with NEK-5000.

  13. Comprehensive panel of real-time TaqMan polymerase chain reaction assays for detection and absolute quantification of filoviruses, arenaviruses, and New World hantaviruses.

    PubMed

    Trombley, Adrienne R; Wachter, Leslie; Garrison, Jeffrey; Buckley-Beason, Valerie A; Jahrling, Jordan; Hensley, Lisa E; Schoepp, Randal J; Norwood, David A; Goba, Augustine; Fair, Joseph N; Kulesh, David A

    2010-05-01

    Viral hemorrhagic fever is caused by a diverse group of single-stranded, negative-sense or positive-sense RNA viruses belonging to the families Filoviridae (Ebola and Marburg), Arenaviridae (Lassa, Junin, Machupo, Sabia, and Guanarito), and Bunyaviridae (hantavirus). Disease characteristics in these families mark each with the potential to be used as a biological threat agent. Because other diseases have similar clinical symptoms, specific laboratory diagnostic tests are necessary to provide the differential diagnosis during outbreaks and for instituting acceptable quarantine procedures. We designed 48 TaqMan-based polymerase chain reaction (PCR) assays for specific and absolute quantitative detection of multiple hemorrhagic fever viruses. Forty-six assays were determined to be virus-specific, and two were designated as pan assays for Marburg virus. The limit of detection for the assays ranged from 10 to 0.001 plaque-forming units (PFU)/PCR. Although these real-time hemorrhagic fever virus assays are qualitative (presence of target), they are also quantitative (measure a single DNA/RNA target sequence in an unknown sample and express the final results as an absolute value (e.g., viral load, PFUs, or copies/mL) on the basis of concentration of standard samples and can be used in viral load, vaccine, and antiviral drug studies.

  14. Determination of the Absolute Configuration of the Pseudo-Symmetric Natural Product Elatenyne by the Crystalline Sponge Method.

    PubMed

    Urban, Sylvia; Brkljača, Robert; Hoshino, Manabu; Lee, Shoukou; Fujita, Makoto

    2016-02-18

    Elatenyne is a marine natural product that was isolated in 1986. Despite its simple 2,2'-bifuranyl backbone, its relative structure was only recently determined. The absolute configuration of elatenyne has still not been unequivocally confirmed because of its pseudo-meso core structure, which results in a specific rotation, [α]D  , of almost zero. In this work, the structure of natural elatenyne was determined by the crystalline sponge method and the use of a porous coordination network (a crystalline sponge) capable of absorbing organic guests; in the sponge, the absorbed guests are ordered and crystallographically observable. The crystalline sponge could differentiate between the two very similar alkyl side chains, and the absolute structure of elatenyne was thus reliably determined. The total amount required for the experiments was only approximately 100 μg, and the majority (95 μg) could be recovered after the experiments.

  15. Methods for Quantification of Soil-Transmitted Helminths in Environmental Media: Current Techniques and Recent Advances.

    PubMed

    Collender, Philip A; Kirby, Amy E; Addiss, David G; Freeman, Matthew C; Remais, Justin V

    2015-12-01

    Limiting the environmental transmission of soil-transmitted helminths (STHs), which infect 1.5 billion people worldwide, will require sensitive, reliable, and cost-effective methods to detect and quantify STHs in the environment. We review the state-of-the-art of STH quantification in soil, biosolids, water, produce, and vegetation with regard to four major methodological issues: environmental sampling; recovery of STHs from environmental matrices; quantification of recovered STHs; and viability assessment of STH ova. We conclude that methods for sampling and recovering STHs require substantial advances to provide reliable measurements for STH control. Recent innovations in the use of automated image identification and developments in molecular genetic assays offer considerable promise for improving quantification and viability assessment.

  16. Quantification of activated carbon contents in soils and sediments using chemothermal and wet oxidation methods.

    PubMed

    Brändli, Rahel C; Bergsli, Anders; Ghosh, Upal; Hartnik, Thomas; Breedveld, Gijs D; Cornelissen, Gerard

    2009-12-01

    Activated carbon (AC) strongly sorbs organic pollutants and can be used for remediation of soils and sediments. A method for AC quantification is essential to monitor AC (re)distribution. Since AC is black carbon (BC), two methods for BC quantification were tested for AC mixed in different soils and sediments: i) chemothermal oxidation (CTO) at a range of temperatures and ii) wet-chemical oxidation with a potassium dichromate/sulfuric acid solution. For three soils, the amount of AC was accurately determined by CTO at 375 degrees C. For two sediments, however, much of the AC disappeared during combustion at 375 degrees C, which could probably be explained by catalytic effects by sediment constituents. CTO at lower temperatures (325-350 degrees C) was a feasible alternative for one of the sediments. Wet oxidation effectively functioned for AC quantification in sediments, with almost complete AC recovery (81-92%) and low remaining amounts of native organic carbon (5-16%).

  17. Methods for quantification of soil-transmitted helminths in environmental media: current techniques and recent advances

    PubMed Central

    Collender, Philip A.; Kirby, Amy E.; Addiss, David G.; Freeman, Matthew C.; Remais, Justin V.

    2015-01-01

    Limiting the environmental transmission of soil-transmitted helminths (STH), which infect 1.5 billion people worldwide, will require sensitive, reliable, and cost effective methods to detect and quantify STH in the environment. We review the state of the art of STH quantification in soil, biosolids, water, produce, and vegetation with respect to four major methodological issues: environmental sampling; recovery of STH from environmental matrices; quantification of recovered STH; and viability assessment of STH ova. We conclude that methods for sampling and recovering STH require substantial advances to provide reliable measurements for STH control. Recent innovations in the use of automated image identification and developments in molecular genetic assays offer considerable promise for improving quantification and viability assessment. PMID:26440788

  18. Large-Scale Measurement of Absolute Protein Glycosylation Stoichiometry.

    PubMed

    Sun, Shisheng; Zhang, Hui

    2015-07-07

    Protein glycosylation is one of the most important protein modifications. Glycosylation site occupancy alteration has been implicated in human diseases and cancers. However, current glycoproteomic methods focus on the identification and quantification of glycosylated peptides and glycosylation sites but not glycosylation occupancy or glycoform stoichiometry. Here we describe a method for large-scale determination of the absolute glycosylation stoichiometry using three independent relative ratios. Using this method, we determined 117 absolute N-glycosylation occupancies in OVCAR-3 cells. Finally, we investigated the possible functions and the determinants for partial glycosylation.

  19. Large-scale multiplex absolute protein quantification of drug-metabolizing enzymes and transporters in human intestine, liver, and kidney microsomes by SWATH-MS: Comparison with MRM/SRM and HR-MRM/PRM.

    PubMed

    Nakamura, Kenji; Hirayama-Kurogi, Mio; Ito, Shingo; Kuno, Takuya; Yoneyama, Toshihiro; Obuchi, Wataru; Terasaki, Tetsuya; Ohtsuki, Sumio

    2016-08-01

    The purpose of the present study was to examine simultaneously the absolute protein amounts of 152 membrane and membrane-associated proteins, including 30 metabolizing enzymes and 107 transporters, in pooled microsomal fractions of human liver, kidney, and intestine by means of SWATH-MS with stable isotope-labeled internal standard peptides, and to compare the results with those obtained by MRM/SRM and high resolution (HR)-MRM/PRM. The protein expression levels of 27 metabolizing enzymes, 54 transporters, and six other membrane proteins were quantitated by SWATH-MS; other targets were below the lower limits of quantitation. Most of the values determined by SWATH-MS differed by less than 50% from those obtained by MRM/SRM or HR-MRM/PRM. Various metabolizing enzymes were expressed in liver microsomes more abundantly than in other microsomes. Ten, 13, and eight transporters listed as important for drugs by International Transporter Consortium were quantified in liver, kidney, and intestinal microsomes, respectively. Our results indicate that SWATH-MS enables large-scale multiplex absolute protein quantification while retaining similar quantitative capability to MRM/SRM or HR-MRM/PRM. SWATH-MS is expected to be useful methodology in the context of drug development for elucidating the molecular mechanisms of drug absorption, metabolism, and excretion in the human body based on protein profile information.

  20. Method and apparatus for ultra-high-sensitivity, incremental and absolute optical encoding

    NASA Technical Reports Server (NTRS)

    Leviton, Douglas B. (Inventor)

    1999-01-01

    An absolute optical linear or rotary encoder which encodes the motion of an object (3) with increased resolution and encoding range and decreased sensitivity to damage to the scale includes a scale (5), which moves with the object and is illuminated by a light source (11). The scale carries a pattern (9) which is imaged by a microscope optical system (13) on a CCD array (17) in a camera head (15). The pattern includes both fiducial markings (31) which are identical for each period of the pattern and code areas (33) which include binary codings of numbers identifying the individual periods of the pattern. The image of the pattern formed on the CCD array is analyzed by an image processor (23) to locate the fiducial marking, decode the information encoded in the code area, and thereby determine the position of the object.

  1. The revised human liver cytochrome P450 "Pie": absolute protein quantification of CYP4F and CYP3A enzymes using targeted quantitative proteomics.

    PubMed

    Michaels, Scott; Wang, Michael Zhuo

    2014-08-01

    The CYP4F subfamily of enzymes has been identified recently to be involved in the metabolism of endogenous compounds (arachidonic acid and leukotriene B4), nutrients (vitamins K1 and E), and xenobiotics (pafuramidine and fingolimod). CYP4F2 and CYP4F3B are reported to be expressed in the human liver. However, absolute concentrations of these enzymes in human liver microsomes (HLMs) and their interindividual variability have yet to be determined because of the lack of specific antibodies. Here, an liquid chromatography with tandem mass spectrometry (LC-MS/MS)-based targeted quantitative proteomic approach was employed to determine the absolute protein concentrations of CYP4F2 and CYP4F3B compared with CYP3A in two panels of HLMs (n = 31). As a result, the human hepatic cytochrome P450 (P450) "pie" has been revised to include the contribution of CYP4F enzymes, which amounts to 15% of the total hepatic cytochrome P450 enzymes. CYP4F3B displayed low interindividual variability (3.3-fold) in the HLM panels whereas CYP4F2 displayed large variability (21-fold). However, CYP4F2 variability decreased to 3.4-fold if the two donors with the lowest expression were excluded. In contrast, CYP3A exhibited 29-fold interindividual variability in the same HLM panels. The proposed marker reaction for CYP4F enzymes pafuramidine/DB289 M1 formation did not correlate with CYP4F protein content, suggesting alternate metabolic pathways for DB289 M1 formation in HLMs. In conclusion, CYP4F enzymes are highly expressed in the human liver and their physiologic and pharmacologic roles warrant further investigation.

  2. Comparing model-based and model-free analysis methods for QUASAR arterial spin labeling perfusion quantification.

    PubMed

    Chappell, Michael A; Woolrich, Mark W; Petersen, Esben T; Golay, Xavier; Payne, Stephen J

    2013-05-01

    Amongst the various implementations of arterial spin labeling MRI methods for quantifying cerebral perfusion, the QUASAR method is unique. By using a combination of labeling with and without flow suppression gradients, the QUASAR method offers the separation of macrovascular and tissue signals. This permits local arterial input functions to be defined and "model-free" analysis, using numerical deconvolution, to be used. However, it remains unclear whether arterial spin labeling data are best treated using model-free or model-based analysis. This work provides a critical comparison of these two approaches for QUASAR arterial spin labeling in the healthy brain. An existing two-component (arterial and tissue) model was extended to the mixed flow suppression scheme of QUASAR to provide an optimal model-based analysis. The model-based analysis was extended to incorporate dispersion of the labeled bolus, generally regarded as the major source of discrepancy between the two analysis approaches. Model-free and model-based analyses were compared for perfusion quantification including absolute measurements, uncertainty estimation, and spatial variation in cerebral blood flow estimates. Major sources of discrepancies between model-free and model-based analysis were attributed to the effects of dispersion and the degree to which the two methods can separate macrovascular and tissue signal.

  3. Combining sub-Nyquist sampling and chirp decomposition for a high-precision and speed absolute distance measurement method.

    PubMed

    Liu, Guodong; Lu, Cheng; Liu, Bingguo; Chen, Fengdong; Gan, Yu

    2016-12-10

    A high-precision and speed absolute distance measurement based on swept-wavelength interferometry is reported. A powerful method combining sub-Nyquist sampling and chirp decomposition for dispersion mismatch compensation is proposed. A standard deviation of 0.72 μm is obtained for the measurement of a target located at 3.9 m, which is better than the traditional method. The measurement can be completed in 1.9 s when the frequency range is 4.26 THz, which is much better than chirp decomposition without sub-Nyquist sampling.

  4. Optimization of a gas chromatography-mass spectrometry method with methyl chloroformate derivatization for quantification of amino acids in plant tissue.

    PubMed

    Vancompernolle, Bram; Croes, Kim; Angenon, Geert

    2016-04-01

    Rapid, easy and reliable quantification of amino acids is crucial in research on plant amino acid metabolism and nutritional improvement of crops via enrichment of essential amino acids. A recently reported analysis method, based on solid phase extraction (SPE), derivatization with methyl chloroformate and gas chromatography-mass spectrometry was optimized and tested on three-week-old Arabidopsis thaliana leaf tissues. Optimization of the SPE cleanup yielded recovery rates of minimum 95% for all amino acids (except arginine). Variations in accuracy and precision did not exceed 12.5%, except for cysteine, histidine and tryptophane, which were excluded from analysis. Quantification of overlapping peaks for isoleucine/threonine and proline/asparagine was possible by selection of two specific fragment ions for each amino acid. Of the 16 selected amino acids, 14 were quantified successfully in at least 75% of the samples, while methionine and tyrosine were only quantifiable in 6% and 42%, respectively. A case study on the aspartate super pathway confirmed the applicability of the optimized method on wild type and genetically modified plants: external supplementation of methionine or lysine yielded a 146-fold or 27-fold increase in the respective absolute amino acid levels compared with the control treatment. Induced expression of dhdps-r1 (a mutated lysine biosynthesis gene encoding a feedback insensitive enzyme) caused an 83-fold increase in absolute lysine levels.

  5. The quest for an absolute chronology in human prehistory: anthropologists, chemists and the fluorine dating method in palaeoanthropology.

    PubMed

    Goodrum, Matthew R; Olson, Cora

    2009-03-01

    By the early twentieth century there was a growing need within palaeoanthropology and prehistoric archaeology to find a way of dating fossils and artefacts in order to know the age of specific specimens, but more importantly to establish an absolute chronology for human prehistory. The radiocarbon and potassium-argon dating methods revolutionized palaeoanthropology during the last half of the twentieth century. However, prior to the invention of these methods there were attempts to devise chemical means of dating fossil bone. Collaborations between Emile Rivière and Adolphe Carnot in the 1890s led to the development of the fluorine dating method, but it was not until the 1940s that this method was improved and widely implemented by Kenneth Oakley to resolve a number of problems in palaeoanthropology, including the Piltdown Man controversy. The invention of the fluorine dating method marked a significant advance in the quest for absolute dating in palaeoanthropology, but it also highlights interesting problems and issues relating to the ability of palaeoanthropologists and chemists to bring together different skills and bodies of knowledge in order successfully to develop and apply the fluorine dating method.

  6. A probabilistic crack size quantification method using in-situ Lamb wave test and Bayesian updating

    NASA Astrophysics Data System (ADS)

    Yang, Jinsong; He, Jingjing; Guan, Xuefei; Wang, Dengjiang; Chen, Huipeng; Zhang, Weifang; Liu, Yongming

    2016-10-01

    This paper presents a new crack size quantification method based on in-situ Lamb wave testing and Bayesian method. The proposed method uses coupon test to develop a baseline quantification model between the crack size and damage sensitive features. In-situ Lamb wave testing data on actual structures are used to update the baseline model parameters using Bayesian method to achieve more accurate crack size predictions. To demonstrate the proposed method, Lamb wave testing on simple plates with artificial cracks of different sizes is performed using surface-bonded piezoelectric wafers, and the data are used to obtain the baseline model. Two damage sensitive features, namely, the phase change and normalized amplitude are identified using signal processing techniques and used in the model. To validate the effectiveness of the method, the damage data from an in-situ fatigue testing on a realistic lap-joint component are used to update the baseline model using Bayesian method.

  7. A Method to Access Absolute fIPAR fo Vegetation in Spatially Complex Ecosystems

    NASA Technical Reports Server (NTRS)

    Wessman, Carol A.; Nel, Elizabeth M.; Bateson, C. Ann; Asner, Gregory P.

    1998-01-01

    Arid and semi-arid lands compose a large fraction of the earth's terrestrial vegetation, and thereby contribute significantly to global atmospheric-biospheric interactions. The thorny shrubs and small trees in these semi-arid shrub lands have counterparts throughout much of the world's tropical and subtropical zones and have captured substantial areas of the world's former grasslands. The objective of our field and remotely sensed measurements in the semi-arid shrublands of Texas is to monitor interannual variability and directional change in landscape structure, ecosystem processes and atmosphere-biosphere exchanges. To understand the role ecosystems play in controlling the composition of the atmosphere, it is necessary to quantify processes such as photosynthesis and primary production, decomposition and soil carbon storage, and trace gas exchanges. Photosynthesis is the link whereby surface-atmosphere exchanges such as the radiation balance and exchange of heat, moisture, and gas can be inferred. It also describes the efficiency of carbon dioxide exchange and is directly related to the primary production of vegetation. Our efforts in this paper focus on the indirect, quantification of photosynthesis, and thereby carbon flux and net primary production, via remote sensing and direct measurements of intercepted photosynthetically active radiation (IPAR).

  8. Quantification of fungicides in snow-melt runoff from turf: A comparison of four extraction methods

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A variety of pesticides are used to control diverse stressors to turf. These pesticides have a wide range in physical and chemical properties. The objective of this project was to develop an extraction and analysis method for quantification of chlorothalonil and PCNB (pentachloronitrobenzene), two p...

  9. 21 CFR 530.24 - Procedure for announcing analytical methods for drug residue quantification.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ..., DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS EXTRALABEL DRUG USE IN ANIMALS Specific Provisions Relating to Extralabel Use of Animal and Human Drugs in Food-Producing Animals § 530.24 Procedure for announcing analytical methods for drug residue quantification....

  10. 21 CFR 530.24 - Procedure for announcing analytical methods for drug residue quantification.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ..., DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS EXTRALABEL DRUG USE IN ANIMALS Specific Provisions Relating to Extralabel Use of Animal and Human Drugs in Food-Producing Animals § 530.24 Procedure for announcing analytical methods for drug residue quantification....

  11. 21 CFR 530.24 - Procedure for announcing analytical methods for drug residue quantification.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ..., DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS EXTRALABEL DRUG USE IN ANIMALS Specific Provisions Relating to Extralabel Use of Animal and Human Drugs in Food-Producing Animals § 530.24 Procedure for announcing analytical methods for drug residue quantification....

  12. 21 CFR 530.24 - Procedure for announcing analytical methods for drug residue quantification.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ..., DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS EXTRALABEL DRUG USE IN ANIMALS Specific Provisions Relating to Extralabel Use of Animal and Human Drugs in Food-Producing Animals § 530.24 Procedure for announcing analytical methods for drug residue quantification....

  13. 21 CFR 530.24 - Procedure for announcing analytical methods for drug residue quantification.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ..., DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS EXTRALABEL DRUG USE IN ANIMALS Specific Provisions Relating to Extralabel Use of Animal and Human Drugs in Food-Producing Animals § 530.24 Procedure for announcing analytical methods for drug residue quantification....

  14. THE QUANTIFICATION OF AQUEOUS TRACERS IN LABORATORY AQUIFER MODELS USING LIGHT TRANSMISSION VISUALIZATION METHOD

    EPA Science Inventory

    The quantification of solute concentrations in laboratory aquifer models has been largely limited to the use of sampling ports, from which samples are collected for external analysis. One of the drawbacks to this method is that the act of sampling may disturb plume dynamics and ...

  15. THE QUANTIFICATION OF AQUEOUS TRACERS IN LABORATORY AQUIFER MODELS USING A LIGHT TRANSMISSION VISUALIZATION METHOD - 1

    EPA Science Inventory

    The quantification of solute concentrations in laboratory aquifer models has been largely limited to the use of sampling ports, from which samples are collected for external analysis. One of the drawbacks to this method is that the act of sampling may disturb plume dynamics and ...

  16. THE QUANTIFICATION OF AQUEOUS TRACERS IN LABORATORY AQUIFER MODELS USING A LIGHT TRANSMISSION VISUALIZATION METHOD - 3

    EPA Science Inventory

    The quantification of solute concentrations in laboratory aquifer models has been largely limited to the use of sampling ports, from which samples are collected for external analysis. One of the drawbacks to this method is that the act of sampling may disturb plume dynamics and ...

  17. THE QUANTIFICATION OF AQUEOUS TRACERS IN LABORATORY AQUIFER MODELS USING A LIGHT TRANSMISSION VISUALIZATION METHOD - 2

    EPA Science Inventory

    The quantification of solute concentrations in laboratory aquifer models has been largely limited to the use of sampling ports, from which samples are collected for external analysis. One of the drawbacks to this method is that the act of sampling may disturb plume dynamics and ...

  18. A reversed-phase high performance liquid chromatography method for quantification of methotrexate in cancer patients serum.

    PubMed

    Li, Yuan-dong; Li, Yan; Liang, Ning-sheng; Yang, Fan; Kuang, Zhi-peng

    2015-10-01

    A simple, rapid and sensitive reversed-phase high performance liquid chromatography (HPLC) method has been developed for the determination of methotrexate in human serum. After deproteinization of the serum with 40% silver nitrate solution, methotrexate and internal standard (IS) were separated on a reversed-phase column with a mobile phase consisting of 10mM sodium phosphate buffer (pH6.40)-methanol (78:22%, v/v) and ultraviolet detection at 310nm. The linearity is evaluated by a calibration curve in the concentration range of 0.05-10.0μg/mL and presented a correlation coefficient of 0.9995. The absolute recoveries were 97.52±3.9% and 96.87±3.7% for methotrexate and ferulic acid (internal standard), respectively. The intra- and inter-day precision were less 6.19 and 5.89%, respectively (n=6). The limit of quantitation was 0.02μg/mL and the limit of detection was 0.006μg/mL. The complete analysis was achieved less than 10min with no interference from endogenous components or 22 examined drugs. This method was validated by using serum samples from high-dose methotrexate treated patients with osteosarcoma, breast cancer, acute leukemia and lymphoma. The method was demonstrated to be a simple, rapid and reliable approach in quantification of methotrexate in serum samples from patients with high-dose methotrexate therapy.

  19. Quantification of geopolymers production by chemical methods- A short review

    NASA Astrophysics Data System (ADS)

    Siyal, Ahmer Ali; Azizli, Khairun Azizi; Ismail, Lukman; Man, Zakaria; Khan, Muhammad Irfan

    2015-07-01

    Inorganic polymers are the aluminosilicate materials possessing properties superior than ordinary Portland cement. In this review paper the chemical techniques used for determining degree of reaction of fly ash or the quantity of geopolymer material produced have been discussed. These methods determine the amount of product formed in percentages. The methods include HCl method, salicylic acid method, and picric acid method. These methods are not only used for fly ash but they are being used for determining the degree of reactions of metakaolin and other pozzolanic materials. The picric acid is an explosive material and its transportation in high concentration is dangerous. During its use in laboratory there is also the risk of fire associated with it. According to the microscopic analysis results the picric acid attack dissolves small amount of fine unreacted fly ash particles also. The salicylic acid is easily available but the residue from its treatment contains unreacted fly ash particles, hydration phases, and certain parts of unreacted OPC. The residue from HCl and salicylic acid attack contains MgO particles which is the part of the hydration product. The HCl method is mostly used due to simple process and lower standard deviation.

  20. Microwave-assisted nile red method for in vivo quantification of neutral lipids in microalgae.

    PubMed

    Chen, Wei; Sommerfeld, Milton; Hu, Qiang

    2011-01-01

    In vivo determination of neutral lipids with Nile red fluorescence has been used as a rapid screening method for certain types of microalgae, but has been unsuccessful in others, particularly those with thick, rigid cell walls that prevent penetration of the fluorescence dye into the cell. To solve the problem, a microwave-assisted Nile red staining method for microalgal lipid determination was developed. In a two-step staining protocol, 50 and 60s were selected as the optimal microwave times for the pretreatment and staining process, respectively. Moreover, several calibration methods for quantitative analysis of neutral lipids in microalgae were investigated and compared with conventional gravimetric methods. Factors that affected the in vivo quantification of cellular neutral lipids were also investigated. Application of the new method for detection and quantification of neutral lipids in a number of green microalgae was demonstrated.

  1. 76 FR 29752 - Nomination of In Vitro Test Methods for Detection and Quantification of Botulinum Neurotoxins and...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-05-23

    .... NICEATM and ICCVAM work collaboratively to evaluate new and improved test methods and strategies... HUMAN SERVICES Nomination of In Vitro Test Methods for Detection and Quantification of Botulinum... Methods AGENCY: Division of National Toxicology Program (NTP), National Institute of Environmental...

  2. Quantification of sudomotor innervation: a comparison of 3 methods

    PubMed Central

    Gibbons, Christopher H; Illigens, Ben MW; Wang, Ningshan; Freeman, Roy

    2011-01-01

    Introduction Peripheral sudomotor dysfunction is present in many peripheral neuropathies, but structural assessments of sudomotor fibers rarely occur. Methods We evaluated 36 diabetic and 72 healthy control subjects who underwent detailed neurologic examinations and punch skin biopsies. Physical exam findings were quantified by neuropathy impairment score in the lower limb. Skin biopsies quantified intra-epidermal nerve fiber density (IENFD) and sweat gland nerve fiber density (SGNFD) by a manual, automated and semi-quantitative method. Results The automated and manual SGNFD correlated with the IENFD at the same site (r=0.62, P<0.05 automated method, r=0.67, P<0.05 manual method). As neuropathy worsened the SGNFD at the distal leg declined (automated counting r=−0.81, P<0.001; manual counting r=−0.88, P<0.001). The semi-quantitative method displayed poor inter- and intra-reviewer reliability and correlated poorly with standard neuropathy evaluation scores. Discussion Our results suggest that sudomotor fibers can be rapidly and reproducibly quantified, and results correlate well with physical exam findings. PMID:20544913

  3. Development and validation of an ESI-LC-MS/MS method for simultaneous identification and quantification of 24 analytes of forensic relevance in vitreous humour, whole blood and plasma.

    PubMed

    Arora, Beauty; Velpandian, Thirumurthy; Saxena, Rohit; Lalwani, Sanjeev; Dogra, T D; Ghose, Supriyo

    2016-01-01

    Detection and quantification of drugs from various biological matrices are of immense importance in forensic toxicological analysis. Despite the various reported methods, development of a new method for the detection and quantification of drugs is still an active area of research. However, every method and biological matrix has its own limitation, which further encourage forensic toxicologists to develop new methods and to explore new matrices for the analysis of drugs. In this study, an electrospray ionization-liquid chromatograph-tandem mass spectrometry (ESI-LC-MS/MS) method is developed and validated for simultaneous identification and quantification of 24 drugs of forensic relevance in various body fluids, namely, whole blood, plasma and vitreous humour. The newly developed method has been validated for intra-day and inter-day accuracy, precision, selectivity and sensitivity. Absolute recovery shows a mean of 84.5, 86.2, and 103% in the vitreous humour, whole blood and plasma respectively, which is suitable for the screening procedure. Further, the absolute matrix effect (AME) shows a mean of 105, 96.5, and 109% in the vitreous humour, whole blood and plasma, respectively. In addition, to examine the practical utility of this method, it has been applied for screening of drugs in post-mortem samples of the vitreous humour, whole blood and plasma collected at autopsy from ten cadavers. Experimental results show that the newly developed method is well applicable for screening of analytes in all the three matrices.

  4. Method based on chirp decomposition for dispersion mismatch compensation in precision absolute distance measurement using swept-wavelength interferometry.

    PubMed

    Lu, Cheng; Liu, Guodong; Liu, Bingguo; Chen, Fengdong; Hu, Tao; Zhuang, Zhitao; Xu, Xinke; Gan, Yu

    2015-12-14

    We establish a theoretical model of dispersion mismatch in absolute distance measurements using swept-wavelength interferometry (SWI) and propose a novel dispersion mismatch compensation method called chirp decomposition. This method separates the dispersion coefficient and distance under test, which ensures dispersion mismatch compensation without introducing additional random errors. In the measurement of a target located at 3.9 m, a measurement resolution of 45.9 μm is obtained, which is close to the theoretical resolution, and a standard deviation of 0.74 μm is obtained, which is better than the traditional method. The measurement results are compared to a single-frequency laser interferometer. The target moves from 1 m to 3.7 m, and the measurement precision using the new method is less than 0.81 μm.

  5. An improved method for quantification of Vibrio vulnificus in oysters.

    PubMed

    Cruz, C D; Win, J K; Fletcher, G C

    2013-12-01

    Chromogenic agar was compared with the FDA recommended cellobiose-colistin agar for assessment of Vibrio vulnificus in oysters. A two-step culture confirmation method was also evaluated. The inclusion of CA gave a 33% increase in the detection rate and the two-step culture confirmation eliminated 62.5% of false positives.

  6. Quantification of sudomotor innervation: a comparison of three methods.

    PubMed

    Gibbons, Christopher H; Illigens, Ben M W; Wang, Ningshan; Freeman, Roy

    2010-07-01

    Peripheral sudomotor dysfunction is present in many peripheral neuropathies, but structural assessments of sudomotor fibers rarely occur. We evaluated 36 diabetic and 72 healthy control subjects who underwent detailed neurologic examinations and punch skin biopsies. Physical exam findings were quantified by neuropathy impairment score in the lower limb. Skin biopsies quantified intraepidermal nerve fiber density (IENFD) and sweat gland nerve fiber density (SGNFD) by a manual, automated, and semiquantitative method. The automated and manual SGNFD correlated with the IENFD at the same site (r = 0.62, P < 0.05 automated method, r = 0.67, P < 0.05 manual method). As neuropathy worsened, the SGNFD at the distal leg declined (automated counting r = -0.81, P < 0.001; manual counting r = -0.88, P < 0.001). The semiquantitative method displayed poor inter- and intrareviewer reliability and correlated poorly with standard neuropathy evaluation scores. Our results suggest that sudomotor fibers can be rapidly and reproducibly quantified, and results correlate well with physical exam findings.

  7. Method validation for methanol quantification present in working places

    NASA Astrophysics Data System (ADS)

    Muna, E. D. M.; Bizarri, C. H. B.; Maciel, J. R. M.; da Rocha, G. P.; de Araújo, I. O.

    2015-01-01

    Given the widespread use of methanol by different industry sectors and high toxicity associated with this substance, it is necessary to use an analytical method able to determine in a sensitive, precise and accurate levels of methanol in the air of working environments. Based on the methodology established by the National Institute for Occupational Safety and Health (NIOSH), it was validated a methodology for determination of methanol in silica gel tubes which had demonstrated its effectiveness based on the participation of the international collaborative program sponsored by the American Industrial Hygiene Association (AIHA).

  8. [Study on quantificational analysis method for the non-crystalline content in blast furnace slag].

    PubMed

    Yan, Ding-Liu; Guo, Pei-Min; Qi, Yuan-Hong; Zhang, Chun-Xia; Wang, Hai-Feng; Dai, Xiao-Tian

    2008-02-01

    Quantificational analysis method for the non-crystalline and crystalline contents in blast furnace slag was studied by means of X-ray diffraction. The process of quantificational analysis method includes standard samples preparation, samples preparation, X-ray diffraction measurement and data treatment. The data treatment includes integration areas of non-crystalline curve and crystalline peaks in certain diffraction angle range, linear fitting and quantificational coefficient determination. The preparation methods of standard samples for X-ray diffraction of blast furnace slag were proposed, including 100% crystalline sample and 100% non-crystalline sample. The 100% crystalline sample can be obtained by heating blast furnace slag for 12 h at 1 000-1 200 degrees C, and the 100% non-crystalline sample can be obtained by quenching the molten slag with enough water. The X-ray diffraction method of quantificational analysis of non-crystalline content in blast furnace slag was proposed with the 100% non-crystalline and 100% crystalline standard samples, and the quantificational coefficient can be obtained by linear regression on the integration areas of non-crystalline curve and crystalline peaks of X-ray diffraction in the 2-theta range 20 degrees-40 degrees. This method is suitable for the blast furnace slag with the non-crystalline content over 80%. The non-crystalline and crystalline contents of original blast furnace slag are obtained by combining the X-ray diffraction results and mathematical treatment, and this method is suitable for the blast furnace slag with the non-crystalline content over 90%, whose process includes preparing the 100% crystalline standard sample by heating blast furnace slag for 12 h at 1000-1200 degrees C, samples preparation with the 0.02 interval in the 0-0.1 mass ratio range of 100% crystalline to original slag, X-ray diffraction measurement of the samples prepared and data treatment using iterative linear regression. The

  9. Quantification of emissions from knapsack sprayers: 'the weight method

    NASA Astrophysics Data System (ADS)

    Garcia-Santos, Glenda; Binder, Claudia R.

    2010-05-01

    Misuse of pesticides kill or seriously sicken thousands of people every year and poison the natural environment. Investigations of occupational and environmental risk have received considerable interest over the last decades. And yet, lack of staff and analytical equipments as well the costs of chemical analyses make difficult, if not impossible, the control of the pesticide contamination and residues in humans, air, water, and soils in developing countries. To assess emissions of pesticides (transport and deposition) during spray application and the risk for the human health and the environment, tracers can be useful tools. Uranine was used to quantify drift airborne and later deposition on the neighbouring field and clothes of the applicator after spraying with a knapsack sprayer in one of the biggest areas of potato production in Colombia. Keeping the same setup the amount of wet drift was measured by difference in the weight of high absorbent papers used to collect the tracer. Surprisingly this weight method (Weight-HAP) was able to explain 71% of the drift variance measured with the tracer. Therefore the weight method is presented as a suitable rapid low cost screening tool, complementary to toxicological tests, to assess air pollution, occupational and environmental exposure generated by the emissions from knapsack sprayers during pesticide application. This technique might be important in places were there is lack of analytical instruments.

  10. Improvement of biochemical methods of polyP quantification

    PubMed Central

    Bru, Samuel; Jiménez, Javier; Canadell, David; Ariño, Joaquín; Clotet, Josep

    2016-01-01

    Polyphosphate (polyP) is an abundant and physiologically important biomolecule for virtually any living cell. Therefore, determination of changes in cellular content of polyP is crucial for its functional characterization. Determination of cellular polyP has been performed by many different methods, and the lack of a standardized procedure is possibly responsible for the large dispersion of results found in the relevant literature. For a relatively simple organism, such as the yeast Saccharomyces cerevisiae, this variation can be up to 12-fold. polyP extraction and determination of free phosphate released by enzymatic degradation of the polymer is a method quite common and relatively straightforward for polyP determination. By using the yeast S. cerevisiae as model, we have experimentally evaluated the different steps in this procedure in order to identify critical issues that might explain the disparate reported results. As the main output of this evaluation we propose a straightforward and robust procedure that can be used as gold standard protocol for cellular polyP purification and determination from unicellular organisms, thus providing consistency to measurements and facilitating inter-laboratory comparisons and biological interpretation of the results. PMID:28357384

  11. Validation of a spectrophotometric method for quantification of carboxyhemoglobin.

    PubMed

    Luchini, Paulo D; Leyton, Jaime F; Strombech, Maria de Lourdes C; Ponce, Julio C; Jesus, Maria das Graças S; Leyton, Vilma

    2009-10-01

    The measurement of carboxyhemoglobin (COHb) levels in blood is a valuable procedure to confirm exposure to carbon monoxide (CO) either for forensic or occupational matters. A previously described method using spectrophotometric readings at 420 and 432 nm after reduction of oxyhemoglobin (O(2)Hb) and methemoglobin with sodium hydrosulfite solution leads to an exponential curve. This curve, used with pre-established factors, serves well for lower concentrations (1-7%) or for high concentrations (> 20%) but very rarely for both. The authors have observed that small variations on the previously described factors F1, F2, and F3, obtained from readings for 100% COHb and 100% O(2)Hb, turn into significant changes in COHb% results and propose that these factors should be determined every time COHb is measured by reading CO and O(2) saturated samples. This practice leads to an increase in accuracy and precision.

  12. Quantification of detergent using colorimetric methods in membrane protein crystallography.

    PubMed

    Prince, Chelsy; Jia, Zongchao

    2015-01-01

    Membrane protein crystallography has the potential to greatly aid our understanding of membrane protein biology. Yet, membrane protein crystals remain challenging to produce. Although robust methods for the expression and purification of membrane proteins continue to be developed, the detergent component of membrane protein samples is equally important to crystallization efforts. This chapter describes the development of three colorimetric assays for the quantitation of detergent in membrane protein samples and provides detailed protocols. All of these techniques use small sample volumes and have potential applications in crystallography. The application of these techniques in crystallization prescreening, detergent concentration modification, and detergent exchange experiments is demonstrated. It has been observed that the concentration of detergent in a membrane protein sample can be just as important as the protein concentration when attempting to reproduce crystallization lead conditions.

  13. Optical phase step method for absolute ranging interferometry using computer-generated holograms

    NASA Astrophysics Data System (ADS)

    Deininger, Martin; Wang, Lingli; Gerstner, Klaus; Tschudi, Theo

    1995-09-01

    One main problem of an interferometric measurement is to evaluate the object distance from the interference function. One of the known methods that delivers the object phase is the phase step method. Here we introduce computer-generated holograms to realize parallel phase steps without phase modulation of the reference path.

  14. Absolute Zero

    NASA Astrophysics Data System (ADS)

    Donnelly, Russell J.; Sheibley, D.; Belloni, M.; Stamper-Kurn, D.; Vinen, W. F.

    2006-12-01

    Absolute Zero is a two hour PBS special attempting to bring to the general public some of the advances made in 400 years of thermodynamics. It is based on the book “Absolute Zero and the Conquest of Cold” by Tom Shachtman. Absolute Zero will call long-overdue attention to the remarkable strides that have been made in low-temperature physics, a field that has produced 27 Nobel Prizes. It will explore the ongoing interplay between science and technology through historical examples including refrigerators, ice machines, frozen foods, liquid oxygen and nitrogen as well as much colder fluids such as liquid hydrogen and liquid helium. A website has been established to promote the series: www.absolutezerocampaign.org. It contains information on the series, aimed primarily at students at the middle school level. There is a wealth of material here and we hope interested teachers will draw their student’s attention to this website and its substantial contents, which have been carefully vetted for accuracy.

  15. Quantification of immobilized Candida antarctica lipase B (CALB) using ICP-AES combined with Bradford method.

    PubMed

    Nicolás, Paula; Lassalle, Verónica L; Ferreira, María L

    2017-02-01

    The aim of this manuscript was to study the application of a new method of protein quantification in Candida antarctica lipase B commercial solutions. Error sources associated to the traditional Bradford technique were demonstrated. Eight biocatalysts based on C. antarctica lipase B (CALB) immobilized onto magnetite nanoparticles were used. Magnetite nanoparticles were coated with chitosan (CHIT) and modified with glutaraldehyde (GLUT) and aminopropyltriethoxysilane (APTS). Later, CALB was adsorbed on the modified support. The proposed novel protein quantification method included the determination of sulfur (from protein in CALB solution) by means of Atomic Emission by Inductive Coupling Plasma (AE-ICP). Four different protocols were applied combining AE-ICP and classical Bradford assays, besides Carbon, Hydrogen and Nitrogen (CHN) analysis. The calculated error in protein content using the "classic" Bradford method with bovine serum albumin as standard ranged from 400 to 1200% when protein in CALB solution was quantified. These errors were calculated considering as "true protein content values" the results of the amount of immobilized protein obtained with the improved method. The optimum quantification procedure involved the combination of Bradford method, ICP and CHN analysis.

  16. Correction to Method of Establishing the Absolute Radiometric Accuracy of Remote Sensing Systems While On-orbit Using Characterized Stellar Sources

    NASA Technical Reports Server (NTRS)

    Bowen, Howard S.; Cunningham, Douglas M.

    2007-01-01

    The contents include: 1) Brief history of related events; 2) Overview of original method used to establish absolute radiometric accuracy of remote sensing instruments using stellar sources; and 3) Considerations to improve the stellar calibration approach.

  17. Comparison of MR-less PiB SUVR quantification methods.

    PubMed

    Bourgeat, Pierrick; Villemagne, Victor L; Dore, Vincent; Brown, Belinda; Macaulay, S Lance; Martins, Ralph; Masters, Colin L; Ames, David; Ellis, Kathryn; Rowe, Christopher C; Salvado, Olivier; Fripp, Jurgen

    2015-01-01

    (11)C-Pittsburgh compound B (PiB) is a positron emission tomography (PET) tracer designed to bind to amyloid-β (Aβ) plaques, one of the hallmarks of Alzheimer's disease (AD). The potential of PiB as an early marker of AD led to the increasing use of PiB in clinical research studies and development of several F-18-labeled Aβ radiotracers. Automatic quantification of PiB images requires an accurate parcellation of the brain's gray matter (GM). Typically, this relies on a coregistered magnetic resonance imaging (MRI) to extract the cerebellar GM, compute the standardized uptake value ratio (SUVR), and provide parcellation and segmentation for quantification of regional and global SUVR. However, not all subjects can undergo MRI, in which case, an MR-less method is desirable. In this study, we assess 3 PET-only quantification methods: a mean atlas, an adaptive atlas, and a multi-atlas approaches on a database of 237 subjects having been imaged with both PiB PET and MRI. The PET-only methods were compared against MR-based SUVR quantification and evaluated in terms of correlation, average error, and performance in classifying subjects with low and high Aβ deposition. The mean atlas method suffered from a significant bias between the estimated neocortical SUVR and the PiB status, resulting in an overall error of 5.6% (R(2) = 0.98), compared with the adaptive and multi-atlas approaches that had errors of 3.06% and 2.74%, respectively (R(2) = 0.98), and no significant bias. In classifying PiB-negative from PiB-positive subjects, the mean atlas had 10 misclassified subjects compared with 0 for the adaptive and 1 for the multi-atlas approach. Overall, the adaptive and the multi-atlas approaches performed similarly well against the MR-based quantification and would be a suitable replacements for PiB quantification when no MRI is available.

  18. Evaluation of Perfusion Quantification Methods with Ultrasound Contrast Agents in a Machine-Perfused Pig Liver.

    PubMed

    Averkiou, M; Keravnou, C P; Izamis, M L; Leen, E

    2016-05-03

    Purpose: To evaluate dynamic contrast-enhanced ultrasound (DCEUS) as a tool for measuring blood flow in the macro- and microcirculation of an ex-vivo machine-perfused pig liver and to confirm the ability of DCEUS to accurately detect induced flow rate changes so that it could then be used clinically for monitoring flow changes in liver tumors. Materials and Methods: Bolus injections of contrast agents in the hepatic artery (HA) and portal vein (PV) were administered to 3 machine-perfused pig livers. Flow changes were induced by the pump of the machine perfusion system. The induced flow rates were of clinical relevance (150 - 400 ml/min for HA and 400 - 1400 ml/min for PV). Quantification parameters from time-intensity curves [rise time (RT), mean transit time (MTT), area under the curve (AUC) and peak intensity (PI)] were extracted in order to evaluate whether the induced flow changes were reflected in these parameters. Results: A linear relationship between the image intensity and the microbubble concentration was confirmed first, while time parameters (RT and MMT) were found to be independent of concentration. The induced flow changes which propagated from the larger vessels to the parenchyma were reflected in the quantification parameters. Specifically, RT, MTT and AUC correlated with flow rate changes. Conclusion Machine-perfused pig liver is an excellent test bed for DCEUS quantification approaches for the study of the hepatic vascular networks. DCEUS quantification parameters (RT, MTT, and AUC) can measure relative flow changes of about 20 % and above in the liver vasculature. DCEUS quantification is a promising tool for real-time monitoring of the vascular network of tumors.

  19. Fluorescence-Based Bacterial Overlay Method for Simultaneous In Situ Quantification of Surface-Attached Bacteria▿

    PubMed Central

    Müller, Rainer; Gröger, Gerhard; Hiller, Karl-Anton; Schmalz, Gottfried; Ruhl, Stefan

    2007-01-01

    For quantification of bacterial adherence to biomaterial surfaces or to other surfaces prone to biofouling, there is a need for methods that allow a comparative analysis of small material specimens. A new method for quantification of surface-attached biotinylated bacteria was established by in situ detection with fluorescence-labeled avidin-D. This method was evaluated utilizing a silicon wafer model system to monitor the influences of surface wettability and roughness on bacterial adhesion. Furthermore, the effects of protein preadsorption from serum, saliva, human serum albumin, and fibronectin were investigated. Streptococcus gordonii, Streptococcus mitis, and Staphylococcus aureus were chosen as model organisms because of their differing adhesion properties and their clinical relevance. To verify the results obtained by this new technique, scanning electron microscopy and agar replica plating were employed. Oxidized and poly(ethylene glycol)-modified silicon wafers were found to be more resistant to bacterial adhesion than wafers coated with hydrocarbon and fluorocarbon moieties. Roughening of the chemically modified surfaces resulted in an overall increase in bacterial attachment. Preadsorption of proteins affected bacterial adherence but did not fully abolish the influence of the original surface chemistry. However, in certain instances, mostly with saliva or serum, masking of the underlying surface chemistry became evident. The new bacterial overlay method allowed a reliable quantification of surface-attached bacteria and could hence be employed for measuring bacterial adherence on material specimens in a variety of applications. PMID:17308176

  20. Development of absolute hot-wire anemometry by the 3omega method.

    PubMed

    Heyd, Rodolphe; Hadaoui, Abdallah; Fliyou, Mohamed; Koumina, Abdelaziz; El Hassani Ameziane, Lahcen; Outzourhit, Abdelkader; Saboungi, Marie-Louise

    2010-04-01

    We have developed hot-wire anemometry applying the 3omega method. The approach is based on the same heat transfer process as traditional anemometry, but substituting the constant current by a sinusoidal current and using synchronous detection to measure the conductive-convective exchange coefficient and the gas flow rate. Our theoretical model is tested with air flow at 300 K under atmospheric pressure: The experimental results are in agreement with the numerical simulation, justifying the technical choices in the 3omega method and the approximations made. The effectiveness of the 3omega method for measuring the flow rate and the conductive-convective exchange coefficient between the hot wire and flowing gas is discussed.

  1. Validation of a HPLC/FLD Method for Quantification of Tocotrienols in Human Plasma

    PubMed Central

    Che, Hui-Ling; Tan, Doryn Meam-Yee; Meganathan, Puvaneswari; Gan, Yee-Lin; Abdul Razak, Ghazali; Fu, Ju-Yen

    2015-01-01

    Quantification of tocotrienols in human plasma is critical when the attention towards tocotrienols on its distinctive properties is arising. We aim to develop a simple and practical normal-phase high performance liquid chromatography method to quantify the amount of four tocotrienol homologues in human plasma. Using both the external and internal standards, tocotrienol homologues were quantified via a normal-phase high performance liquid chromatography with fluorescence detector maintained at the excitation wavelength of 295 nm and the emission wavelength of 325 nm. The four tocotrienol homologues were well separated within 30 minutes. A large interindividual variation between subjects was observed as the absorption of tocotrienols is dependent on food matrix and gut lipolysis. The accuracies of lower and upper limit of quantification ranged between 92% and 109% for intraday assays and 90% and 112% for interday assays. This method was successfully applied to quantify the total amount of four tocotrienol homologues in human plasma. PMID:26604927

  2. Capillary isoelectric focusing-tandem mass spectrometry and reversed-phase liquid chromatography-tandem mass spectrometry for quantitative proteomic analysis of differentiating PC12 cells by eight-plex isobaric tags for relative and absolute quantification.

    PubMed

    Zhu, Guijie; Sun, Liangliang; Keithley, Richard B; Dovichi, Norman J

    2013-08-06

    We report the application of capillary isoelectric focusing for quantitative analysis of a complex proteome. Biological duplicates were generated from PC12 cells at days 0, 3, 7, and 12 following treatment with nerve growth factor. These biological duplicates were digested with trypsin, labeled using eight-plex isobaric tags for relative and absolute quantification (iTRAQ) chemistry, and pooled. The pooled peptides were separated into 25 fractions using reversed-phase liquid chromatography (RPLC). Technical duplicates of each fraction were separated by capillary isoelectric focusing (cIEF) using a set of amino acids as ampholytes. The cIEF column was interfaced to an Orbitrap Velos mass spectrometer with an electrokinetically pumped sheath-flow nanospray interface. This HPLC-cIEF-electrospray-tandem mass spectrometry (ESI-MS/MS) approach identified 835 protein groups and produced 2,329 unique peptides IDs. The biological duplicates were analyzed in parallel using conventional strong-cation exchange (SCX)-RPLC-ESI-MS/MS. The iTRAQ peptides were first separated into eight fractions using SCX. Each fraction was then analyzed by RPLC-ESI-MS/MS. The SCX-RPLC approach generated 1,369 protein groups and 3,494 unique peptide IDs. For protein quantitation, 96 and 198 differentially expressed proteins were obtained with RPLC-cIEF and SCX-RPLC, respectively. The combined set identified 231 proteins. Protein expression changes measured by RPLC-cEIF and SCX-RPLC were highly correlated.

  3. Quantification of Forecasting and Change-Point Detection Methods for Predictive Maintenance

    DTIC Science & Technology

    2015-08-19

    Final 3. DATES COVERED (From - To) 22 Jul 2014 – 21 Jul 2015 4. TITLE AND SUBTITLE Quantification of forecasting and change -point...disadvantages of change detection techniques using Singular Spectral Transform (SST) and Autoregressive Integrated Moving Average (ARIMA) applied to equipment...are used to evaluate the capability of detection of both methods for several types of changes . SST was applied to change detection in rotating

  4. An immunomagnetic separation-real-time PCR method for quantification of Cryptosporidium parvum in water samples.

    PubMed

    Fontaine, Melanie; Guillot, Emmanuelle

    2003-07-01

    The protozoan parasite Cryptosporidium parvum is known to occur widely in both raw and drinking water and is the cause of waterborne outbreaks of gastroenteritis throughout the world. The routinely used method for the detection of Cryptosporidium oocysts in water is based on an immunofluorescence assay (IFA). It is both time-consuming and nonspecific for the human pathogenic species C. parvum. We have developed a TaqMan polymerase chain reaction (PCR) test that accurately quantifies C. parvum oocysts in treated and untreated water samples. The protocol consisted of the following successive steps: Envirochek capsule filtration, immunomagnetic separation (IMS), thermal lysis followed by DNA purification using Nanosep centrifugal devices and, finally, real-time PCR using fluorescent TaqMan technology. Quantification was accomplished by comparing the fluorescence signals obtained from test samples with those from standard dilutions of C. parvum oocysts. This IMS-real-time PCR assay permits rapid and reliable quantification over six orders of magnitude, with a detection limit of five oocysts for purified oocyst solutions and eight oocysts for spiked water samples. Replicate samples of spiked tap water and Seine River water samples (with approximately 78 and 775 oocysts) were tested. C. parvum oocyst recoveries, which ranged from 47.4% to 99% and from 39.1% to 68.3%, respectively, were significantly higher and less variable than those reported using the traditional US Environmental Protection Agency (USEPA) method 1622. This new molecular method offers a rapid, sensitive and specific alternative for C. parvum oocyst quantification in water.

  5. Greenhouse gas emissions from waste management--assessment of quantification methods.

    PubMed

    Mohareb, Eugene A; MacLean, Heather L; Kennedy, Christopher A

    2011-05-01

    Of the many sources of urban greenhouse gas (GHG) emissions, solid waste is the only one for which management decisions are undertaken primarily by municipal governments themselves and is hence often the largest component of cities' corporate inventories. It is essential that decision-makers select an appropriate quantification methodology and have an appreciation of methodological strengths and shortcomings. This work compares four different waste emissions quantification methods, including Intergovernmental Panel on Climate Change (IPCC) 1996 guidelines, IPCC 2006 guidelines, U.S. Environmental Protection Agency (EPA) Waste Reduction Model (WARM), and the Federation of Canadian Municipalities-Partners for Climate Protection (FCM-PCP) quantification tool. Waste disposal data for the greater Toronto area (GTA) in 2005 are used for all methodologies; treatment options (including landfill, incineration, compost, and anaerobic digestion) are examined where available in methodologies. Landfill was shown to be the greatest source of GHG emissions, contributing more than three-quarters of total emissions associated with waste management. Results from the different landfill gas (LFG) quantification approaches ranged from an emissions source of 557 kt carbon dioxide equivalents (CO2e) (FCM-PCP) to a carbon sink of -53 kt CO2e (EPA WARM). Similar values were obtained between IPCC approaches. The IPCC 2006 method was found to be more appropriate for inventorying applications because it uses a waste-in-place (WIP) approach, rather than a methane commitment (MC) approach, despite perceived onerous data requirements for WIP. MC approaches were found to be useful from a planning standpoint; however, uncertainty associated with their projections of future parameter values limits their applicability for GHG inventorying. MC and WIP methods provided similar results in this case study; however, this is case specific because of similarity in assumptions of present and future landfill

  6. Method of excess fractions with application to absolute distance metrology: wavelength selection and the effects of common error sources.

    PubMed

    Falaggis, Konstantinos; Towers, David P; Towers, Catherine E

    2012-09-20

    Multiwavelength interferometry (MWI) is a well established technique in the field of optical metrology. Previously, we have reported a theoretical analysis of the method of excess fractions that describes the mutual dependence of unambiguous measurement range, reliability, and the measurement wavelengths. In this paper wavelength, selection strategies are introduced that are built on the theoretical description and maximize the reliability in the calculated fringe order for a given measurement range, number of wavelengths, and level of phase noise. Practical implementation issues for an MWI interferometer are analyzed theoretically. It is shown that dispersion compensation is best implemented by use of reference measurements around absolute zero in the interferometer. Furthermore, the effects of wavelength uncertainty allow the ultimate performance of an MWI interferometer to be estimated.

  7. Absolute Summ

    NASA Astrophysics Data System (ADS)

    Phillips, Alfred, Jr.

    Summ means the entirety of the multiverse. It seems clear, from the inflation theories of A. Guth and others, that the creation of many universes is plausible. We argue that Absolute cosmological ideas, not unlike those of I. Newton, may be consistent with dynamic multiverse creations. As suggested in W. Heisenberg's uncertainty principle, and with the Anthropic Principle defended by S. Hawking, et al., human consciousness, buttressed by findings of neuroscience, may have to be considered in our models. Predictability, as A. Einstein realized with Invariants and General Relativity, may be required for new ideas to be part of physics. We present here a two postulate model geared to an Absolute Summ. The seedbed of this work is part of Akhnaton's philosophy (see S. Freud, Moses and Monotheism). Most important, however, is that the structure of human consciousness, manifest in Kenya's Rift Valley 200,000 years ago as Homo sapiens, who were the culmination of the six million year co-creation process of Hominins and Nature in Africa, allows us to do the physics that we do. .

  8. SU-E-T-33: An EPID-Based Method for Testing Absolute Leaf Position for MLC Without Backup Jaws

    SciTech Connect

    Hancock, S; Whitaker, M

    2014-06-01

    Purpose: Methods in common use for MLC leaf position QA are limited to measurements relative to an arbitrary reference position. The authors previously presented an EPID-based method for efficiently testing accuracy of leaf position relative to the mechanical isocenter for MLC with backup jaws. The purpose of this work is to extend that method to the general case of MLC without backup jaws. Methods: A pair of collimator walkout images is used to determine the location of the mechanical isocenter relative to the center of one field using a parameter called X-offset. The method allows for shift of the imager panel to cover subsets of MLC leaves within the limited field of view of the imager. For a shifted panel position, an image of three beam strips defined by a subset of MLC leaves allows determination of the position of each leaf relative to the isocenter. The location of the isocenter is determined by applying X-offset to an image of a single rectangular field obtained at that panel position. The method can also be used to test backup jaws instead of MLC leaves. A software tool was developed to efficiently analyze the images. Results: The software tool reports leaf position and deviation from nominal position, and provides visual displays to facilitate rapid qualitative interpretation. Test results using this method agree well with results using the previous method requiring backup jaws. Test results have been successfully used to recalibrate one model MLC (Elekta MLCi2™). Work in progress includes extension of the software tool to other MLC models, and quantification of reproducibility of the measurements. Conclusion: This work successfully demonstrates a method to efficiently and accurately measure MLC leaf position, or backup jaw position, relative to the mechanical isocenter of the collimator.

  9. An improved HPLC-DAD method for clavulanic acid quantification in fermentation broths of Streptomyces clavuligerus.

    PubMed

    Ramirez-Malule, Howard; Junne, Stefan; López, Carlos; Zapata, Julian; Sáez, Alex; Neubauer, Peter; Rios-Estepa, Rigoberto

    2016-02-20

    Clavulanic acid (CA) is an important secondary metabolite commercially produced by cultivation of Streptomyces clavuligerus (Sc). It is a potent inhibitor of bacterial β-lactamases. In this work, a specific and improved high performance liquid chromatography (HPLC) method, using a C-18 reversed phase column, diode array detector and gradient elution for CA quantification in fermentation broths of Sc, was developed and successfully validated. Samples were imidazole-derivatized for the purpose of creating a stable chromophore (clavulanate-imidazole). The calibration curve was linear over a typical range of CA concentration between 0.2 and 400mg/L. The detection and quantification limits were 0.01 and 0.02mg/L, respectively. The precision of the method was evaluated for CA spiked into production media and a recovery of 103.8%, on average, was obtained. The clavulanate-imidazole complex was not stable when the samples were not cooled during the analysis. The recovery rate was 39.3% on average. This assay was successfully tested for CA quantification in samples from Sc fermentation, using both, a chemically defined and a complex medium.

  10. Dietary Sugars Analysis: Quantification of Fructooligossacharides during Fermentation by HPLC-RI Method

    PubMed Central

    Correia, Daniela M.; Dias, Luís G.; Veloso, Ana C. A.; Dias, Teresa; Rocha, Isabel; Rodrigues, Lígia R.; Peres, António M.

    2014-01-01

    In this work, a simple chromatographic method is proposed and in-house validated for the quantification of total and individual fructooligossacharides (e.g., 1-kestose, nystose, and 1F-fructofuranosylnystose). It was shown that a high-performance liquid chromatography with refractive index detector could be used to monitor the dynamic of fructooligossacharides production via sucrose fermentation using Aspergillus aculeatus. This analytical technique may be easily implemented at laboratorial or industrial scale for fructooligossacharides mass-production monitoring allowing also controlling the main substrate (sucrose) and the secondary by-products (glucose and fructose). The proposed chromatographic method had a satisfactory intra- and inter-day variability (in general, with a relative standard deviation lower than 5%), high sensitivity for each sugar (usually, with a relative error lower than 5%), and low detection (lower than 0.06 ± 0.04 g/L) and quantification (lower than 0.2 ± 0.1 g/L) limits. The correct quantification of fructooligossacharides in fermentative media may allow a more precise nutritional formulation of new functional foods, since it is reported that different fructooligossacharides exhibit different biological activities and effects. PMID:25988114

  11. Assessment of methods for organic and inorganic carbon quantification in carbonate-containing Mediterranean soils

    NASA Astrophysics Data System (ADS)

    Apesteguia, Marcos; Virto, Iñigo; Plante, Alain

    2014-05-01

    Quantification of soil organic matter (SOM) stocks and fluxes continues to be an important endeavor in assessments of soil quality, and more broadly in assessments of ecosystem functioning. The quantification of SOM in alkaline, carbonate-containing soils, such as those found in Mediterranean areas, is complicated by the need to differentiate between organic carbon (OC) and inorganic carbon (IC), which continues to present methodological challenges. Acidification is frequently used to eliminate carbonates prior to soil OC quantification, but when performed in the liquid phase, can promote the dissolution and loss of a portion of the OC. Acid fumigation (AF) is increasingly preferred for carbonate removal, but its effectiveness is difficult to assess using conventional elemental and isotopic analyses. In addition, the potential effects of AF on SOM are not well characterized. The objective of the current study was to apply a multi-method approach to determine the efficacy of carbonate removal by AF and its effects on the residual SOM. We selected a set of 24 surface agricultural soils representing a large range of textures, SOM contents and presumed carbonate contents. For each soil, OC was determined using wet combustion (Walkley-Black) and IC was determined using the calcimeter method. Samples were then subjected to elemental (total C) and isotopic (δ13C) analyses by dry combustion using a Costech autoanalyzer coupled to a Thermo Finnigan Delta Plus isotope ratio mass spectrometer (IRMS) before and after AF. IC was equated to total C determined after fumigation, and OC was estimated as the different in total C before and after AF. Samples were also subjected to ramped oxidation using a Netzsch STA109 PC Luxx thermal analyzer coupled to a LICOR 820A infrared gas analyzer (IRGA). Quantification of OC was performed using evolved gas analysis of CO2 (CO2-EGA) in the exothermic region 200-500° C associated with organic matter combustion. IC was quantified by CO2-EGA

  12. Mapping the African thunderstorm center in absolute units using Schumann resonance spectral decomposition method

    NASA Astrophysics Data System (ADS)

    Dyrda, Michal; Kulak, Andrzej; Mlynarczyk, Janusz

    2015-04-01

    Monitoring of the global lightning activity provides a very useful tool to study the global warming phenomenon and the other longer-scale climate changes induced by humans. The lightning activity is measured using various observational methods form space (optical satellite observations) as well as from the ground mostly by VLF /LF lightning detection networks, i.e. World Wide Lightning Location Network (WWLLN) or lightning detection network (LINET) in Europe. However, the global lightning activity measurements are possible only in the ELF range. Here we examine the African thunderstorm activity center, which is the most violent and active one. In a spherical damped resonator, such as the Earth-ionosphere cavity, the electromagnetic field is described by the solution of an inhomogeneous wave equation. For such equation the general solution can be expressed by the superposition of the solutions of the homogeneous equation, describing the resonance field, and the component, which is quite strong close to the source and weakens with source-observer separation. Thus, the superposition of the standing wave field with the field of traveling waves, which supply the energy from the lighting discharges to the global resonator, is a main reason for an asymmetric shape of the observational Schumann resonance (SR) power spectra, which highly deviate from the Lorentz curves. It is possible to separate this component from the signal using the spectrum decomposition method proposed by Kułak et al. [2006]. In our approach, we apply the inverse problem solution for determining the distance of the dominant lightning source. The distances to the thunderstorm centers are calculated using the analytical models for the electromagnetic waves propagation in the Earth-ionosphere cavity. Such forms of analytic solutions of the resonant field in the spherical cavity is the zonal harmonic series representation, described by Mushtak and Williams [2002] and we calculated the sets of such curves

  13. Absolute Photometry

    NASA Astrophysics Data System (ADS)

    Hartig, George

    1990-12-01

    The absolute sensitivity of the FOS will be determined in SV by observing 2 stars at 3 epochs, first in 3 apertures (1.0", 0.5", and 0.3" circular) and then in 1 aperture (1.0" circular). In cycle 1, one star, BD+28D4211 will be observed in the 1.0" aperture to establish the stability of the sensitivity and flat field characteristics and improve the accuracy obtained in SV. This star will also be observed through the paired apertures since these are not calibrated in SV. The stars will be observed in most detector/grating combinations. The data will be averaged to form the inverse sensitivity functions required by RSDP.

  14. A versatile targeted metabolomics method for the rapid quantification of multiple classes of phenolics in fruits and beverages.

    PubMed

    Vrhovsek, Urska; Masuero, Domenico; Gasperotti, Mattia; Franceschi, Pietro; Caputi, Lorenzo; Viola, Roberto; Mattivi, Fulvio

    2012-09-12

    Compelling evidence of the health benefits of phenolic compounds and their impact on food quality have stimulated the development of analytical methods for the identification and quantification of these compounds in different matrices in recent years. A targeted metabolomics method has been developed for the quantification of 135 phenolics, such as benzoates, phenylpropanoids, coumarins, stilbenes, dihydrochalcones, and flavonoids, in fruit and tea extracts and wine using UPLC/QqQ-MS/MS. Chromatography was optimized to achieve separation of the compounds over a period of 15 min, and MRM transitions were selected for accurate quantification. The method was validated by studying the detection and quantification limits, the linearity ranges, and the intraday and interday repeatability of the analysis. The validated method was applied to the analysis of apples, berries, green tea, and red wine, providing a valuable tool for food quality evaluation and breeding studies.

  15. Quantification of SPIO nanoparticles in vivo using the finite perturber method.

    PubMed

    Langley, Jason; Liu, Wei; Jordan, E Kay; Frank, J A; Zhao, Qun

    2011-05-01

    The susceptibility gradients generated by super-paramagnetic iron oxide (SPIO) nanoparticles make them an ideal contrast agent in magnetic resonance imaging. Traditional quantification methods for SPIO nanoparticle-based contrast agents rely on either mapping T₂* values within a region or by modeling the magnetic field inhomogeneities generated by the contrast agent. In this study, a new model-based SPIO quantification method is introduced. The proposed method models magnetic field inhomogeneities by approximating regions containing SPIOs as ensembles of magnetic dipoles, referred to as the finite perturber method. The proposed method was verified using data acquired from a phantom and in vivo mouse models. The phantom consisted of an agar solution with four embedded vials, each vial containing known but different concentrations of SPIO nanoparticles. Gaussian noise was also added to the phantom data to test performance of the proposed method. The in vivo dataset was acquired using five mice, each of which was subcutaneously implanted in the flanks with 1 × 10(5) labeled and 1 × 10(6) unlabeled C6 glioma cells. For the phantom data set, the proposed algorithm was generate accurate estimations of the concentration of SPIOs. For the in vivo dataset, the method was able to give estimations of the concentration within SPIO-labeled tumors that are reasonably close to the known concentration.

  16. An LC-MS/MS method for multi-mycotoxin quantification in cow milk.

    PubMed

    Flores-Flores, Myra E; González-Peñas, Elena

    2017-03-01

    The simultaneous quantification of 15 mycotoxins in cow milk by liquid chromatography-mass spectrometry, is presented. Extraction was performed with acidified acetonitrile, followed by a cleanup step with sodium acetate. During validation limits of detection (LOD) and quantification, linearity, precision, accuracy, recovery, matrix effect, and stability were studied. LOD values were between 0.02 and 10.14ng/mL for aflatoxins M1, B1, B2, G1, G2, ochratoxins A and B, HT-2 and T-2 toxins, deepoxy-deoxynivalenol, zearalenone, sterigmatocystin and fumonisins B1, B2 and B3. Recovery values were between 82.6 and 94.4% for all the mycotoxins, except for fumonisins. The recovery values for fumonisins were between 42.1% and 64.6%. Matrix effect, between 25.5 and 96.8%, appeared for all of the mycotoxins, especially for deepoxy-deoxynivalenol, zearalenone and sterigmatocystin. The validated method achieves the quantification of those mycotoxins of major concern and mycotoxins that are not frequently studied in milk, such as fumonisins, sterigmatocystin or ochratoxin B.

  17. GBIQ: a non-arbitrary, non-biased method for quantification of fluorescent images

    PubMed Central

    Ninomiya, Youichirou; Zhao, Wei; Saga, Yumiko

    2016-01-01

    Non-arbitrary and non-biased quantification of fluorescent images is an essential tool for the data-centric approach to biological systems. Typical application is high-content analysis, where various phenotypic changes in cellular components and/or morphology are measured from fluorescent image data. A standard protocol to detect cellular phenotypes is cell-segmentation, in which boundaries of cellular components, such as cell nucleus and plasma membrane, are first identified to define cell segments, then acquiring various phenotypic data of each segment. To achieve reliable outcome, cell-segmentation requires manual adjustments of many parameters; this requirement could hamper automated image processing in high-throughput workflow, whose quantification must be non-arbitrary and non-biased. As a practical alternative to the segmentation-based method, we developed GBIQ (Grid Based Image Quantification), which allows comparison of cellular information without identification of single cells. GBIQ divides an image with tiles of fixed size grids and records statistics of the grids with their location coordinates, minimizing arbitrary intervenes. GBIQ requires only one parameter (size of grid) to be set; nonetheless it robustly produces results suitable for further statistical evaluation. The simplicity of GBIQ allows it to be readily implemented in an automated high-throughput image analysis workflow. PMID:27211912

  18. MPQ-cytometry: a magnetism-based method for quantification of nanoparticle-cell interactions

    NASA Astrophysics Data System (ADS)

    Shipunova, V. O.; Nikitin, M. P.; Nikitin, P. I.; Deyev, S. M.

    2016-06-01

    Precise quantification of interactions between nanoparticles and living cells is among the imperative tasks for research in nanobiotechnology, nanotoxicology and biomedicine. To meet the challenge, a rapid method called MPQ-cytometry is developed, which measures the integral non-linear response produced by magnetically labeled nanoparticles in a cell sample with an original magnetic particle quantification (MPQ) technique. MPQ-cytometry provides a sensitivity limit 0.33 ng of nanoparticles and is devoid of a background signal present in many label-based assays. Each measurement takes only a few seconds, and no complicated sample preparation or data processing is required. The capabilities of the method have been demonstrated by quantification of interactions of iron oxide nanoparticles with eukaryotic cells. The total amount of targeted nanoparticles that specifically recognized the HER2/neu oncomarker on the human cancer cell surface was successfully measured, the specificity of interaction permitting the detection of HER2/neu positive cells in a cell mixture. Moreover, it has been shown that MPQ-cytometry analysis of a HER2/neu-specific iron oxide nanoparticle interaction with six cell lines of different tissue origins quantitatively reflects the HER2/neu status of the cells. High correlation of MPQ-cytometry data with those obtained by three other commonly used in molecular and cell biology methods supports consideration of this method as a prospective alternative for both quantifying cell-bound nanoparticles and estimating the expression level of cell surface antigens. The proposed method does not require expensive sophisticated equipment or highly skilled personnel and it can be easily applied for rapid diagnostics, especially under field conditions.Precise quantification of interactions between nanoparticles and living cells is among the imperative tasks for research in nanobiotechnology, nanotoxicology and biomedicine. To meet the challenge, a rapid method

  19. Preliminary Method for Direct Quantification of Colistin Methanesulfonate by Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy

    PubMed Central

    Niece, Krista L.

    2015-01-01

    Colistin use has increased in response to the advent of infections caused by multidrug-resistant organisms. It is administered parenterally as an inactive prodrug, colistin methanesulfonate (CMS). Various formulations of CMS and labeling conventions can lead to confusion about colistin dosing, and questions remain about the pharmacokinetics of CMS. Since CMS does not have strong UV absorbance, current methods employ a laborious process of chemical conversion to colistin followed by precolumn derivatization to detect formed colistin by high-performance liquid chromatography. Here, we report a method for direct quantification of colistin methanesulfonate by attenuated total reflectance Fourier transform infrared spectroscopy (ATR FTIR). PMID:26124160

  20. Methods for calculating the absolute entropy and free energy of biological systems based on ideas from polymer physics.

    PubMed

    Meirovitch, Hagai

    2010-01-01

    The commonly used simulation techniques, Metropolis Monte Carlo (MC) and molecular dynamics (MD) are of a dynamical type which enables one to sample system configurations i correctly with the Boltzmann probability, P(i)(B), while the value of P(i)(B) is not provided directly; therefore, it is difficult to obtain the absolute entropy, S approximately -ln P(i)(B), and the Helmholtz free energy, F. With a different simulation approach developed in polymer physics, a chain is grown step-by-step with transition probabilities (TPs), and thus their product is the value of the construction probability; therefore, the entropy is known. Because all exact simulation methods are equivalent, i.e. they lead to the same averages and fluctuations of physical properties, one can treat an MC or MD sample as if its members have rather been generated step-by-step. Thus, each configuration i of the sample can be reconstructed (from nothing) by calculating the TPs with which it could have been constructed. This idea applies also to bulk systems such as fluids or magnets. This approach has led earlier to the "local states" (LS) and the "hypothetical scanning" (HS) methods, which are approximate in nature. A recent development is the hypothetical scanning Monte Carlo (HSMC) (or molecular dynamics, HSMD) method which is based on stochastic TPs where all interactions are taken into account. In this respect, HSMC(D) can be viewed as exact and the only approximation involved is due to insufficient MC(MD) sampling for calculating the TPs. The validity of HSMC has been established by applying it first to liquid argon, TIP3P water, self-avoiding walks (SAW), and polyglycine models, where the results for F were found to agree with those obtained by other methods. Subsequently, HSMD was applied to mobile loops of the enzymes porcine pancreatic alpha-amylase and acetylcholinesterase in explicit water, where the difference in F between the bound and free states of the loop was calculated. Currently

  1. Protein inference: A protein quantification perspective.

    PubMed

    He, Zengyou; Huang, Ting; Liu, Xiaoqing; Zhu, Peijun; Teng, Ben; Deng, Shengchun

    2016-08-01

    In mass spectrometry-based shotgun proteomics, protein quantification and protein identification are two major computational problems. To quantify the protein abundance, a list of proteins must be firstly inferred from the raw data. Then the relative or absolute protein abundance is estimated with quantification methods, such as spectral counting. Until now, most researchers have been dealing with these two processes separately. In fact, the protein inference problem can be regarded as a special protein quantification problem in the sense that truly present proteins are those proteins whose abundance values are not zero. Some recent published papers have conceptually discussed this possibility. However, there is still a lack of rigorous experimental studies to test this hypothesis. In this paper, we investigate the feasibility of using protein quantification methods to solve the protein inference problem. Protein inference methods aim to determine whether each candidate protein is present in the sample or not. Protein quantification methods estimate the abundance value of each inferred protein. Naturally, the abundance value of an absent protein should be zero. Thus, we argue that the protein inference problem can be viewed as a special protein quantification problem in which one protein is considered to be present if its abundance is not zero. Based on this idea, our paper tries to use three simple protein quantification methods to solve the protein inference problem effectively. The experimental results on six data sets show that these three methods are competitive with previous protein inference algorithms. This demonstrates that it is plausible to model the protein inference problem as a special protein quantification task, which opens the door of devising more effective protein inference algorithms from a quantification perspective. The source codes of our methods are available at: http://code.google.com/p/protein-inference/.

  2. A method for non-invasive full-field imaging and quantification of chemical species.

    PubMed

    Shkolnikov, Viktor; Santiago, Juan G

    2013-04-21

    We present a novel method for full-field scalar visualization and quantification of species concentration fields. We term this method species-altered fluorescence imaging (SAFI). The method employs electrically neutral fluorescent dyes whose quantum yields are selectively quenched or enhanced by species of interest. SAFI enables simultaneous imaging of material interfaces and provides non-invasive, scalar-field quantitation of two-dimensional species concentration fields. We describe criteria for choosing SAFI dyes and tabulate 35 promising SAFI dyes and their relevant properties. Next, we describe species concentration quantification with SAFI via Stern-Volmer quenching and discuss the sensitivity and resolution of our method. We demonstrate this method with two dyes, 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ) and 10-(3-sulfopropyl)acridinium betaine (SAB). We demonstrate our method in full-field visualization of several challenging electrokinetic flows: isotachophoresis (ITP) in both cationic and anionic modes, and in a convective electrokinetic instability (EKI) flow. Through these experiments we collectively quantify ion concentration shock velocities, simultaneously measure concentrations of five species, and quantify the development of an unsteady, chaotic, 2D flow.

  3. Quantification of SPIO Nanoparticles in vivo Using the Finite Perturber Method

    PubMed Central

    Langley, Jason; Liu, Wei; Jordan, E Kay; Frank, J. A.; Zhao, Qun

    2013-01-01

    The susceptibility gradients generated by super-paramagnetic iron oxide (SPIO) nanoparticles make them an ideal contrast agent in magnetic resonance imaging. Traditional quantification methods for SPIO nanoparticle-based contrast agents rely on either mapping T2* values within a region of interest or by modeling the magnetic field inhomogeneities generated by the contrast agent. In this study, a new model-based SPIO quantification method is introduced. The proposed method models magnetic field inhomogeneities by approximating regions containing SPIO nanoparticles as ensembles of magnetic dipoles, referred to as the finite perturber method. The proposed method was verified using data acquired from a phantom and in vivo mouse models. The phantom consisted of an agar solution with four embedded vials, each vial containing known but different concentrations of SPIO nanoparticles. Gaussian noise was also added to the phantom data to test performance of the proposed method. The in vivo dataset was acquired using five mice, each of which was subcutaneously implanted in the flanks with 1×105 labeled and 1×106 unlabeled C6 glioma cells. For both datasets, the proposed algorithm was found to model the gradient of the magnetic field inhomogeneities well and generate accurate estimations of the concentration of SPIOs. PMID:21500271

  4. Development and Validation of an UPLC-Q-TOF-MS Method for Quantification of Fuziline in Beagle Dog After Intragastric and Intravenous Administration.

    PubMed

    Gong, Xiao-hong; Li, Yan; Li, Yun-xia; Yuan, An; Zhao, Meng-jie; Zhang, Ruo-qi; Zeng, Dai-wen; Peng, Cheng

    2016-03-01

    A specific and sensitive UPLC-Q-TOF-MS method operated in the positive ion mode was developed and validated for the quantification of Fuziline in Beagle dog plasma. Fuziline and Neoline internal standard were separated on an Acquity UPLC BEH C18 column with the total running time of 4 min using gradient elution at the flow rate of 0.25 mL/min. The calibration curves for Fuziline showed good linearity in the concentrations ranging from 2 to 400 ng/mL with correlation coefficients (r) greater than 0.9971. The lower limit of quantification was 0.8 ng/mL. Intra- and interbatch relative standard deviations ranged from 2.11 to 3.11% and 3.12 to 3.81%, respectively. Fuziline was stable under different sample storage and processing conditions. The developed method was successfully applied to the comparative pharmacokinetic study of Fuziline in Beagle dog after intravenous and oral administration. Low absolute bioavailability of Fuziline (1.45 ± 0.76%) suggested a significant metabolism transformation extent in Beagle dog.

  5. Susceptibility and size quantification of small human veins from an MRI method.

    PubMed

    Hsieh, Ching-Yi; Cheng, Yu-Chung N; Xie, He; Haacke, E Mark; Neelavalli, Jaladhar

    2015-12-01

    Recently a method called CISSCO (Complex Image Summation around a Spherical or a Cylindrical Object) was introduced for accurately quantifying the susceptibility and the radius of any narrow cylindrical object at any orientation using a typical two-echo gradient echo sequence. This work further optimizes the method for quantifying oxygen saturation in small cerebral veins in the human brain. The revised method is first validated through numerical simulations and then applied to data from phantom and human brain. The effect of phase high pass filtering on the quantified parameters is studied and procedures for mitigating its adverse effects are suggested. Uncertainty of each measurement is estimated from the error propagation method. It is shown that the revised method allows for accurate quantification of both the vessel size and its oxygen saturation even in the case of a low SNR (signal to noise ratio) in the vein. The results are self consistent across different veins within a given subject with a variation of less than 6%. Finally, imaging parameters and some procedures are suggested for accurate susceptibility and radius quantifications of small human veins.

  6. Absolute x-ray energy calibration over a wide energy range using a diffraction-based iterative method.

    PubMed

    Hong, Xinguo; Chen, Zhiqiang; Duffy, Thomas S

    2012-06-01

    In this paper, we report a method of precise and fast absolute x-ray energy calibration over a wide energy range using an iterative x-ray diffraction based method. Although accurate x-ray energy calibration is indispensable for x-ray energy-sensitive scattering and diffraction experiments, there is still a lack of effective methods to precisely calibrate energy over a wide range, especially when normal transmission monitoring is not an option and complicated micro-focusing optics are fixed in place. It is found that by using an iterative algorithm the x-ray energy is only tied to the relative offset of sample-to-detector distance, which can be readily varied with high precision of the order of 10(-5) -10(-6) spatial resolution using gauge blocks. Even starting with arbitrary initial values of 0.1 Å, 0.3 Å, and 0.4 Å, the iteration process converges to a value within 3.5 eV for 31.122 keV x-rays after three iterations. Different common diffraction standards CeO(2), Au, and Si show an energy deviation of 14 eV. As an application, the proposed method has been applied to determine the energy-sensitive first sharp diffraction peak of network forming GeO(2) glass at high pressure, exhibiting a distinct behavior in the pressure range of 2-4 GPa. Another application presented is pair distribution function measurement using calibrated high-energy x-rays at 82.273 keV. Unlike the traditional x-ray absorption-based calibration method, the proposed approach does not rely on any edges of specific elements, and is applicable to the hard x-ray region where no appropriate absorption edge is available.

  7. Geomorphological Dating Using an Improved Scarp Degradation Model: Is This a Reliable Approach Compared With Common Absolute Dating Methods?

    NASA Astrophysics Data System (ADS)

    Oemisch, M.; Hergarten, S.; Neugebauer, H. J.

    2002-12-01

    Geomorphological dating of a certain landform or geomorphological structure is based on the evolution of the landscape itself. In this context it is difficult to use common absolute dating techniques such as luminescence and radiocarbon dating because they require datable material which is often not available. Additionally these methods do not always date the time since the formation of these structures. For these reasons the application of geomorphological dating seems one reliable possibility to date certain geomorphological features. The aim of our work is to relate present-day shapes of fault scarps and terrace risers to their ages. The time span since scarp formation ceased is reflected by the stage of degradation as well as the rounding of the profile edges due to erosive processes. It is assumed that the average rate of downslope soil movement depends on the local slope angle and can be described in terms of a diffusion equation. On the basis of these assumptions we present a model to simulate the temporal development of scarp degradation by erosion. A diffusivity reflecting the effects of soil erosion, surface runoff and detachability of particles as well as present-day shapes of scarps are included in the model. As observations of present-day scarps suggest a higher diffusivity at the toe than at the head of a slope, we suggest a linear approach with increasing diffusivities in downslope direction. First results show a better match between simulated and observed profiles of the Upper Rhine Graben in comparison to models using a constant diffusivity. To date the scarps the model has to be calibrated. For this purpose we estimate diffusivities by fitting modelled profiles to observed ones of known age. Field data have been collected in the area around Bonn, Germany and in the Alps, Switzerland. It is a matter of current research to assess the quality of this dating technique and to compare the results and the applicability with some of the absolute dating

  8. Ceramic materials lead to underestimated DNA quantifications: a method for reliable measurements.

    PubMed

    Piccinini, E; Sadr, N; Martin, I

    2010-07-22

    In the context of investigating cell-material interactions or of material-guided generation of tissues, DNA quantification represents an elective method to precisely assess the number of cells attached or embedded within different substrates. Nonetheless, nucleic acids are known to electrostatically bind to ceramics, a class of materials commonly employed in orthopaedic implants and bone tissue engineering scaffolds. This phenomenon is expected to lead to a relevant underestimation of the DNA amount, resulting in erroneous experimental readouts. The present work aims at *lpar;i) investigating the effects of DNA-ceramic bond occurrence on DNA quantification, and (ii) developing a method to reliably extract and accurately quantify DNA in ceramic-containing specimens. A cell-free model was adopted to study DNA-ceramic binding, highlighting an evident DNA loss (up to 90%) over a wide range of DNA/ceramic ratios (w/w). A phosphate buffer-based (800 mM) enzymatic extraction protocol was developed and its efficacy in terms of reliable DNA extraction and measurement was confirmed with commonly used fluorometric assays, for various ceramic substrates. The proposed buffered DNA extraction technique was validated in a cell-based experiment showing 95% DNA retrieval in a cell seeding experiment, demonstrating a 3.5-fold increase in measured DNA amount as compared to a conventional enzymatic extraction protocol. In conclusion, the proposed phosphate buffer method consistently improves the DNA extraction process assuring unbiased analysis of samples and allowing accurate and sensitive cell number quantification on ceramic containing substrates.

  9. New method for quantification of dye sorption using SBA mesoporous silica as a target sorbent.

    PubMed

    Nesic, Aleksandra R; Kokunesoski, Maja J; Volkov-Husovic, Tatjana D; Velickovic, Sava J

    2016-03-01

    In this work, a new method for the quantification of methyl violet cationic dye sorption onto SBA-15 mesoporous silica was developed. This method related the intensity of coloration of SBA-15 samples (after reached equilibrium sorption) within dye concentration in aqueous solution using Image-Pro Plus software. The sorption process of methyl violet dye onto SBA-15 was analyzed varying different initial parameters (dye concentration, mass of sorbent, pH of dye solution, and contact sorption time). SBA-15 proved as efficient sorbent for removal of methyl violet dye in contact time of 5 min, with maximum percentage of dye removal 99% at pH 8. The results obtained from Image-Pro Plus showed to be in good agreement with the sorption parameters obtained by UV/Vis spectroscopy, which has been the most commonly used instrument for quantification of dye sorption. The image analysis method proved well prediction of dye concentrations with maximum relative error of 1.83%. The advantages of this method are low cost and reliable quantitative evaluation with minimum of time.

  10. Quantification of sugars in wheat flours with an HPAEC-PAD method.

    PubMed

    Pico, Joana; Martínez, Mario M; Martín, M Teresa; Gómez, Manuel

    2015-04-15

    An HPAEC-PAD method has been developed and validated for the simultaneous determination and quantification of six sugars (glucose, isomaltose, maltose, maltotriose, maltotetraose and maltopentaose) in wheat flours, by extraction with water and precipitation of proteins with Carrez II. Analyses were carried out on a Hamilton RCX-30 column with a gradient elution of NaOH 50mM (A) and NaOH 50mM+NaAcO 500 mM (B). Total run time was 38 min. Detector conditions were as follows: E1, +100 mV; E2, +550 mV; E3, -100 mV. The method was validated, with LODs ranging between 0.03-0.21 mg L(-1) and LOQs between 0.10-0.71 mg L(-1), R(2) between 0.9941 and 0.9983; recoveries were from 74.16% to 110.86% and RSDs for intraday repeatability, interday repeatability and reproducibility between 0.35-8.34%, 2.34-6.64% and 1.90-5.68%, respectively. The method was successfully applied to quantification of these sugars in wheat flours.

  11. Flow Cytometric Methods for Indirect Analysis and Quantification of Gametogenesis in Chlamydomonas reinhardtii (Chlorophyceae)

    PubMed Central

    Tomkins, Joseph L.

    2016-01-01

    Induction of sexual reproduction in the facultatively sexual Chlamydomonas reinhardtii is cued by depletion of nitrogen. We explore the capacity for indirect monitoring of population variation in the gametogenic process using flow cytometry. We describe a high-throughput method capable of identifying fluorescence, ploidy and scatter profiles that track vegetative cells entering and undergoing gametogenesis. We demonstrate for the first time, that very early and late growth phases reduce the capacity to distinguish putative gametes from vegetative cells based on scatter and fluorescence profiles, and that early/mid-logarithmic cultures show the optimal distinction between vegetative cells and gamete scatter profiles. We argue that early/mid logarithmic cultures are valuable in such high throughput comparative approaches when investigating optimisation or quantification of gametogenesis based on scatter and fluorescence profiles. This approach provides new insights into the impact of culture conditions on gametogenesis, while documenting novel scatter and fluorescence profile shifts which typify the process. This method has potential applications to; enabling quick high-throughput monitoring, uses in increasing efficiency in the quantification of gametogenesis, as a method of comparing the switch between vegetative and gametic states across treatments, and as criteria for enrichment of gametic phenotypes in cell sorting assays. PMID:27676075

  12. Surrogate-based Multi-Objective Optimization and Uncertainty Quantification Methods for Large, Complex Geophysical Models

    NASA Astrophysics Data System (ADS)

    Gong, Wei; Duan, Qingyun

    2016-04-01

    Parameterization scheme has significant influence to the simulation ability of large, complex dynamic geophysical models, such as distributed hydrological models, land surface models, weather and climate models, etc. with the growing knowledge of physical processes, the dynamic geophysical models include more and more processes and producing more output variables. Consequently the parameter optimization / uncertainty quantification algorithms should also be multi-objective compatible. Although such algorithms have long been available, they usually require a large number of model runs and are therefore computationally expensive for large, complex dynamic models. In this research, we have developed surrogate-based multi-objective optimization method (MO-ASMO) and Markov Chain Monte Carlo method (MC-ASMO) for uncertainty quantification for these expensive dynamic models. The aim of MO-ASMO and MC-ASMO is to reduce the total number of model runs with appropriate adaptive sampling strategy assisted by surrogate modeling. Moreover, we also developed a method that can steer the search process with the help of prior parameterization scheme derived from the physical processes involved, so that all of the objectives can be improved simultaneously. The proposed algorithms have been evaluated with test problems and a land surface model - the Common Land Model (CoLM). The results demonstrated their effectiveness and efficiency.

  13. Simultaneous quantification of ten cytotoxic drugs by a validated LC-ESI-MS/MS method.

    PubMed

    Nussbaumer, Susanne; Fleury-Souverain, Sandrine; Antinori, Paola; Sadeghipour, Farshid; Hochstrasser, Denis F; Bonnabry, Pascal; Veuthey, Jean-Luc; Geiser, Laurent

    2010-12-01

    A liquid chromatography separation with electrospray ionisation and tandem mass spectrometry detection method was developed for the simultaneous quantification of ten commonly handled cytotoxic drugs in a hospital pharmacy. These cytotoxic drugs are cytarabine, gemcitabine, methotrexate, etoposide phosphate, cyclophosphamide, ifosfamide, irinotecan, doxorubicin, epirubicin and vincristine. The chromatographic separation was carried out by RPLC in less than 21 min, applying a gradient elution of water and acetonitrile in the presence of 0.1% formic acid. MS/MS was performed on a triple quadrupole in selected reaction monitoring mode. The analytical method was validated to determine the limit of quantification (LOQ) and quantitative performance: lowest LOQs were between 0.25 and 2 ng mL(-1) for the ten investigated cytotoxic drugs; trueness values (i.e. recovery) were between 85% and 110%, and relative standard deviations for both repeatability and intermediate precision were always inferior to 15%. The multi-compound method was successfully applied for the quality control of pharmaceutical formulations and for analyses of spiked samples on potentially contaminated surfaces.

  14. Development and validation of a microemulsion electrokinetic chromatography method for patulin quantification in commercial apple juice.

    PubMed

    Murillo-Arbizu, M; González-Peñas, E; Hansen, S H; Amézqueta, S; Ostergaard, J

    2008-06-01

    A microemulsion electrokinetic chromatography (MEECK) method for patulin (PAT) quantification in apple juice samples has been developed. The effects of several important factors such as co-surfactant type, concentration of surfactant, acetonitrile percentage in the microemulsion, and running voltage and temperature were investigated to determine the optimum conditions. They resulted to be: a background electrolyte (BGE) composed of 25mM of sodium tetraborate, SDS (2.16%w/w), ethanol (6.49%w/w), n-octanol (0.82%w/w) and 2%v/v acetonitrile; applied voltage of +15kV; and a capillary temperature of 35 degrees C. PAT was detected at 276nm. Quantification and detection limits (LOQ and LOD) in apple juice samples were 8.0microgL(-1) and 3.2microgL(-1), respectively. Patulin was extracted from apple juice using ethyl acetate with a mean recovery value of 75.3% (RSD=4.5). This method was applied to the measurement of patulin in twenty commercial apple juice samples obtained from different Danish supermarkets. The PAT apple juice mean and median levels obtained were 35.9 and 10.9microgL(-1), respectively. The comparison with a previously validated micellar electrokinetic chromatography (MEKC) method for PAT analysis showed the suitability of using MEEKC for this mycotoxin analysis. However, the expectations of obtaining a higher efficiency and thus lower limits of detection and quantitation when using MEEKC were not met.

  15. Quantification method for the appearance of melanin pigmentation using independent component analysis

    NASA Astrophysics Data System (ADS)

    Ojima, Nobutoshi; Okiyama, Natsuko; Okaguchi, Saya; Tsumura, Norimichi; Nakaguchi, Toshiya; Hori, Kimihiko; Miyake, Yoichi

    2005-04-01

    In the cosmetics industry, skin color is very important because skin color gives a direct impression of the face. In particular, many people suffer from melanin pigmentation such as liver spots and freckles. However, it is very difficult to evaluate melanin pigmentation using conventional colorimetric values because these values contain information on various skin chromophores simultaneously. Therefore, it is necessary to extract information of the chromophore of individual skins independently as density information. The isolation of the melanin component image based on independent component analysis (ICA) from a single skin image was reported in 2003. However, this technique has not developed a quantification method for melanin pigmentation. This paper introduces a quantification method based on the ICA of a skin color image to isolate melanin pigmentation. The image acquisition system we used consists of commercially available equipment such as digital cameras and lighting sources with polarized light. The images taken were analyzed using ICA to extract the melanin component images, and Laplacian of Gaussian (LOG) filter was applied to extract the pigmented area. As a result, for skin images including those showing melanin pigmentation and acne, the method worked well. Finally, the total amount of extracted area had a strong correspondence to the subjective rating values for the appearance of pigmentation. Further analysis is needed to recognize the appearance of pigmentation concerning the size of the pigmented area and its spatial gradation.

  16. A simple identification method for vaginal secretions using relative quantification of Lactobacillus DNA.

    PubMed

    Doi, Masanori; Gamo, Shinsuke; Okiura, Tatsuyuki; Nishimukai, Hiroaki; Asano, Migiwa

    2014-09-01

    In criminal investigations there are some cases in which identifying the presence of vaginal secretions provides crucial evidence in proving sexual assault. However, there are no methods for definitively identifying vaginal secretions. In the present study, we focused on Lactobacillus levels in vaginal secretions and developed a novel identification method for vaginal secretions by relative quantification based on real time PCR. We designed a Lactobacillus conserved region primer pair (LCP) by aligning 16S rRNA gene sequences from major vaginal Lactobacillus species (Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus iners and Lactobacillus jensenii), and selected the human specific primer pair (HSP) as an endogenous control for relative quantification. As a result, the ΔCt (ΔCt=Ct[LCP]-Ct[HSP]) values of vaginal secretions (11 out of 12 samples) were significantly lower than those of saliva, semen and skin surface samples, and it was possible to discriminate between vaginal secretions and other body fluids. For the one remaining sample, it was confirmed that the predominant species in the microflora was not of the Lactobacillus genus. The ΔCt values in this study were calculated when the total DNA input used from the vaginal secretions was 10pg or more. Additionally, the ΔCt values of samples up to 6-months-old, which were kept at room temperature, remained unchanged. Thus, we concluded in this study that the simple ΔCt method by real time PCR is a useful tool for detecting the presence of vaginal secretions.

  17. Automated measurement and quantification of heterotrophic bacteria in water samples based on the MPN method.

    PubMed

    Fuchsluger, C; Preims, M; Fritz, I

    2011-01-01

    Quantification of heterotrophic bacteria is a widely used measure for water analysis. Especially in terms of drinking water analysis, testing for microorganisms is strictly regulated by the European Drinking Water Directive, including quality criteria and detection limits. The quantification procedure presented in this study is based on the most probable number (MPN) method, which was adapted to comply with the need for a quick and easy screening tool for different kinds of water samples as well as varying microbial loads. Replacing tubes with 24-well titer plates for cultivation of bacteria drastically reduces the amount of culture media and also simplifies incubation. Automated photometric measurement of turbidity instead of visual evaluation of bacterial growth avoids misinterpretation by operators. Definition of a threshold ensures definite and user-independent determination of microbial growth. Calculation of the MPN itself is done using a program provided by the US Food and Drug Administration (FDA). For evaluation of the method, real water samples of different origins as well as pure cultures of bacteria were analyzed in parallel with the conventional plating methods. Thus, the procedure described requires less preparation time, reduces costs and ensures both stable and reliable results for water samples.

  18. A high throughput method for quantification of cell surface bound and internalized chitosan nanoparticles.

    PubMed

    Tammam, Salma N; Azzazy, Hassan M E; Lamprecht, Alf

    2015-11-01

    Chitosan has become a popular polymer for drug delivery. It's hydro solubility and mild formulation conditions have made it an attractive polymer for macromolecular delivery. Accurate quantification of internalized chitosan nanoparticles (NPs) is imperative for fair assessment of the nano-formulation where it is important to determine the exact amount of drug actually being delivered into the cell, especially for macromolecular drugs where cellular entry is limited by molecule size and/or charge. The preferential affinity of wheat germ agglutinin tagged with fluorescein isothiocyanate (WGA-FITC) to chitosan is exploited in the development of a simple and rapid method for the differentiation between and quantification of cell surface bound and internalized chitosan NPs. The percentage of cell surface bound NPs could be easily determined and corrected NP uptake could be calculated accordingly. The developed method is applicable in several cell lines and has successfully been tested with NPs with different sizes (25 and 150nm) and with very low NP concentrations (20μg/mL). The method will allow for the correct evaluation of chitosan NP uptake and could be further used to evaluate chitosan based nanomedicine and provide guidelines on how to modify NPs for enhanced internalization, and improved drug delivery.

  19. New Method for Simultaneous Quantification of 12 Ginsenosides in Red Ginseng Powder and Extract: In-house Method Validation.

    PubMed

    In, Gyo; Ahn, Nam-Geun; Bae, Bong-Seok; Han, Sung-Tai; Noh, Kil-Bong; Kim, Cheon-Suk

    2012-04-01

    For quality control of components in Korean red ginseng powder and extract, a new method for simultaneous quantification of 12 ginsenosides (Rg1, Re, Rf, Rh1, Rg2[S], Rg2[R], Rb1, Rc, Rb2, Rd, Rg3[S], and Rg3[R]) was studied. Compared to the official method for quantification of marker substances (ginsenosides Rg1 and Rb1), the proposed methods were guaranteed by in-house method validation. Several criteria such as linearity, specificity, precision and accuracy were evaluated. For red ginseng powder, recovery (averaging 95% to 105%) was calculated, and analysis of variance was carried out to estimate the relative standard deviation (0.20% to 2.12%). For red ginseng extract, the average recovery rate was 90% to 99% and the relative standard deviation was 0.39% to 2.40%. These results indicate that the proposed method could be used in the laboratory for determination of 12 ginsenosides in red ginseng powder and extract. In addition, this method was found to be suitable for quality control of ginseng products and potentially offer time and cost benefits.

  20. Effect of various methods for rectum delineation on relative and absolute dose-volume histograms for prostate IMRT treatment planning

    SciTech Connect

    Kusumoto, Chiaki; Ohira, Shingo; Miyazaki, Masayoshi; Ueda, Yoshihiro; Isono, Masaru; Teshima, Teruki

    2016-07-01

    Several reports have dealt with correlations of late rectal toxicity with rectal dose-volume histograms (DVHs) for high dose levels. There are 2 techniques to assess rectal volume for reception of a specific dose: relative-DVH (R-DVH, %) that indicates relative volume for a vertical axis, and absolute-DVH (A-DVH, cc) with its vertical axis showing absolute volume of the rectum. The parameters of DVH vary depending on the rectum delineation method, but the literature does not present any standardization of such methods. The aim of the present study was to evaluate the effects of different delineation methods on rectal DVHs. The enrollment for this study comprised 28 patients with high-risk localized prostate cancer, who had undergone intensity-modulated radiation therapy (IMRT) with the prescription dose of 78 Gy. The rectum was contoured with 4 different methods using 2 lengths, short (Sh) and long (Lg), and 2 cross sections, rectum (Rec) and rectal wall (Rw). Sh means the length from 1 cm above the seminal vesicles to 1 cm below the prostate and Lg the length from the rectosigmoid junction to the anus. Rec represents the entire rectal volume including the rectal contents and Rw the rectal volume of the area with a wall thickness of 4 mm. We compared dose-volume parameters by using 4 rectal contour methods for the same plan with the R-DVHs as well as the A-DVHs. For the high dose levels, the R-DVH parameters varied widely. The mean of V{sub 70} for Sh-Rw was the highest (19.4%) and nearly twice as high as that for Lg-Rec (10.4%). On the contrary, only small variations were observed in the A-DVH parameters (4.3, 4.3, 5.5, and 5.5 cc for Sh-Rw, Lg-Rw, Sh-Rec, and Lg-Rec, respectively). As for R-DVHs, the parameters of V{sub 70} varied depending on the rectal lengths (Sh-Rec vs Lg-Rec: R = 0.76; Sh-Rw vs Lg-Rw: R = 0.85) and cross sections (Sh-Rec vs Sh-Rw: R = 0.49; Lg-Rec vs Lg-Rw: R = 0.65). For A-DVHs, however, the parameters of Sh rectal A-DVHs hardly changed

  1. A comparison of conventional methods for the quantification of bacterial cells after exposure to metal oxide nanoparticles

    PubMed Central

    2014-01-01

    Background Due to potential interference of nanoparticles on bacterial quantification, there is a challenge to develop a fast, accurate and reproducible method for bacterial quantification. Currently various bacterial quantification methods are used by researchers performing nanoparticles study, but there has been no efficacy evaluation of these methods. Here we study interference of nanoparticles on three most commonly used conventional bacterial quantification methods, including colony counting to determine the colony-forming units (CFU), spectrophotometer method of optical density (OD) measurement, and flow cytometry (FCM). Results Three oxide nanoparticles including ZnO, TiO2, and SiO2 and four bacterial species including Salmonella enterica serovar Newport, Staphylococcus epidermidis, Enterococcus faecalis, and Escherichia coli were included in the test. Results showed that there is no apparent interference of the oxide nanoparticles on quantifications of all four bacterial species by FCM measurement; CFU counting is time consuming, less accurate and not suitable for automation; and the spectrophotometer method using OD measurement was the most unreliable method to quantify and detect the bacteria in the presence of the nanoparticles. Conclusion In summary, FCM measurement proved to be the best method, which is suitable for rapid, accurate and automatic detection of bacteria in the presence of the nanoparticles. PMID:25138641

  2. Emphysema quantification from CT scans using novel application of diaphragm curvature estimation: comparison with standard quantification methods and pulmonary function data

    NASA Astrophysics Data System (ADS)

    Keller, Brad M.; Reeves, Anthony P.; Yankelevitz, David F.; Henschke, Claudia I.; Barr, R. Graham

    2009-02-01

    Emphysema is a disease of the lungs that destroys the alveolar air sacs and induces long-term respiratory dysfunction. CT scans allow for the imaging of the anatomical basis of emphysema and quantification of the underlying disease state. Several measures have been introduced for the quantification emphysema directly from CT data; most,however, are based on the analysis of density information provided by the CT scans, which vary by scanner and can be hard to standardize across sites and time. Given that one of the anatomical variations associated with the progression of emphysema is the flatting of the diaphragm due to the loss of elasticity in the lung parenchyma, curvature analysis of the diaphragm would provide information about emphysema from CT. Therefore, we propose a new, non-density based measure of the curvature of the diaphragm that would allow for further quantification methods in a robust manner. To evaluate the new method, 24 whole-lung scans were analyzed using the ratios of the lung height and diaphragm width to diaphragm height as curvature estimates as well as using the emphysema index as comparison. Pearson correlation coefficients showed a strong trend of several of the proposed diaphragm curvature measures to have higher correlations, of up to r=0.57, with DLCO% and VA than did the emphysema index. Furthermore, we found emphysema index to have only a 0.27 correlation to the proposed measures, indicating that the proposed measures evaluate different aspects of the disease.

  3. Rapid quantification of microalgal lipids in aqueous medium by a simple colorimetric method.

    PubMed

    Mishra, Sanjiv K; Suh, William I; Farooq, Wasif; Moon, Myounghoon; Shrivastav, Anupama; Park, Min S; Yang, Ji-Won

    2014-03-01

    Identification of novel microalgal strains with high lipid productivity is one of the most important research topics in renewable biofuel research. However, the major bottleneck in the strain screening process is that currently known methods for the estimation of microalgal lipid are laborious and time-consuming. The present study successfully employed sulpho-phospho-vanillin (SPV) colorimetric method for direct quantitative measurement of lipids within liquid microalgal culture. The SPV reacts with lipids to produce a distinct pink color, and its intensity can be quantified using spectrophotometric methods by measuring absorbance at 530nm. This method was employed for a rapid quantification of intracellular lipid contents within Chlorella sp., Monoraphidium sp., Ettlia sp. and Nannochloropsis sp., all of which were found to have lipid contents ranging in between 10% and 30%. Subsequent analysis of the biomass using gas chromatography confirmed that our protocol is highly accurate (R(2)=0.99).

  4. Simultaneous Quantification of Antidiabetic Agents in Human Plasma by a UPLC–QToF-MS Method

    PubMed Central

    Fachi, Mariana Millan; Cerqueira, Letícia Bonancio; Leonart, Letícia Paula; de Francisco, Thais Martins Guimarães

    2016-01-01

    An ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry method for the simultaneous quantification of chlorpropamide, glibenclamide, gliclazide, glimepiride, metformin, nateglinide, pioglitazone, rosiglitazone, and vildagliptin in human plasma was developed and validated, using isoniazid and sulfaquinoxaline as internal standards. Following plasma protein precipitation using acetonitrile with 1% formic acid, chromatographic separation was performed on a cyano column using gradient elution with water and acetonitrile, both containing 0.1% formic acid. Detection was performed in a quadrupole time-of-flight analyzer, using electrospray ionization operated in the positive mode. Data from validation studies demonstrated that the new method is highly sensitive, selective, precise (RSD < 10%), accurate (RE < 12%), linear (r > 0.99), free of matrix and has no residual effects. The developed method was successfully applied to volunteers’ plasma samples. Hence, this method was demonstrated to be appropriate for clinical monitoring of antidiabetic agents. PMID:27930700

  5. Solid-state evaluation and polymorphic quantification of venlafaxine hydrochloride raw materials using the Rietveld method.

    PubMed

    Bernardi, Larissa S; Ferreira, Fábio F; Cuffini, Silvia L; Campos, Carlos E M; Monti, Gustavo A; Kuminek, Gislaine; Oliveira, Paulo R; Cardoso, Simone G

    2013-12-15

    Venlafaxine hydrochloride (VEN) is an antidepressant drug widely used for the treatment of depression. The purpose of this study was to carry out the preparation and solid state characterization of the pure polymorphs (Forms 1 and 2) and the polymorphic identification and quantification of four commercially-available VEN raw materials. These two polymorphic forms were obtained from different crystallization methods and characterized by X-ray Powder Diffraction (XRPD), Diffuse Reflectance Infrared Fourier Transform (DRIFT), Raman Spectroscopy (RS), liquid and solid state Nuclear Magnetic Resonance (NMR and ssNMR) spectroscopies, Differential Scanning Calorimetry (DSC), and Scanning Electron Microscopy (SEM) techniques. The main differences were observed by DSC and XRPD and the latter was chosen as the standard technique for the identification and quantification studies in combination with the Rietveld method for the commercial raw materials (VEN1-VEN4) acquired from different manufacturers. Additionally Form 1 and Form 2 can be clearly distinguished from their (13)C ssNMR spectra. Through the analysis, it was possible to conclude that VEN1 and VEN2 were composed only of Form 1, while VEN3 and VEN4 were a mixture of Forms 1 and 2. Additionally, the Rietveld refinement was successfully applied to quantify the polymorphic ratio for VEN3 and VEN4.

  6. The parallel reaction monitoring method contributes to a highly sensitive polyubiquitin chain quantification

    SciTech Connect

    Tsuchiya, Hikaru; Tanaka, Keiji Saeki, Yasushi

    2013-06-28

    Highlights: •The parallel reaction monitoring method was applied to ubiquitin quantification. •The ubiquitin PRM method is highly sensitive even in biological samples. •Using the method, we revealed that Ufd4 assembles the K29-linked ubiquitin chain. -- Abstract: Ubiquitylation is an essential posttranslational protein modification that is implicated in a diverse array of cellular functions. Although cells contain eight structurally distinct types of polyubiquitin chains, detailed function of several chain types including K29-linked chains has remained largely unclear. Current mass spectrometry (MS)-based quantification methods are highly inefficient for low abundant atypical chains, such as K29- and M1-linked chains, in complex mixtures that typically contain highly abundant proteins. In this study, we applied parallel reaction monitoring (PRM), a quantitative, high-resolution MS method, to quantify ubiquitin chains. The ubiquitin PRM method allows us to quantify 100 attomole amounts of all possible ubiquitin chains in cell extracts. Furthermore, we quantified ubiquitylation levels of ubiquitin-proline-β-galactosidase (Ub-P-βgal), a historically known model substrate of the ubiquitin fusion degradation (UFD) pathway. In wild-type cells, Ub-P-βgal is modified with ubiquitin chains consisting of 21% K29- and 78% K48-linked chains. In contrast, K29-linked chains are not detected in UFD4 knockout cells, suggesting that Ufd4 assembles the K29-linked ubiquitin chain(s) on Ub-P-βgal in vivo. Thus, the ubiquitin PRM is a novel, useful, quantitative method for analyzing the highly complicated ubiquitin system.

  7. SU-E-T-804: Verification of the BJR-25 Method of KQ Determination for CyberKnife Absolute Dosimetry

    SciTech Connect

    Gersh, J; Willett, B

    2015-06-15

    Purpose: Absolute calibration of the CyberKnife is performed using a 6cm-diameter cone defined at 80cm SAD. Since kQ is defined using PDD values determined using 10×10 cm fields at 100cm SSD, the PDD must be corrected in order to correctly apply the quality conversion factor. The accepted method is based on equivalent field-size conversions of PDD values using BJR25. Using the new InCise MLC system, the CK is capable of generating a rectangular field equivalent to 10×10 cm square field. In this study, a comparison is made between kQ values determined using the traditional BJR25 method and the MLC method introduced herein. Methods: First, kQ(BJR) is determined: a PDD is acquired using a 6cm circular field at 100cm SSD, its field size converted to an equivalent square, and PDD converted to a 10×10cm field using the appropriate BJR25 table. Maintaining a consistent setup, the collimator is changed, and the MLC method is used. Finally, kQ is determined using PDDs acquired with a 9.71×10.31cm at 100cm SSD. This field is produced by setting the field to a size of 7.77×8.25cm (since it is defined at 80cm SAD). An exact 10×10cm field since field size is relegated to increments of its leaf width (0.25cm). This comparison is made using an Exradin A1SL, IBA CC08, IBA CC13, and an Exradin A19. For each detector and collimator type, the beam injector was adjusted to give 5 different beam qualities; representing a range of clinical systems. Results: Averaging across all beam qualities, kQ(MLC) differed from kQ(BJR) by less than 0.15%. The difference between the values increased with detector volume. Conclusion: For CK users with standard cone collimators, the BJR25 method has been verified. For CK users the MLC system, a technique is described to determine kQ. Primary author is the President/Owner of Spectrum Medical Physics, LLC, a company which maintains contracts with Siemens Healthcare and Standard Imaging, Inc.

  8. APR1400 LBLOCA uncertainty quantification by Monte Carlo method and comparison with Wilks' formula

    SciTech Connect

    Hwang, M.; Bae, S.; Chung, B. D.

    2012-07-01

    An analysis of the uncertainty quantification for the PWR LBLOCA by the Monte Carlo calculation has been performed and compared with the tolerance level determined by Wilks' formula. The uncertainty range and distribution of each input parameter associated with the LBLOCA accident were determined by the PIRT results from the BEMUSE project. The Monte-Carlo method shows that the 95. percentile PCT value can be obtained reliably with a 95% confidence level using the Wilks' formula. The extra margin by the Wilks' formula over the true 95. percentile PCT by the Monte-Carlo method was rather large. Even using the 3 rd order formula, the calculated value using the Wilks' formula is nearly 100 K over the true value. It is shown that, with the ever increasing computational capability, the Monte-Carlo method is accessible for the nuclear power plant safety analysis within a realistic time frame. (authors)

  9. HUMAN ERROR QUANTIFICATION USING PERFORMANCE SHAPING FACTORS IN THE SPAR-H METHOD

    SciTech Connect

    Harold S. Blackman; David I. Gertman; Ronald L. Boring

    2008-09-01

    This paper describes a cognitively based human reliability analysis (HRA) quantification technique for estimating the human error probabilities (HEPs) associated with operator and crew actions at nuclear power plants. The method described here, Standardized Plant Analysis Risk-Human Reliability Analysis (SPAR-H) method, was developed to aid in characterizing and quantifying human performance at nuclear power plants. The intent was to develop a defensible method that would consider all factors that may influence performance. In the SPAR-H approach, calculation of HEP rates is especially straightforward, starting with pre-defined nominal error rates for cognitive vs. action-oriented tasks, and incorporating performance shaping factor multipliers upon those nominal error rates.

  10. Application of Photothermal Methods for Quantification of Carotenoids in Apricot Jams

    NASA Astrophysics Data System (ADS)

    Dóka, O.; Bicanic, D.; Stéger-Máté, M.; Végvári, Gy.

    2015-09-01

    Carotenes, found in a diversity of fruit-containing foods, are important sources of antioxidants; a good example is apricot jam. In the study described in this paper, both the total carotenoid content ( TCC) as well as the content of \\upbeta -carotene in six different apricot jams were quantified using traditional (UV-VIS) spectrophotometry (SP), high-performance liquid chromatography (HPLC), laser photoacoustic spectroscopy (LPAS), and the optothermal window (OW) method. Unlike SP and HPLC, LPAS and the OW methods require the minimum of sample preparation and only a one time calibration step which enables practically direct quantification of the TCC. Results were verified versus data obtained with SP as the reference technique. It was shown that LPAS and the OW method (at 473 nm) provide satisfactory results with R2=0.9884 and 0.9766 for LPAS and OW, respectively.

  11. Freshwater phytoplankton quantification by chlorophyll a: a comparative study of in vitro, in vivo and in situ methods.

    PubMed

    Gregor, J; Marsálek, B

    2004-02-01

    Standard ISO method for chlorophyll a quantification (extraction into ethanol, spectrophotometrical quantification at 665 and 750 nm), spectrofluorometry (reader for 96 wells, excitation 410 nm, emission 670 nm), and a submersible fluorescence probe for in situ phytoplankton quantification (excitation 410, 525, 570, 590, and 610 nm, emission 685 nm) were compared in different freshwater environments-reservoirs and rivers. The ISO method is accepted as a standard method but requires sample handling and transport to the laboratory. Spectrofluorometry is a sensitive method, even for natural phytoplankton populations. Nevertheless, it cannot be recommended for the quantification of cyanobacterial water blooms because colonial and filamentous species such as Microcystis, Anabaena, or Aphanizomenon display unacceptable variability (18-33%). The submersible probe featured high correlation with a standard ISO method (r=0.97, P<0.05). This probe can provide the selective measurement of technologically important phytoplankton groups like cyanobacteria, diatoms, green algae, and cryptophytes in lake vertical profiles of up to 100 m. The limitation of this instrument is the possible reabsorption of the light signal, e.g. in the presence of humic substances, or dense algal blooms. The use of submersible probes for in situ phytoplankton quantification can be recommended as a sensitive tool for water management, especially in the case of drinking water resources.

  12. Development of a method for detection and quantification of B. brongniartii and B. bassiana in soil

    PubMed Central

    Canfora, L.; Malusà, E.; Tkaczuk, C.; Tartanus, M.; Łabanowska, B.H.; Pinzari, F.

    2016-01-01

    A culture independent method based on qPCR was developed for the detection and quantification of two fungal inoculants in soil. The aim was to adapt a genotyping approach based on SSR (Simple Sequence Repeat) marker to a discriminating tracing of two different species of bioinoculants in soil, after their in-field release. Two entomopathogenic fungi, Beauveria bassiana and B. brongniartii, were traced and quantified in soil samples obtained from field trials. These two fungal species were used as biological agents in Poland to control Melolontha melolontha (European cockchafer), whose larvae live in soil menacing horticultural crops. Specificity of SSR markers was verified using controls consisting of: i) soil samples containing fungal spores of B. bassiana and B. brongniartii in known dilutions; ii) the DNA of the fungal microorganisms; iii) soil samples singly inoculated with each fungus species. An initial evaluation of the protocol was performed with analyses of soil DNA and mycelial DNA. Further, the simultaneous detection and quantification of B. bassiana and B. brongniartii in soil was achieved in field samples after application of the bio-inoculants. The protocol can be considered as a relatively low cost solution for the detection, identification and traceability of fungal bio-inoculants in soil. PMID:26975931

  13. Development of a method for detection and quantification of B. brongniartii and B. bassiana in soil

    NASA Astrophysics Data System (ADS)

    Canfora, L.; Malusà, E.; Tkaczuk, C.; Tartanus, M.; Łabanowska, B. H.; Pinzari, F.

    2016-03-01

    A culture independent method based on qPCR was developed for the detection and quantification of two fungal inoculants in soil. The aim was to adapt a genotyping approach based on SSR (Simple Sequence Repeat) marker to a discriminating tracing of two different species of bioinoculants in soil, after their in-field release. Two entomopathogenic fungi, Beauveria bassiana and B. brongniartii, were traced and quantified in soil samples obtained from field trials. These two fungal species were used as biological agents in Poland to control Melolontha melolontha (European cockchafer), whose larvae live in soil menacing horticultural crops. Specificity of SSR markers was verified using controls consisting of: i) soil samples containing fungal spores of B. bassiana and B. brongniartii in known dilutions; ii) the DNA of the fungal microorganisms; iii) soil samples singly inoculated with each fungus species. An initial evaluation of the protocol was performed with analyses of soil DNA and mycelial DNA. Further, the simultaneous detection and quantification of B. bassiana and B. brongniartii in soil was achieved in field samples after application of the bio-inoculants. The protocol can be considered as a relatively low cost solution for the detection, identification and traceability of fungal bio-inoculants in soil.

  14. A HPLC method for the quantification of butyramide and acetamide at ppb levels in hydrogeothermal waters

    SciTech Connect

    Gracy Elias; Earl D. Mattson; Jessica E. Little

    2012-01-01

    A quantitative analytical method to determine butyramide and acetamide concentrations at the low ppb levels in geothermal waters has been developed. The analytes are concentrated in a preparation step by evaporation and analyzed using HPLC-UV. Chromatographic separation is achieved isocratically with a RP C-18 column using a 30 mM phosphate buffer solution with 5 mM heptane sulfonic acid and methanol (98:2 ratio) as the mobile phase. Absorbance is measured at 200 nm. The limit of detection (LOD) for BA and AA were 2.0 {mu}g L{sup -1} and 2.5 {mu}g L{sup -1}, respectively. The limit of quantification (LOQ) for BA and AA were 5.7 {mu}g L{sup -1} and 7.7 {mu}g L{sup -1}, respectively, at the detection wavelength of 200 nm. Attaining these levels of quantification better allows these amides to be used as thermally reactive tracers in low-temperature hydrogeothermal systems.

  15. Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs

    PubMed Central

    Varkonyi-Gasic, Erika; Wu, Rongmei; Wood, Marion; Walton, Eric F; Hellens, Roger P

    2007-01-01

    MicroRNAs (miRNAs) are a class of small non-coding RNAs with a critical role in development and environmental responses. Efficient and reliable detection of miRNAs is an essential step towards understanding their roles in specific cells and tissues. However, gel-based assays currently used to detect miRNAs are very limited in terms of throughput, sensitivity and specificity. Here we provide protocols for detection and quantification of miRNAs by RT-PCR. We describe an end-point and real-time looped RT-PCR procedure and demonstrate detection of miRNAs from as little as 20 pg of plant tissue total RNA and from total RNA isolated from as little as 0.1 μl of phloem sap. In addition, we have developed an alternative real-time PCR assay that can further improve specificity when detecting low abundant miRNAs. Using this assay, we have demonstrated that miRNAs are differentially expressed in the phloem sap and the surrounding vascular tissue. This method enables fast, sensitive and specific miRNA expression profiling and is suitable for facilitation of high-throughput detection and quantification of miRNA expression. PMID:17931426

  16. Simultaneous determination of the absolute configuration of twelve monosaccharide enantiomers from natural products in a single injection by UPLC-UV/MS method

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In natural product chemistry, it is often crucial to determine sugar composition as well as the absolute configuration of each monosaccharide in glycosides. An ultra-performance liquid chromatography method using both photodiode array (PDA) and mass spectrometry detectors (UPLC-UV/MS) was developed....

  17. A rapid, sensitive and economic method for the detection, quantification and confirmation of aflatoxins.

    PubMed

    Majerus, P; Zakaria, Z

    1992-10-01

    A rapid, sensitive and economic method for the detection, quantification and confirmation of aflatoxins is described. Aflatoxins B1, B2, G1, and G2, are extracted by methanol/water (85 + 15) and partitioned into methylene dichloride. The methylene dichloride solution is cleaned up on a polypropylene column, filled with 0.5 g silica gel 60. The aflatoxins are eluted with chloroform-acetone (90:10) and are detected using bidirectional thin-layer chromatography (TLC) with aluminium silica gel foil. The mean recovery of aflatoxins B1, B2, G1, and G2 in corn samples was 73, 78, 80, and 64%, respectively; the limit of detection was 0.5 micrograms/kg. The results can also be confirmed by derivative formation using trifluoroacetic acid on the TLC plate. The method has been applied to a wide range of foods with good results.

  18. Development of a real-time PCR method for the differential detection and quantification of four solanaceae in GMO analysis: potato (Solanum tuberosum), tomato (Solanum lycopersicum), eggplant (Solanum melongena), and pepper (Capsicum annuum).

    PubMed

    Chaouachi, Maher; El Malki, Redouane; Berard, Aurélie; Romaniuk, Marcel; Laval, Valérie; Brunel, Dominique; Bertheau, Yves

    2008-03-26

    The labeling of products containing genetically modified organisms (GMO) is linked to their quantification since a threshold for the presence of fortuitous GMOs in food has been established. This threshold is calculated from a combination of two absolute quantification values: one for the specific GMO target and the second for an endogenous reference gene specific to the taxon. Thus, the development of reliable methods to quantify GMOs using endogenous reference genes in complex matrixes such as food and feed is needed. Plant identification can be difficult in the case of closely related taxa, which moreover are subject to introgression events. Based on the homology of beta-fructosidase sequences obtained from public databases, two couples of consensus primers were designed for the detection, quantification, and differentiation of four Solanaceae: potato (Solanum tuberosum), tomato (Solanum lycopersicum), pepper (Capsicum annuum), and eggplant (Solanum melongena). Sequence variability was studied first using lines and cultivars (intraspecies sequence variability), then using taxa involved in gene introgressions, and finally, using taxonomically close taxa (interspecies sequence variability). This study allowed us to design four highly specific TaqMan-MGB probes. A duplex real time PCR assay was developed for simultaneous quantification of tomato and potato. For eggplant and pepper, only simplex real time PCR tests were developed. The results demonstrated the high specificity and sensitivity of the assays. We therefore conclude that beta-fructosidase can be used as an endogenous reference gene for GMO analysis.

  19. Ascertaining year of birth/age at death in forensic cases: A review of conventional methods and methods allowing for absolute chronology.

    PubMed

    Lynnerup, Niels; Kjeldsen, Henrik; Zweihoff, Ralf; Heegaard, Steffen; Jacobsen, Christina; Heinemeier, Jan

    2010-09-10

    Based on an actual case, where we were able to ascertain the year of birth of three dead babies found in a deep-freezer to within 1-2 years (1986, 1988 and 2004, respectively), we review the current state of forensic age determination/year of birth determination. The age of an individual (year of birth) is often a fundamental piece of data in connection with forensic identification of unidentified bodies. The methods most often used are based on determining various morphological, age-related, changes on the skeleton (or teeth, although odontological methods are not reviewed in this paper). As such, these methods are all relative, i.e. they do not furnish calendar ages or years, but an estimate of the age at death, with a rather large range, i.e. the methods rely on biological aging following the chronological aging. More recently, methods have been proposed using more direct ascertainment of age at death, e.g. protein racemisation, or, as in our case, radiocarbon methods. Especially the latter method may in fact yield absolute ages (years of birth), because (14)C activity, as measured in specific proteins in specific cells or tissues in the body, were in equilibrium with the so-called bomb-pulse, when these proteins were formed (at birth). The bomb pulse reflects a dramatic change in atmospheric (14)C content due to nuclear bomb testing, and these dramatic changes can be rather tightly related to single calendar years.

  20. A BAYESIAN METHOD FOR CALCULATING REAL-TIME QUANTITATIVE PCR CALIBRATION CURVES USING ABSOLUTE PLASMID DNA STANDARDS

    EPA Science Inventory

    In real-time quantitative PCR studies using absolute plasmid DNA standards, a calibration curve is developed to estimate an unknown DNA concentration. However, potential differences in the amplification performance of plasmid DNA compared to genomic DNA standards are often ignore...

  1. Efficient uncertainty quantification of large two-dimensional optical systems with a parallelized stochastic Galerkin method.

    PubMed

    Zubac, Z; Fostier, J; De Zutter, D; Vande Ginste, D

    2015-11-30

    It is well-known that geometrical variations due to manufacturing tolerances can degrade the performance of optical devices. In recent literature, polynomial chaos expansion (PCE) methods were proposed to model this statistical behavior. Nonetheless, traditional PCE solvers require a lot of memory and their computational complexity leads to prohibitively long simulation times, making these methods non-tractable for large optical systems. The uncertainty quantification (UQ) of various types of large, two-dimensional lens systems is presented in this paper, leveraging a novel parallelized Multilevel Fast Multipole Method (MLFMM) based Stochastic Galerkin Method (SGM). It is demonstrated that this technique can handle large optical structures in reasonable time, e.g., a stochastic lens system with more than 10 million unknowns was solved in less than an hour by using 3 compute nodes. The SGM, which is an intrusive PCE method, guarantees the accuracy of the method. By conjunction with MLFMM, usage of a preconditioner and by constructing and implementing a parallelized algorithm, a high efficiency is achieved. This is demonstrated with parallel scalability graphs. The novel approach is illustrated for different types of lens system and numerical results are validated against a collocation method, which relies on reusing a traditional deterministic solver. The last example concerns a Cassegrain system with five random variables, for which a speed-up of more than 12× compared to a collocation method is achieved.

  2. A feasible method for indirect quantification of L-T4 in drugs by iodine determination.

    PubMed

    Mesko, Marcia Foster; Teotonio, Alessandra Cortes; Teixeira Oliveira, Dirce Taina; Rosa Novo, Diogo La; Costa, Vanize Caldeira

    2017-05-01

    In this work, a method combining microwave-induced combustion (MIC) for sample preparation of commercial levothyroxine sodium (L-T4) drugs (L-T4: 25-200µg/tablet), and potentiometry with ion selective electrode (ISE) for iodine determination and subsequent indirect quantification of L-T4 was proposed. The type and concentration of the absorbing solution were evaluated to select the most suitable conditions for this study. Using the MIC method, it was possible to use solutions as diluted as 150mmolL(-1) (NH4)2CO3 (for samples containing 25-200µg of L-T4/tablet) for I absorption. In these conditions, recoveries for L-T4 were between 99% and 101%, and relative standard deviations were lower than 10%. The limit of detection for L-T4 was 11.2µg/tablet, which is almost two times lower than the minimum concentration of L-T4 in commercially available drugs. Thus, the MIC was suitable for the digestion of several L-T4 drugs for subsequent I determination by ISE and indirect quantification of L-T4. Furthermore, the proposed method presents high throughput with low reagent consumption and consequently lower waste generation, making it suitable for routine determination of L-T4 in drugs. From the obtained results, it was possible to observe that one of the analyzed samples is not in agreement with the limits established by the United States Pharmacopeia, indicating the importance of the drug quality control. The United States Pharmacopeia establishes that each tablet must contain between 90% and 110% of the amount of active substance declared by the manufacturer.

  3. A probabilistic decomposition-synthesis method for the quantification of rare events due to internal instabilities

    NASA Astrophysics Data System (ADS)

    Mohamad, Mustafa A.; Cousins, Will; Sapsis, Themistoklis P.

    2016-10-01

    We consider the problem of the probabilistic quantification of dynamical systems that have heavy-tailed characteristics. These heavy-tailed features are associated with rare transient responses due to the occurrence of internal instabilities. Systems with these properties can be found in a variety of areas including mechanics, fluids, and waves. Here we develop a computational method, a probabilistic decomposition-synthesis technique, that takes into account the nature of internal instabilities to inexpensively determine the non-Gaussian probability density function for any arbitrary quantity of interest. Our approach relies on the decomposition of the statistics into a 'non-extreme core', typically Gaussian, and a heavy-tailed component. This decomposition is in full correspondence with a partition of the phase space into a 'stable' region where we have no internal instabilities, and a region where non-linear instabilities lead to rare transitions with high probability. We quantify the statistics in the stable region using a Gaussian approximation approach, while the non-Gaussian distribution associated with the intermittently unstable regions of phase space is inexpensively computed through order-reduction methods that take into account the strongly nonlinear character of the dynamics. The probabilistic information in the two domains is analytically synthesized through a total probability argument. The proposed approach allows for the accurate quantification of non-Gaussian tails at more than 10 standard deviations, at a fraction of the cost associated with the direct Monte-Carlo simulations. We demonstrate the probabilistic decomposition-synthesis method for rare events for two dynamical systems exhibiting extreme events: a two-degree-of-freedom system of nonlinearly coupled oscillators, and in a nonlinear envelope equation characterizing the propagation of unidirectional water waves.

  4. Leak Rate Quantification Method for Gas Pressure Seals with Controlled Pressure Differential

    NASA Technical Reports Server (NTRS)

    Daniels, Christopher C.; Braun, Minel J.; Oravec, Heather A.; Mather, Janice L.; Taylor, Shawn C.

    2015-01-01

    An enhancement to the pressure decay leak rate method with mass point analysis solved deficiencies in the standard method. By adding a control system, a constant gas pressure differential across the test article was maintained. As a result, the desired pressure condition was met at the onset of the test, and the mass leak rate and measurement uncertainty were computed in real-time. The data acquisition and control system were programmed to automatically stop when specified criteria were met. Typically, the test was stopped when a specified level of measurement uncertainty was attained. Using silicone O-ring test articles, the new method was compared with the standard method that permitted the downstream pressure to be non-constant atmospheric pressure. The two methods recorded comparable leak rates, but the new method recorded leak rates with significantly lower measurement uncertainty, statistical variance, and test duration. Utilizing this new method in leak rate quantification, projects will reduce cost and schedule, improve test results, and ease interpretation between data sets.

  5. A simple method for simultaneous quantification of total arabinoxylans and fructans in wheat flour.

    PubMed

    Liu, Zhiqian; Rochfort, Simone

    2014-08-20

    Current methods for measuring fructan and arabinoxylan concentrations in wheat flour are time-consuming, and each type of the polymers requires a separate method. Here, we report the development of a new method that allows simultaneous determination of arabinoxylan and fructan contents in wheat flour. The new method is based on a single hydrolysis procedure for both arabinoxylans and fructans and an optimized separation technique for all monomers released. Owing to the use of milder hydrolysis conditions that afforded higher recovery of pentoses, the level of arabinoxylans in wheat flour determined by this new method is slightly higher than that measured with the widely used hydrolysis protocol. On the basis of the finding that, for a given flour sample, the total fructose concentration after hydrolysis is highly correlated with its total fructan concentration, the fructan content of a wheat flour sample can thus be estimated directly by the total fructose content. By simplifying and combining the two separate methods used for arabonoxylan and fructan analysis, this new method enables the quantification of arabinoxylans and fructans in wheat flour using a single acid hydrolysis step and a single high-performance liquid chromatography run.

  6. 78 FR 52898 - Science-Based Methods for Entity-Scale Quantification of Greenhouse Gas Sources and Sinks From...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-08-27

    ... Science-Based Methods for Entity-Scale Quantification of Greenhouse Gas Sources and Sinks From Agriculture... methods for quantifying entity-scale greenhouse gas (GHG) emissions and removals from the agriculture and... emissions and carbon sequestration at the farm-, forest- and entity-scale. The report and other...

  7. Overview of the AVT-191 Project to Assess Sensitivity Analysis and Uncertainty Quantification Methods for Military Vehicle Design

    NASA Technical Reports Server (NTRS)

    Benek, John A.; Luckring, James M.

    2017-01-01

    A NATO symposium held in 2008 identified many promising sensitivity analysis and un-certainty quantification technologies, but the maturity and suitability of these methods for realistic applications was not known. The STO Task Group AVT-191 was established to evaluate the maturity and suitability of various sensitivity analysis and uncertainty quantification methods for application to realistic problems of interest to NATO. The program ran from 2011 to 2015, and the work was organized into four discipline-centric teams: external aerodynamics, internal aerodynamics, aeroelasticity, and hydrodynamics. This paper presents an overview of the AVT-191 program content.

  8. Summary Findings from the AVT-191 Project to Assess Sensitivity Analysis and Uncertainty Quantification Methods for Military Vehicle Design

    NASA Technical Reports Server (NTRS)

    Benek, John A.; Luckring, James M.

    2017-01-01

    A NATO symposium held in Greece in 2008 identified many promising sensitivity analysis and uncertainty quantification technologies, but the maturity and suitability of these methods for realistic applications was not clear. The NATO Science and Technology Organization, Task Group AVT-191 was established to evaluate the maturity and suitability of various sensitivity analysis and uncertainty quantification methods for application to realistic vehicle development problems. The program ran from 2011 to 2015, and the work was organized into four discipline-centric teams: external aerodynamics, internal aerodynamics, aeroelasticity, and hydrodynamics. This paper summarizes findings and lessons learned from the task group.

  9. Improved quantification of protein in vaccines containing aluminum hydroxide by simple modification of the Lowry method.

    PubMed

    Lee, Naery; Shin, SukJin; Chung, Hye Joo; Kim, Do Keun; Lim, Jong-Mi; Park, Hyunsung; Oh, Ho Jung

    2015-09-22

    Aluminum (Al) components in vaccines are known to act as adsorbents that interfere with accurate protein quantification by the Lowry method. Therefore, certain modifications based on the characteristics and compositions of the vaccine are required for determination of protein contents. We investigated the effects of an additional centrifugal separation and found that protein contents were overestimated by up to 238% without centrifugation through a collaborative study performed with hepatitis B vaccines containing Al. However, addition of a centrifugation step yielded protein concentrations that were similar to the actual values, with small coefficients of variation (CVs). Proficiency testing performed in 11 laboratories showed that four laboratories did not have satisfactory results for vaccines containing aluminum hydroxide, although all laboratories were proficient in protein analysis when samples did not contain aluminum hydroxide. Incomplete resuspension of aluminum hydroxide solution with alkaline copper solution was the major cause of insufficient proficiency in these laboratories.

  10. COMPARISON OF TOTAL LEUKOCYTE QUANTIFICATION METHODS IN FREE-LIVING GALAPAGOS TORTOISES (CHELONOIDIS SPP.).

    PubMed

    Sheldon, Julie D; Stacy, Nicole I; Blake, Stephen; Cabrera, Fredy; Deem, Sharon L

    2016-03-01

    Reptile hematologic data provide important health information for conservation efforts of vulnerable wildlife species such as the Galapagos tortoise (Chelonoidis spp.). Given the reported discrepancies between manual leukocyte counts for nonmammalian species, two manual leukocyte quantification methods, the Natt and Herrick's (NH) and the Eopette (EO), were compared to white blood cell (WBC) estimates from blood films of 42 free-living, clinically healthy, adult female Galapagos tortoises. To investigate the effects of delay in sample processing, estimated WBC counts and leukocyte differentials were compared for blood films prepared at time of collection under field conditions (T0) to blood films prepared from samples that were stored for 18-23 hr at 4°C in the laboratory (T1). Passing-Bablok regression analysis revealed no constant or proportional error between the NH and WBC estimates (T0 and T1) with slopes of 1.1 and 0.9, respectively. However both constant and proportional errors were present between EO and WBC estimates (T0 and T1) with slopes of 3.1 and 2.7, respectively. Bland Altman plots also showed agreement between the NH and WBC estimates where the points fell within the confidence-interval limit lines and were evenly distributed about the mean. In contrast, the EO and WBC estimate comparisons showed numerous points above the upper limit line, especially at higher concentrations. WBC estimates obtained from T0 and T1 films were in agreement, whereas heterophil and monocyte percentages based on differentials were not. Cell morphology and preservation were superior in T0 blood films because thrombocytes exhibited swelling after storage, becoming difficult to differentiate from lymphocytes. In this study, the highest quality and most reliable hematologic data in Galapagos tortoises were obtained by combining immediate blood film preparation with the NH leukocyte quantification method and a confirmatory WBC estimate from the blood film.

  11. The Quantification of Glycosaminoglycans: A Comparison of HPLC, Carbazole, and Alcian Blue Methods

    PubMed Central

    Frazier, Sarah B.; Roodhouse, Kevin A.; Hourcade, Dennis E.; Zhang, Lijuan

    2010-01-01

    Glycosaminoglycans (GAGs) are linear polysaccharides that are found in the extracellular matrix and biological fluids of animals where they interact with hundreds of proteins and perform a variety of critical roles. There are five classes of animal GAGs: heparan sulfate (HS), chondroitin sulfate (CS), dermatan sulfate (DS), keratan sulfate (KS), and hyaluronan (HA). Many biological functions can be monitored directly by their impact on GAG quantity. Thus, simple, sensitive, and robust GAG quantification methods are needed for the development of biomarkers. We have systematically compared three available GAG quantification assays including an HPLC-based assay, a simplified Alcian Blue assay, and a miniaturized carbazole assay. The carbazole and Alcian Blue assays were reproducible and simple to perform in general lab settings, but had important limitations: The carbazole assay could not detect KS and it overestimated GAGs that were contaminated with salts or dissolved in PBS. The Alcian Blue assay detected only those GAGs that were sulfated. In contrast, while the HPLC method was time-consuming, it was a robust and sensitive assay that not only detected all GAGs but also quantified glucosamine-GAGs and galactosamine-GAGs simultaneously. The HPLC assay was not affected by salt or level of GAG sulfation and it yielded reproducible values for all types of GAGs tested. These results suggest that an automated HPLC assay would be generally useful for the routine measurement of a panel of GAG-based biomarkers while the carbazole assay and the Alcian Blue assays could prove valuable for more specific purposes. PMID:20640171

  12. Automated quantification of budding Saccharomyces cerevisiae using a novel image cytometry method.

    PubMed

    Laverty, Daniel J; Kury, Alexandria L; Kuksin, Dmitry; Pirani, Alnoor; Flanagan, Kevin; Chan, Leo Li-Ying

    2013-06-01

    The measurements of concentration, viability, and budding percentages of Saccharomyces cerevisiae are performed on a routine basis in the brewing and biofuel industries. Generation of these parameters is of great importance in a manufacturing setting, where they can aid in the estimation of product quality, quantity, and fermentation time of the manufacturing process. Specifically, budding percentages can be used to estimate the reproduction rate of yeast populations, which directly correlates with metabolism of polysaccharides and bioethanol production, and can be monitored to maximize production of bioethanol during fermentation. The traditional method involves manual counting using a hemacytometer, but this is time-consuming and prone to human error. In this study, we developed a novel automated method for the quantification of yeast budding percentages using Cellometer image cytometry. The automated method utilizes a dual-fluorescent nucleic acid dye to specifically stain live cells for imaging analysis of unique morphological characteristics of budding yeast. In addition, cell cycle analysis is performed as an alternative method for budding analysis. We were able to show comparable yeast budding percentages between manual and automated counting, as well as cell cycle analysis. The automated image cytometry method is used to analyze and characterize corn mash samples directly from fermenters during standard fermentation. Since concentration, viability, and budding percentages can be obtained simultaneously, the automated method can be integrated into the fermentation quality assurance protocol, which may improve the quality and efficiency of beer and bioethanol production processes.

  13. Hyperspectral materials detection/identification/quantification using the residual correlation method

    NASA Astrophysics Data System (ADS)

    Hudgins, Lonnie H.; Hayashi, Joan; Blake, Pamela L.; Tran, Luong V.; Kilday, Elizabeth

    2001-08-01

    Remote detection, identification, and quantification of materials is an important problem in earth resource assessment. Satellite-based hyperspectral imaging sensors currently being developed by government and industry partnerships (e.g. the Coastal Ocean Imaging Spectrometer aboard the Naval EarthMap Observer) appear to be uniquely qualified for this purpose. Obtaining accurate estimates of material abundance on a pixel-by-pixel basis poses many challenging algorithmic and computational difficulties. A significant issue that must be addressed is how to efficiently select endmembers from a library when that library is spectrally redundant. In this paper, we demonstrate how an improved version of the Residual Correlation Method (RCM+) can provide a flexible solution to this problem. The RCM+ offers a robust treatment for selecting endmembers from spectrally redundant libraries in a one-at-a-time fashion. We discuss alternative methods such as two-at-a-time, or more generally, N-at-a-time methods within a unified mathematical framework for analysis. Certain theorems apply to all such methods, and help to define a trade space for endmember selection methods in general. We demonstrate our results using synthetic test cases, and discuss how all endmember selection methods may be affected by redundancy within the library as well as specific properties of the data.

  14. Time lapse imaging of water content with geoelectrical methods: on the interest of working with absolute water content data

    NASA Astrophysics Data System (ADS)

    Dumont, Gaël; Pilawski, Tamara; Robert, Tanguy; Hermans, Thomas; Garré, Sarah; Nguyen, Frederic

    2016-04-01

    The electrical resistivity tomography is a suitable method to estimate the water content of a waste material and detect changes in water content. Various ERT profiles, both static data and time-lapse, where acquired on a landfill during the Minerve project. In the literature, the relative change of resistivity (Δρ/ρ) is generally computed. For saline or heat tracer tests in the saturated zone, the Δρ/ρ can be easily translated into pore water conductivity or underground temperature changes (provided that the initial salinity or temperature condition is homogeneous over the ERT panel extension). For water content changes in the vadose zone resulting of an infiltration event or injection experiment, many authors also work with the Δρ/ρ or relative changes of water content Δθ/θ (linked to the change of resistivity through one single parameter: the Archie's law exponent "m"). This parameter is not influenced by the underground temperature and pore fluid conductivity (ρ¬w) condition but is influenced by the initial water content distribution. Therefore, you never know if the loss of Δθ/θ signal is representative of the limit of the infiltration front or more humid initial condition. Another approach for the understanding of the infiltration process is the assessment of the absolute change of water content (Δθ). This requires the direct computation of the water content of the waste from the resistivity data. For that purpose, we used petrophysical laws calibrated with laboratory experiments and our knowledge of the in situ temperature and pore fluid conductivity parameters. Then, we investigated water content changes in the waste material after a rainfall event (Δθ= Δθ/θ* θ). This new observation is really representatives of the quantity of water infiltrated in the waste material. However, the uncertainty in the pore fluid conductivity value may influence the computed water changes (Δθ=k*m√(ρw) ; where "m" is the Archie's law exponent

  15. Comparative quantification of dietary supplemented neural creatine concentrations with (1)H-MRS peak fitting and basis spectrum methods.

    PubMed

    Turner, Clare E; Russell, Bruce R; Gant, Nicholas

    2015-11-01

    Magnetic resonance spectroscopy (MRS) is an analytical procedure that can be used to non-invasively measure the concentration of a range of neural metabolites. Creatine is an important neurometabolite with dietary supplementation offering therapeutic potential for neurological disorders with dysfunctional energetic processes. Neural creatine concentrations can be probed using proton MRS and quantified using a range of software packages based on different analytical methods. This experiment examines the differences in quantification performance of two commonly used analysis packages following a creatine supplementation strategy with potential therapeutic application. Human participants followed a seven day dietary supplementation regime in a placebo-controlled, cross-over design interspersed with a five week wash-out period. Spectroscopy data were acquired the day immediately following supplementation and analyzed with two commonly-used software packages which employ vastly different quantification methods. Results demonstrate that neural creatine concentration was augmented following creatine supplementation when analyzed using the peak fitting method of quantification (105.9%±10.1). In contrast, no change in neural creatine levels were detected with supplementation when analysis was conducted using the basis spectrum method of quantification (102.6%±8.6). Results suggest that software packages that employ the peak fitting procedure for spectral quantification are possibly more sensitive to subtle changes in neural creatine concentrations. The relative simplicity of the spectroscopy sequence and the data analysis procedure suggest that peak fitting procedures may be the most effective means of metabolite quantification when detection of subtle alterations in neural metabolites is necessary. The straightforward technique can be used on a clinical magnetic resonance imaging system.

  16. An HPLC-ECD method for monoamines and metabolites quantification in cuttlefish (cephalopod) brain tissue.

    PubMed

    Bidel, Flavie; Corvaisier, Sophie; Jozet-Alves, Christelle; Pottier, Ivannah; Dauphin, François; Naud, Nadège; Bellanger, Cécile

    2016-08-01

    The cuttlefish belongs to the mollusk class Cephalopoda, considered as the most advanced marine invertebrates and thus widely used as models to study the biology of complex behaviors and cognition, as well as their related neurochemical mechanisms. Surprisingly, methods to quantify the biogenic monoamines and their metabolites in cuttlefish brain remain sparse and measure a limited number of analytes. This work aims to validate an HPLC-ECD method for the simultaneous quantification of dopamine, serotonin, norepinephrine and their main metabolites in cuttlefish brain. In comparison and in order to develop a method suitable to answer both ecological and biomedical questions, the validation was also carried out on a phylogenetically remote species: mouse (mammals). The method was shown to be accurate, precise, selective, repeatable and sensitive over a wide range of concentrations for 5-hydroxyindole-3-acetic acid, serotonin, dopamine, 3,4-dihydroxyphenylacetic acid and norepinephrine in the both extracts of cuttlefish and mouse brain, though with low precision and recovery for 4-hydroxy-3-methoxyphenylethylene glycol. Homovanillic acid, accurately studied in rodents, was not detectable in the brain of cuttlefish. Overall, we described here the first fully validated HPLC method for the routine measurement of both monoamines and metabolites in cuttlefish brain. Copyright © 2016 John Wiley & Sons, Ltd.

  17. Quantification of Rifaximin in Tablets by Spectrophotometric Method Ecofriendly in Ultraviolet Region

    PubMed Central

    2016-01-01

    Rifaximin is an oral nonabsorbable antibiotic that acts locally in the gastrointestinal tract with minimal systemic adverse effects. It does not have spectrophotometric method ecofriendly in the ultraviolet region described in official compendiums and literature. The analytical techniques for determination of rifaximin reported in the literature require large amount of time to release results and are significantly onerous. Furthermore, they use toxic reagents both for the operator and environment and, therefore, cannot be considered environmentally friendly analytical techniques. The objective of this study was to develop and validate an ecofriendly spectrophotometric method in the ultraviolet region to quantify rifaximin in tablets. The method was validated, showing linearity, selectivity, precision, accuracy, and robustness. It was linear over the concentration range of 10–30 mg L−1 with correlation coefficients greater than 0.9999 and limits of detection and quantification of 1.39 and 4.22 mg L−1, respectively. The validated method is useful and applied for the routine quality control of rifaximin, since it is simple with inexpensive conditions and fast in the release of results, optimizes analysts and equipment, and uses environmentally friendly solvents, being considered a green method, which does not prejudice either the operator or the environment. PMID:27429835

  18. Quantification of Aconitum alkaloids in aconite roots by a modified RP-HPLC method.

    PubMed

    Jiang, Zhi-Hong; Xie, Ying; Zhou, Hua; Wang, Jing-Rong; Liu, Zhong-Qiu; Wong, Yuen-Fan; Cai, Xiong; Xu, Hong-Xi; Liu, Liang

    2005-01-01

    The three Aconitum alkaloids, aconitine (1), mesaconitine (2) and hypaconitine (3), are pharmacologically active but also highly toxic. A standardised method is needed for assessing the levels of these alkaloids in aconite roots in order to ensure the safe use of these plant materials as medicinal herbs. By optimising extraction, separation and measurement conditions, a reliable, reproducible and accurate method for the quantitative determination of all three Aconitum alkaloids in unprocessed and processed aconite roots has been developed. This method should be appropriate for use in the quality control of Aconitum products. The three Aconitum alkaloids were separated by a modified HPLC method employing a C18 column gradient eluted with acetonitrile and ammonium bicarbonate buffer. Quantification of Aconitum alkaloids, detected at 240 nm, in different batches of samples showed that the content of 1, 2 and 3 varied significantly. In general, the alkaloid content of unprocessed roots was higher than that of processed roots. These variations were considered to be the result of differences in species, processing methods and places of origin of the samples.

  19. Quantification of Rifaximin in Tablets by Spectrophotometric Method Ecofriendly in Ultraviolet Region.

    PubMed

    Kogawa, Ana Carolina; Salgado, Hérida Regina Nunes

    2016-01-01

    Rifaximin is an oral nonabsorbable antibiotic that acts locally in the gastrointestinal tract with minimal systemic adverse effects. It does not have spectrophotometric method ecofriendly in the ultraviolet region described in official compendiums and literature. The analytical techniques for determination of rifaximin reported in the literature require large amount of time to release results and are significantly onerous. Furthermore, they use toxic reagents both for the operator and environment and, therefore, cannot be considered environmentally friendly analytical techniques. The objective of this study was to develop and validate an ecofriendly spectrophotometric method in the ultraviolet region to quantify rifaximin in tablets. The method was validated, showing linearity, selectivity, precision, accuracy, and robustness. It was linear over the concentration range of 10-30 mg L(-1) with correlation coefficients greater than 0.9999 and limits of detection and quantification of 1.39 and 4.22 mg L(-1), respectively. The validated method is useful and applied for the routine quality control of rifaximin, since it is simple with inexpensive conditions and fast in the release of results, optimizes analysts and equipment, and uses environmentally friendly solvents, being considered a green method, which does not prejudice either the operator or the environment.

  20. Critical assessment of three high performance liquid chromatography analytical methods for food carotenoid quantification.

    PubMed

    Dias, M Graça; Oliveira, Luísa; Camões, M Filomena G F C; Nunes, Baltazar; Versloot, Pieter; Hulshof, Paul J M

    2010-05-21

    Three sets of extraction/saponification/HPLC conditions for food carotenoid quantification were technically and economically compared. Samples were analysed for carotenoids alpha-carotene, beta-carotene, beta-cryptoxanthin, lutein, lycopene, and zeaxanthin. All methods demonstrated good performance in the analysis of a composite food standard reference material for the analytes they are applicable to. Methods using two serial connected C(18) columns and a mobile phase based on acetonitrile, achieved a better carotenoid separation than the method using a mobile phase based on methanol and one C(18)-column. Carotenoids from leafy green vegetable matrices appeared to be better extracted with a mixture of methanol and tetrahydrofuran than with tetrahydrofuran alone. Costs of carotenoid determination in foods were lower for the method with mobile phase based on methanol. However for some food matrices and in the case of E-Z isomer separations, this was not technically satisfactory. Food extraction with methanol and tetrahydrofuran with direct evaporation of these solvents, and saponification (when needed) using pyrogallol as antioxidant, combined with a HPLC system using a slight gradient mobile phase based on acetonitrile and a stationary phase composed by two serial connected C(18) columns was the most technically and economically favourable method.

  1. Video meteor light curve analysis of Orionids and Geminids and developing a method for obtaining the absolute light curves of shower meteors from the single station data

    NASA Astrophysics Data System (ADS)

    Grašić, L.; Milanović, N.; Pavlović, D.

    2016-01-01

    We developed a method for obtaining the absolute light curves of the shower meteors from single station video data. We found that even though the height of a meteor atmospheric trajectory obtained by using this method may have a large error, the absolute light curve shape is preserved. We used our method to calculate the F parameters of the Orionid and Geminid light curves. The light curves were obtained from the single station video data by the instrument with a limiting sensitivity of 3.5m. We found that for our sample of the light curves the zenith distance of meteor radiant does not affect the F parameter for either of the two showers. The value of F parameter of the Orionids obtained in this paper matches the values obtained by other authors, whilst for the Geminids it is significantly different.

  2. Leakage detection and quantification techniques using various methods of nearfield acoustic holography

    NASA Astrophysics Data System (ADS)

    Chelliah, Kanthasamy

    This thesis proposes an acoustic technique to detect and relatively quantify leakages in buildings and enclosures using various methods of nearfield acoustic holography (NAH). This laboratory study was performed on a scaled, wooden building model. Known leakages can be created in the wooden model and the acoustic method was tested to localize and relatively quantify these known leakage areas. An acoustic source was placed inside the building model and a planar hologram measurement was performed near the surface of the building model. Various methods of NAH were applied on the hologram data to reconstruct the sound pressure field on the wall of the building model. The detection and quantification capabilities of four different NAH methods, namely, discrete Fourier transform (DFT) based NAH, equivalent source model (ESM) based NAH, boundary element method (BEM) based NAH and statistically optimized NAH (SONAH), were compared in this study. It was shown that the NAH methods were able to successfully locate and relatively quantify the area of the leakages using the reconstructions. Although all the four algorithms produced comparable results in the very nearfield, at larger hologram distances, ESM and SONAH reconstructions were more accurate than the reconstructions using the other methods. Although, ESM and SONAH produced similar results for most of the cases, ESM is more preferable due to its simplicity in implementation and less computational time requirements. Lower frequency reconstructions were found to be more accurate and advantageous in the context of leakage detection and quantification. When the hologram distance was increased more than a particular limit, all the four algorithms arrive at inaccurate reconstructions due to the very ill-conditioned propagation matrices. New filtering methods to alleviate these larger reconstruction errors were introduced and the results were demonstrated. Effects of large sensor phase mismatch were also studied. It was

  3. Quantification of methane emissions from 15 Danish landfills using the mobile tracer dispersion method

    SciTech Connect

    Mønster, Jacob; Samuelsson, Jerker; Scheutz, Charlotte

    2015-01-15

    Highlights: • Quantification of whole landfill site methane emission at 15 landfills. • Multiple on-site source identification and quantification. • Quantified methane emission from shredder waste and composting. • Large difference between measured and reported methane emissions. - Abstract: Whole-site methane emissions from 15 Danish landfills were assessed using a mobile tracer dispersion method with either Fourier transform infrared spectroscopy (FTIR), using nitrous oxide as a tracer gas, or cavity ring-down spectrometry (CRDS), using acetylene as a tracer gas. The landfills were chosen to represent the different stages of the lifetime of a landfill, including open, active, and closed covered landfills, as well as those with and without gas extraction for utilisation or flaring. Measurements also included landfills with biocover for oxidizing any fugitive methane. Methane emission rates ranged from 2.6 to 60.8 kg h{sup −1}, corresponding to 0.7–13.2 g m{sup −2} d{sup −1}, with the largest emission rates per area coming from landfills with malfunctioning gas extraction systems installed, and the smallest emission rates from landfills closed decades ago and landfills with an engineered biocover installed. Landfills with gas collection and recovery systems had a recovery efficiency of 41–81%. Landfills where shredder waste was deposited showed significant methane emissions, with the largest emission from newly deposited shredder waste. The average methane emission from the landfills was 154 tons y{sup −1}. This average was obtained from a few measurement campaigns conducted at each of the 15 landfills and extrapolating to annual emissions requires more measurements. Assuming that these landfills are representative of the average Danish landfill, the total emission from Danish landfills were calculated at 20,600 tons y{sup −1}, which is significantly lower than the 33,300 tons y{sup −1} estimated for the national greenhouse gas inventory for

  4. Comparison of Methods for Quantification of Global DNA Methylation in Human Cells and Tissues

    PubMed Central

    Tomaszewski, Bartłomiej; De Prins, Sofie; Jacobs, Griet; Koppen, Gudrun; Mathers, John C.; Langie, Sabine A. S.

    2013-01-01

    DNA methylation is a key epigenetic modification which, in mammals, occurs mainly at CpG dinucleotides. Most of the CpG methylation in the genome is found in repetitive regions, rich in dormant transposons and endogenous retroviruses. Global DNA hypomethylation, which is a common feature of several conditions such as ageing and cancer, can cause the undesirable activation of dormant repeat elements and lead to altered expression of associated genes. DNA hypomethylation can cause genomic instability and may contribute to mutations and chromosomal recombinations. Various approaches for quantification of global DNA methylation are widely used. Several of these approaches measure a surrogate for total genomic methyl cytosine and there is uncertainty about the comparability of these methods. Here we have applied 3 different approaches (luminometric methylation assay, pyrosequencing of the methylation status of the Alu repeat element and of the LINE1 repeat element) for estimating global DNA methylation in the same human cell and tissue samples and have compared these estimates with the “gold standard” of methyl cytosine quantification by HPLC. Next to HPLC, the LINE1 approach shows the smallest variation between samples, followed by Alu. Pearson correlations and Bland-Altman analyses confirmed that global DNA methylation estimates obtained via the LINE1 approach corresponded best with HPLC-based measurements. Although, we did not find compelling evidence that the gold standard measurement by HPLC could be substituted with confidence by any of the surrogate assays for detecting global DNA methylation investigated here, the LINE1 assay seems likely to be an acceptable surrogate in many cases. PMID:24260150

  5. Method validation using weighted linear regression models for quantification of UV filters in water samples.

    PubMed

    da Silva, Claudia Pereira; Emídio, Elissandro Soares; de Marchi, Mary Rosa Rodrigues

    2015-01-01

    This paper describes the validation of a method consisting of solid-phase extraction followed by gas chromatography-tandem mass spectrometry for the analysis of the ultraviolet (UV) filters benzophenone-3, ethylhexyl salicylate, ethylhexyl methoxycinnamate and octocrylene. The method validation criteria included evaluation of selectivity, analytical curve, trueness, precision, limits of detection and limits of quantification. The non-weighted linear regression model has traditionally been used for calibration, but it is not necessarily the optimal model in all cases. Because the assumption of homoscedasticity was not met for the analytical data in this work, a weighted least squares linear regression was used for the calibration method. The evaluated analytical parameters were satisfactory for the analytes and showed recoveries at four fortification levels between 62% and 107%, with relative standard deviations less than 14%. The detection limits ranged from 7.6 to 24.1 ng L(-1). The proposed method was used to determine the amount of UV filters in water samples from water treatment plants in Araraquara and Jau in São Paulo, Brazil.

  6. Quantification of designer nuclease induced mutation rates: a direct comparison of different methods

    PubMed Central

    Ehrke-Schulz, Eric; Bergmann, Thorsten; Schiwon, Maren; Doerner, Johannes; Saydaminova, Kamola; Lieber, Andre; Ehrhardt, Anja

    2016-01-01

    Designer nucleases are broadly applied to induce site-specific DNA double-strand breaks (DSB) in genomic DNA. These are repaired by nonhomologous end joining leading to insertions or deletions (in/dels) at the respective DNA-locus. To detect in/del mutations, the heteroduplex based T7-endonuclease I -assay is widely used. However, it only provides semi-quantitative evidence regarding the number of mutated alleles. Here we compared T7-endonuclease I- and heteroduplex mobility assays, with a quantitative polymerase chain reaction mutation detection method. A zinc finger nuclease pair specific for the human adeno-associated virus integration site 1 (AAVS1), a transcription activator-like effector nuclease pair specific for the human DMD gene, and a zinc finger nuclease- and a transcription activator-like effector nuclease pair specific for the human CCR5 gene were explored. We found that the heteroduplex mobility assays and T7-endonuclease I - assays detected mutations but the relative number of mutated cells/alleles can only be estimated. In contrast, the quantitative polymerase chain reaction based method provided quantitative results which allow calculating mutation and homologous recombination rates in different eukaryotic cell types including human peripheral blood mononuclear cells. In conclusion, our quantitative polymerase chain reaction based mutation detection method expands the array of methods for in/del mutation detection and facilitates quantification of introduced in/del mutations for a genomic locus containing a mixture of mutated and unmutated DNA. PMID:27419195

  7. HPLC-UV method validation for the identification and quantification of bioactive amines in commercial eggs.

    PubMed

    de Figueiredo, Tadeu Chaves; de Assis, Débora Cristina Sampaio; Menezes, Liliane Denize Miranda; da Silva, Guilherme Resende; Lanza, Isabela Pereira; Heneine, Luiz Guilherme Dias; Cançado, Silvana de Vasconcelos

    2015-09-01

    A quantitative and confirmatory high-performance liquid chromatography with ultraviolet detection (HPLC-UV) method for the determination of bioactive amines in the albumen and yolk of commercial eggs was developed, optimized and validated by analyte extraction with trichloroacetic acid and pre-column derivatization with dansyl chloride. Phenylethylamine, putrescine, cadaverine, histamine, tyramine, spermidine and spermine standards were used to evaluate the following performance parameters: limit of detection (LoD), limit of quantification (LoQ), selectivity, linearity, precision, recovery and ruggedness. The LoD of the method was defined from 0.2 to 0.3 mg kg(-1) for the yolk matrix and from 0.2 to 0.4 mg kg(-1) for the albumen matrix; the LoQ was from 0.7 to 1.0 mg kg(-1) for the yolk matrix and from 0.7 to 1.1 mg kg(-1) for the albumen matrix. The validated method exhibited excellent selectivity and separation of all amines with coefficients of determination higher than 0.99. The obtained recovery values were from 90.5% to 108.3%, and the relative standard deviation (RSD) was lower than 10% under repeatability conditions for the studied analytes. The performance parameters show the validated method to be adequate for the determination of bioactive amines in egg albumen and yolk.

  8. A Simple Method for Noninvasive Quantification of Pressure Gradient Across the Pulmonary Valve

    PubMed Central

    Zhou, Xueying; Xing, Changyang; Feng, Yang; Duan, Yunyou; Zheng, Qiangsun; Wang, Zuojun; Liu, Jie; Cao, Tiesheng; Yuan, Lijun

    2017-01-01

    Pressure gradient across the pulmonary valve (PVPG) is an important hemodynamic variable used in the management of patients with cardiovascular and pulmonary disease. However, a reliable noninvasive method is unavailable. We hypothesized that a progressive Muller maneuver would elicit the pulmonary valve premature opening (PVPO) in diastole and that this event would be detectable by Doppler echocardiography. The intrathoracic pressure (ITP) decrease during this maneuver equals PVPG, which may be assessed with a custom airway pressure measurement device. A total of 102 subjects were enrolled in the study. At the earliest appearance of PVPO, the ITP decrease was recorded as the PVPG. PVPG was also simultaneously measured and compared by other two methods: right heart catheterization in 43 subjects, and routine Doppler echocardiography (pulmonary regurgitation jet) in the other 59 subjects. The results measured by different approaches were compared using the Bland-Altman analysis. PVPG assessed via PVPO showed strong agreement with PVPG measured by catheterization or routine Doppler echocardiography methods, with Lin concordance correlation coefficients of 0.91 and 0.70, respectively. In conclusion, PVPO provides a new noninvasive method of quantification of PVPG. PMID:28198458

  9. Simultaneous quantification of stemocurtisine, stemocurtisinol and stemofoline in Stemona curtisii (Stemonaceae) by TLC-densitometric method.

    PubMed

    Kongkiatpaiboon, Sumet; Keeratinijakal, Vichien; Gritsanapan, Wandee

    2013-01-01

    Stemona curtisii Hook. F. (Family Stemonaceae), a prominent species distributed in the south and southwest of Thailand, has widely been used as a natural pesticide and as treatment for head lice and skin diseases. This study developed a thin-layer chromatography (TLC)-densitometric method for the simultaneous quantification of major components--stemocurtisine, stemocurtisinol and stemofoline--in the extracts from the roots of S. curtisii collected from 10 locations in Thailand. Components were found in the ranges of 0.0353-0.1949, <0.0121-0.0859 and 0.0733-0.1689 percent dry weight, respectively. The method was validated for linearity, precision, accuracy, robustness, limit of detection and limit of quantitation. The linearity was found over the range of 40-320 ng/spot with a good correlation coefficient (r > 0.9866). Intra-day and inter-day precision showed a relative standard deviation of less than 6%. The accuracy of the method was determined by a recovery study, and the average recoveries were 100.4, 100.2 and 100.3% for stemocurtisine, stemocurtisinol and stemofoline, respectively. The proposed TLC-densitometric method was found to be simple, precise, specific and inexpensive, and can be used simultaneously for the routine quality control of raw materials of S. curtisii roots, extracts and their products, and also other products containing these markers.

  10. Quantification and comparison of extraction methods for alkaloids in Aegle marmelos leaves by HPLC.

    PubMed

    Karmase, Aniket; Prasanna, K; Rasabattula, Sruti; Bhutani, Kamlesh K

    2014-07-01

    The leaves of Aegle marmelos are reported to contain multi-bioactive classes of compounds including coumarins, furanocoumarins and alkaloids. HPLC analysis of the crude extract was challenging due to low concentrations of the compounds in the leaves. Five compounds visible in the HPLC chromatogram were separated and identified by HPLC and further elaborated for quantification as marker compounds of A. marmelos leaves using a C18 column with detection at 275 nm. A gradient mobile phase consisting of acetonitrile and water was used. The developed HPLC method showed good linearity (r2 > 0.994), high precision (RSD<5%), and good recovery (99.27-99.98%) of the compounds. The lowest detection limit was 5 ng and the method was found to be robust. All the validation parameters were within the permissible limits. Therefore, the developed method is accurate and reliable for the quality control of A. marmelos. This is the first report of extensive quantitative HPLC analysis of marker compounds in A. marmelos leaves and method validation.

  11. Quantification of Self Pollution from Two Diesel School Buses using Three Independent Methods

    PubMed Central

    Liu, L.-J. Sally; Phuleria, Harish C.; Webber, Whitney; Davey, Mark; Lawson, Douglas R.; Ireson, Robert G.; Zielinska, Barbara; Ondov, John M.; Weaver, Christopher S.; Lapin, Charles A.; Easter, Michael; Hesterberg, Thomas W.; Larson, Timothy

    2010-01-01

    We monitored two Seattle school buses to quantify the buses’ self pollution using the dual tracers (DT), lead vehicle (LV), and chemical mass balance (CMB) methods. Each bus drove along a residential route simulating stops, with windows closed or open. Particulate matter (PM) and its constituents were monitored in the bus and from a LV. We collected source samples from the tailpipe and crankcase emissions using an on-board dilution tunnel. Concentrations of PM1, ultrafine particle counts, elemental and organic carbon (EC/OC) were higher on the bus than the LV. The DT method estimated that the tailpipe and the crankcase emissions contributed 1.1 and 6.8 μg/m3 of PM2.5 inside the bus, respectively, with significantly higher crankcase self pollution (SP) when windows were closed. Approximately two-thirds of in-cabin PM2.5 originated from background sources. Using the LV approach, SP estimates from the EC and the active personal DataRAM (pDR) measurements correlated well with the DT estimates for tailpipe and crankcase emissions, respectively, although both measurements need further calibration for accurate quantification. CMB results overestimated SP from the DT method but confirmed crankcase emissions as the major SP source. We confirmed buses’ SP using three independent methods and quantified crankcase emissions as the dominant contributor. PMID:20694046

  12. Crack Imaging and Quantification in Aluminum Plates with Guided Wave Wavenumber Analysis Methods

    NASA Technical Reports Server (NTRS)

    Yu, Lingyu; Tian, Zhenhua; Leckey, Cara A. C.

    2015-01-01

    Guided wavefield analysis methods for detection and quantification of crack damage in an aluminum plate are presented in this paper. New wavenumber components created by abrupt wave changes at the structural discontinuity are identified in the frequency-wavenumber spectra. It is shown that the new wavenumbers can be used to detect and characterize the crack dimensions. Two imaging based approaches, filter reconstructed imaging and spatial wavenumber imaging, are used to demonstrate how the cracks can be evaluated with wavenumber analysis. The filter reconstructed imaging is shown to be a rapid method to map the plate and any existing damage, but with less precision in estimating crack dimensions; while the spatial wavenumber imaging provides an intensity image of spatial wavenumber values with enhanced resolution of crack dimensions. These techniques are applied to simulated wavefield data, and the simulation based studies show that spatial wavenumber imaging method is able to distinguish cracks of different severities. Laboratory experimental validation is performed for a single crack case to confirm the methods' capabilities for imaging cracks in plates.

  13. Development of a CZE method for the quantification of pseudoephedrine and cetirizine.

    PubMed

    Alnajjar, Ahmed O; Idris, Abubakr M

    2014-10-01

    Pseudoephedrine and cetirizine have been combined in dosage forms with more therapeutic benefits when compared with single-drug treatment. The current manuscript reports the development of the first capillary zone electrophoresis (CZE) assay method for that combination. The effects of pH and buffer concentration on resolution, noise, migration time and peak area were examined employing experimental design approach. The analytes were electropherographed into a 50.2 cm-long and 50 µm i.d. fused-silica capillary column using 10 mmol/L borate at pH 8.3 with a potential of 25 kV at 25°C and UV detection at 214 nm. The method was successfully validated in order to verify its suitability for pharmaceutical analysis for the purposes of quality control. Over previous high-performance liquid chromatographic methods, the current CZE method features the benefits of the use of cost-effective electrolyte, besides high sample throughput (11 samples/h). Furthermore, other analytical results including linear dynamic ranges, recovery (96.9-98.1%), intra- and interday precision (relative standard deviation ≤ 1.70%) as well as the limits of detection and quantification (≤2.65 µg/mL) were all satisfactory for the intended purpose.

  14. A Simple Method for Noninvasive Quantification of Pressure Gradient Across the Pulmonary Valve.

    PubMed

    Zhou, Xueying; Xing, Changyang; Feng, Yang; Duan, Yunyou; Zheng, Qiangsun; Wang, Zuojun; Liu, Jie; Cao, Tiesheng; Yuan, Lijun

    2017-02-15

    Pressure gradient across the pulmonary valve (PVPG) is an important hemodynamic variable used in the management of patients with cardiovascular and pulmonary disease. However, a reliable noninvasive method is unavailable. We hypothesized that a progressive Muller maneuver would elicit the pulmonary valve premature opening (PVPO) in diastole and that this event would be detectable by Doppler echocardiography. The intrathoracic pressure (ITP) decrease during this maneuver equals PVPG, which may be assessed with a custom airway pressure measurement device. A total of 102 subjects were enrolled in the study. At the earliest appearance of PVPO, the ITP decrease was recorded as the PVPG. PVPG was also simultaneously measured and compared by other two methods: right heart catheterization in 43 subjects, and routine Doppler echocardiography (pulmonary regurgitation jet) in the other 59 subjects. The results measured by different approaches were compared using the Bland-Altman analysis. PVPG assessed via PVPO showed strong agreement with PVPG measured by catheterization or routine Doppler echocardiography methods, with Lin concordance correlation coefficients of 0.91 and 0.70, respectively. In conclusion, PVPO provides a new noninvasive method of quantification of PVPG.

  15. Pre-staining paper chromatography method for quantification of gamma-aminobutyric acid.

    PubMed

    Li, Haixing; Qiu, Ting; Cao, Yusheng; Yang, Jiyan; Huang, Zhibing

    2009-06-19

    The routine method of paper chromatography includes five steps: spotting, separating, drying, spraying/immersing and color development. In this paper, a pre-staining paper chromatography which only consisted of spotting, separating and color development was developed for quantitative analysis of gamma-aminobutyric acid. Compared to the routine paper chromatography, the improved method is clean, rapid, inexpensive and reproducible. The effects of ninhydrin concentration, color temperature, color time and Cu(2+) concentration on the color yield in the ninhydrin reaction were optimized. And then the pre-staining paper chromatography coupled with vis spectrophotometry was applied to gamma-aminobutyric acid quantification. The results indicated that the limit of detection was 0.05 mg mL(-1) and the linear range was from 0.5 to 20.0 mg mL(-1). Furthermore, an excellent correlation coefficient was observed with an R(2)=0.998. The method is accurate (RSD<2.64%), and has good recoveries (102.7-103.9%). The validation of the modified technique was verified by a HPLC method.

  16. Comparison of different immunochemical methods for the detection and quantification of hazelnut proteins in food products.

    PubMed

    Koppelman, S J; Knulst, A C; Koers, W J; Penninks, A H; Peppelman, H; Vlooswijk, R; Pigmans, I; van Duijn, G; Hessing, M

    1999-10-29

    Hazelnuts are widely used in the food industry owing to their nutritive value and taste. The amount of hazelnut present in a recipe is usually considered as a mark of quality. On the other hand, contamination of foods that normally do not contain hazelnuts is a threat for patients with a hazelnut allergy. For this reason, the availability of a method for the detection and quantification of hazelnuts in foods would be desirable. The objective of this study was to develop a method for the detection and quantification of minor amounts of hazelnut protein in food products that is potentially applicable for the food industry. Several immunochemical methods, e.g., immunoblotting and enzyme-linked immunosorbent assay (ELISA), were developed with antibodies from both hazelnut-sensitized patient sera and the sera of rabbits hyperimmunized with hazelnut protein. Immunoblotting appeared to be non-specific when the sera of patients were used as a source of antibodies. Using immunopurified antibodies from rabbits immunized with hazelnuts, immunoblotting became specific, but the sensitivity of this method was limited. Inhibition of IgE binding is a generally used test in clinical laboratories to establish contamination with hazelnuts. This approach is sensitive and specific, but not readily accessible for the food industry since patient serum is needed. Similar results in terms of sensitivity and specificity were obtained with a sandwich ELISA constructed with an immunopurified antibody from rabbits sensitized to hazelnuts. No substantial cross-reactivity with other nuts, legumes or other food constituents was observed, and concentrations as low as 5 ng/ml, corresponding to 1 ppm in food products, were detected. In a field test, several consumer products regarded to be free of hazelnuts were shown to contain traces of hazelnut. This sandwich ELISA constructed with immunopurified antibodies from rabbits sensitized with hazelnut protein is a sensitive and specific method to detect

  17. Testing the Multispecimen Absolute Paleointensity Method with Archaeological Baked Clays and Bricks: New Data for Central Europe

    NASA Astrophysics Data System (ADS)

    Schnepp, Elisabeth; Leonhardt, Roman

    2014-05-01

    The domain-state corrected multiple-specimen paleointensity determination technique (MSP-DSC, Fabian & Leonhardt, EPSL 297, 84, 2010) has been tested for archaeological baked clays and bricks. The following procedure was applied: (1) Exclusion of secondary overprints using alternating field (AF) or thermal demagnetization and assignment of characteristic remanent magnetization (ChRM) direction. (2) Determination of magneto mineralogical alteration using anhysteretic remanent magnetization (ARM) or temperature dependence of susceptibility. (3) Measurement of ARM anisotropy tensor, calculation of the ancient magnetic field direction. (4) Sister specimens were subjected to the MSP-DSC technique aligned (anti-)parallel to the ancient magnetic field direction. (5) Several checks were applied in order to exclude data points from further evaluation: (a) The accuracy of orientation (< 10°), (b) absence of secondary components (< 10°), (c) use of a considerable NRM fraction (20 to 80%), (d) weak alteration (smaller than for domain state change) and finally (e) domain state correction was applied. Bricks and baked clays from archaeological sites with ages between 645 BC and 2003 AD have been subjected to MSP-DSC absolute paleointensity (PI) determination. Aims of study are to check precision and reliability of the method. The obtained PI values are compared with direct field observation, the IGRF, the GUFM1 or Thellier results. The Thellier experiments often show curved lines and pTRM checks fail for higher temperatures. Nevertheless in the low temperature range straight lines have been obtained but they provide scattered paleointensity values. Mean paleointensites have relative errors often exceeding 10%, which are not considered as high quality PI estimates. MSP-DSC experiments for the structures older than 300 years are still under progress. The paleointensities obtained from the MSP-DSC experiments for the young materials (after 1700 AD) have small relative errors of a

  18. Volatile organic silicon compounds in biogases: development of sampling and analytical methods for total silicon quantification by ICP-OES.

    PubMed

    Chottier, Claire; Chatain, Vincent; Julien, Jennifer; Dumont, Nathalie; Lebouil, David; Germain, Patrick

    2014-01-01

    Current waste management policies favor biogases (digester gases (DGs) and landfill gases (LFGs)) valorization as it becomes a way for energy politics. However, volatile organic silicon compounds (VOSiCs) contained into DGs/LFGs severely damage combustion engines and endanger the conversion into electricity by power plants, resulting in a high purification level requirement. Assessing treatment efficiency is still difficult. No consensus has been reached to provide a standardized sampling and quantification of VOSiCs into gases because of their diversity, their physicochemical properties, and the omnipresence of silicon in analytical chains. Usually, samplings are done by adsorption or absorption and quantification made by gas chromatography-mass spectrometry (GC-MS) or inductively coupled plasma-optical emission spectrometry (ICP-OES). In this objective, this paper presents and discusses the optimization of a patented method consisting in VOSiCs sampling by absorption of 100% ethanol and quantification of total Si by ICP-OES.

  19. Volatile Organic Silicon Compounds in Biogases: Development of Sampling and Analytical Methods for Total Silicon Quantification by ICP-OES

    PubMed Central

    Julien, Jennifer; Dumont, Nathalie; Lebouil, David; Germain, Patrick

    2014-01-01

    Current waste management policies favor biogases (digester gases (DGs) and landfill gases (LFGs)) valorization as it becomes a way for energy politics. However, volatile organic silicon compounds (VOSiCs) contained into DGs/LFGs severely damage combustion engines and endanger the conversion into electricity by power plants, resulting in a high purification level requirement. Assessing treatment efficiency is still difficult. No consensus has been reached to provide a standardized sampling and quantification of VOSiCs into gases because of their diversity, their physicochemical properties, and the omnipresence of silicon in analytical chains. Usually, samplings are done by adsorption or absorption and quantification made by gas chromatography-mass spectrometry (GC-MS) or inductively coupled plasma-optical emission spectrometry (ICP-OES). In this objective, this paper presents and discusses the optimization of a patented method consisting in VOSiCs sampling by absorption of 100% ethanol and quantification of total Si by ICP-OES. PMID:25379538

  20. A single chiroptical spectroscopic method may not be able to establish the absolute configurations of diastereomers: dimethylesters of hibiscus and garcinia acids.

    PubMed

    Polavarapu, Prasad L; Donahue, Emily A; Shanmugam, Ganesh; Scalmani, Giovanni; Hawkins, Edward K; Rizzo, Carmelo; Ibnusaud, Ibrahim; Thomas, Grace; Habel, Deenamma; Sebastian, Dellamol

    2011-06-09

    Electronic circular dichroism (ECD), optical rotatory dispersion (ORD), and vibrational circular dichroism (VCD) spectra of hibiscus acid dimethyl ester have been measured and analyzed in combination with quantum chemical calculations of corresponding spectra. These results, along with those reported previously for garcinia acid dimethyl ester, reveal that none of these three (ECD, ORD, or VCD) spectroscopic methods, in isolation, can unequivocally establish the absolute configurations of diastereomers. This deficiency is eliminated when a combined spectral analysis of either ECD and VCD or ORD and VCD methods is used. It is also found that the ambiguities in the assignment of absolute configurations of diastereomers may also be overcome when unpolarized vibrational absorption is included in the spectral analysis.

  1. A Single Chiroptical Spectroscopic Method May Not Be Able To Establish the Absolute Configurations of Diastereomers: Dimethylesters of Hibiscus and Garcinia Acids

    PubMed Central

    Polavarapu, Prasad L.; Donahue, Emily A.; Shanmugam, Ganesh; Scalmani, Giovanni; Hawkins, Edward K.; Rizzo, Carmelo; Ibnusaud, Ibrahim; Thomas, Grace; Habel, Deenamma; Sebastian, Dellamol

    2013-01-01

    Electronic circular dichroism (ECD), optical rotatory dispersion (ORD), and vibrational circular dichroism (VCD) spectra of hibiscus acid dimethyl ester have been measured and analyzed in combination with quantum chemical calculations of corresponding spectra. These results, along with those reported previously for garcinia acid dimethyl ester, reveal that none of these three (ECD, ORD, or VCD) spectroscopic methods, in isolation, can unequivocally establish the absolute configurations of diastereomers. This deficiency is eliminated when a combined spectral analysis of either ECD and VCD or ORD and VCD methods is used. It is also found that the ambiguities in the assignment of absolute configurations of diastereomers may also be overcome when unpolarized vibrational absorption is included in the spectral analysis. PMID:21568330

  2. Method and apparatus using selected superparamagnetic labels for rapid quantification of immunochromatographic tests

    PubMed Central

    Laitinen, Mika PA; Salmela, Jari; Gilbert, Leona; Kaivola, Risto; Tikkala, Topi; Oker-Blom, Christian; Pekola, Jukka; Vuento, Matti

    2009-01-01

    A rapid method and instrumentation for quantification of immunochromatographic tests (ICT) are described. The principle and performance of the method was demonstrated by measuring the levels of human chorionic gonadotropin (hCG) present in urine. The test format was a sandwich assay using two distinct monoclonal antibodies directed against hCG. The first anti-hCG antibody was labeled with superparamagnetic particles whereas the second was immobilized as a narrow detection zone on a porous membrane. The human urine sample was mixed with superparamagnetic particles coated with the first anti-hCG antibody, and the mixture was allowed to migrate past the detection zone containing the second anti-hCG antibody. Capillary forces facilitated migration of the immune complexes along the porous membrane. The amount of superparamagnetic particle-labelled monoclonal anti-hCG bound to the detection zone was directly proportional to the amount of hCG present in the sample as detected by measuring magnetization in the detector coil. The method had a practical detection limit of 20 U/l (54 nM) of hCG per 5 μl of human urine and a linear range of three decades from 20 U/l to 10 000 U/l. In addition, the analysis was completed within less than 10 minutes. Thus, the test format should be suitable for fast detection and monitoring of a large variety of clinically important parameters and analytes. PMID:24198463

  3. Method and apparatus using selected superparamagnetic labels for rapid quantification of immunochromatographic tests.

    PubMed

    Laitinen, Mika Pa; Salmela, Jari; Gilbert, Leona; Kaivola, Risto; Tikkala, Topi; Oker-Blom, Christian; Pekola, Jukka; Vuento, Matti

    2009-01-01

    A rapid method and instrumentation for quantification of immunochromatographic tests (ICT) are described. The principle and performance of the method was demonstrated by measuring the levels of human chorionic gonadotropin (hCG) present in urine. The test format was a sandwich assay using two distinct monoclonal antibodies directed against hCG. The first anti-hCG antibody was labeled with superparamagnetic particles whereas the second was immobilized as a narrow detection zone on a porous membrane. The human urine sample was mixed with superparamagnetic particles coated with the first anti-hCG antibody, and the mixture was allowed to migrate past the detection zone containing the second anti-hCG antibody. Capillary forces facilitated migration of the immune complexes along the porous membrane. The amount of superparamagnetic particle-labelled monoclonal anti-hCG bound to the detection zone was directly proportional to the amount of hCG present in the sample as detected by measuring magnetization in the detector coil. The method had a practical detection limit of 20 U/l (54 nM) of hCG per 5 μl of human urine and a linear range of three decades from 20 U/l to 10 000 U/l. In addition, the analysis was completed within less than 10 minutes. Thus, the test format should be suitable for fast detection and monitoring of a large variety of clinically important parameters and analytes.

  4. Improved method for the quantification of motility in glia and other morphologically complex cells.

    PubMed

    Sild, Mari; Chatelain, Robert P; Ruthazer, Edward S

    2013-01-01

    Cells such as astrocytes and radial glia with many densely ramified, fine processes pose particular challenges for the quantification of structural motility. Here we report the development of a method to calculate a motility index for individual cells with complex, dynamic morphologies. This motility index relies on boxcar averaging of the difference images generated by subtraction of images collected at consecutive time points. An image preprocessing step involving 2D projection, edge detection, and dilation of the raw images is first applied in order to binarize the images. The boxcar averaging of difference images diminishes the impact of artifactual pixel fluctuations while accentuating the group-wise changes in pixel values which are more likely to represent real biological movement. Importantly, this provides a value that correlates with mean process elongation and retraction rates without requiring detailed reconstructions of very complex cells. We also demonstrate that additional increases in the sensitivity of the method can be obtained by denoising images using the temporal frequency power spectra, based on the fact that rapid intensity fluctuations over time are mainly due to imaging artifact. The MATLAB programs implementing these motility analysis methods, complete with user-friendly graphical interfaces, have been made publicly available for download.

  5. Validated method for the quantification of free and total carnitine, butyrobetaine, and acylcarnitines in biological samples.

    PubMed

    Minkler, Paul E; Stoll, Maria S K; Ingalls, Stephen T; Kerner, Janos; Hoppel, Charles L

    2015-09-01

    A validated quantitative method for the determination of free and total carnitine, butyrobetaine, and acylcarnitines is presented. The versatile method has four components: (1) isolation using strong cation-exchange solid-phase extraction, (2) derivatization with pentafluorophenacyl trifluoromethanesulfonate, (3) sequential ion-exchange/reversed-phase (ultra) high-performance liquid chromatography [(U)HPLC] using a strong cation-exchange trap in series with a fused-core HPLC column, and (4) detection with electrospray ionization multiple reaction monitoring (MRM) mass spectrometry (MS). Standardized carnitine along with 65 synthesized, standardized acylcarnitines (including short-chain, medium-chain, long-chain, dicarboxylic, hydroxylated, and unsaturated acyl moieties) were used to construct multiple-point calibration curves, resulting in accurate and precise quantification. Separation of the 65 acylcarnitines was accomplished in a single chromatogram in as little as 14 min. Validation studies were performed showing a high level of accuracy, precision, and reproducibility. The method provides capabilities unavailable by tandem MS procedures, making it an ideal approach for confirmation of newborn screening results and for clinical and basic research projects, including treatment protocol studies, acylcarnitine biomarker studies, and metabolite studies using plasma, urine, tissue, or other sample matrixes.

  6. An optimized whole-body cortisol quantification method for assessing stress levels in larval zebrafish.

    PubMed

    Yeh, Chen-Min; Glöck, Mario; Ryu, Soojin

    2013-01-01

    Glucocorticoids serve important regulatory functions for many physiological processes and are critical mediators of the stress response. The stress response is a set of bodily processes aimed at counteracting a state of threatened homeostasis. Proper stress response is critical for the survival of an animal, however prolonged or abnormal stress response can be detrimental and is implicated in a number of human diseases such as depression and metabolic diseases. To dissect the underlying mechanism of this complex and important response, the zebrafish, Danio rerio offer important advantages such as ease of genetic manipulations and high-throughput behavioral analyses. However, there is a paucity of suitable methods to measure stress level in larval zebrafish. Therefore, an efficient low-cost method to monitor stress hormone levels will greatly facilitate stress research in zebrafish larvae. In this study, we optimized sample collection as well as cortisol extraction methods and developed a home-made ELISA protocol for measuring whole-body cortisol level in zebrafish larvae. Further, using our customized protocols, we characterized the response of larval zebrafish to a variety of stressors. This assay, developed for efficient cortisol quantification, will be useful for systematic and large-scale stress analyses in larval zebrafish.

  7. An Optimized Whole-Body Cortisol Quantification Method for Assessing Stress Levels in Larval Zebrafish

    PubMed Central

    Yeh, Chen-Min; Glöck, Mario; Ryu, Soojin

    2013-01-01

    Glucocorticoids serve important regulatory functions for many physiological processes and are critical mediators of the stress response. The stress response is a set of bodily processes aimed at counteracting a state of threatened homeostasis. Proper stress response is critical for the survival of an animal, however prolonged or abnormal stress response can be detrimental and is implicated in a number of human diseases such as depression and metabolic diseases. To dissect the underlying mechanism of this complex and important response, the zebrafish, Danio rerio offer important advantages such as ease of genetic manipulations and high-throughput behavioral analyses. However, there is a paucity of suitable methods to measure stress level in larval zebrafish. Therefore, an efficient low-cost method to monitor stress hormone levels will greatly facilitate stress research in zebrafish larvae. In this study, we optimized sample collection as well as cortisol extraction methods and developed a home-made ELISA protocol for measuring whole-body cortisol level in zebrafish larvae. Further, using our customized protocols, we characterized the response of larval zebrafish to a variety of stressors. This assay, developed for efficient cortisol quantification, will be useful for systematic and large-scale stress analyses in larval zebrafish. PMID:24223943

  8. Blood flow quantification using optical flow methods in a body fitted coordinate system

    NASA Astrophysics Data System (ADS)

    Maday, Peter; Brosig, Richard; Endres, Jurgen; Kowarschik, Markus; Navab, Nassir

    2014-03-01

    In this paper a blood flow quantification method that is based on a physically motivated dense 2D flow estimation algorithm is outlined. It yields accurate time varying volumetric flow rate measurements based on digital subtraction angiography (DSA) image sequences, with robustness to significant inter-frame displacements. Time varying volumetric flow rates are estimated for individual non-branching vascular segments based on the estimated 2D flow fields and a 3D vessel segmentation from a 3D Rotational Angiography (3DRA) acquisition. The novelty of the approach lies in the use of a vessel aligned coordinate system for the problem formulation. The coordinate functions are generated using the Schwarz-Christoffel1(SC) map that yields a solution with coordinate lines aligned with the vessel boundaries. The use of vessel aligned coordinates enables the easy and accurate handling of boundary conditions in the irregular domain of a vessel lumen while only requiring slight modifications to the used finite difference approach. Unlike traditional coarse to fine methods we use an anisotropic scaling strategy that enables the estimation of flows with larger inter frame displacements. The evaluation of our method is based on highly realistic synthetic DSA datasets for a number of cases. Ground truth volumetric flow rate values are compared against the measurements and a high degree of fidelity is observed. Performance measures are obtained with varying flow velocities and acquisition rates.

  9. A simple and efficient method for poly-3-hydroxybutyrate quantification in diazotrophic bacteria within 5 minutes using flow cytometry.

    PubMed

    Alves, L P S; Almeida, A T; Cruz, L M; Pedrosa, F O; de Souza, E M; Chubatsu, L S; Müller-Santos, M; Valdameri, G

    2017-01-16

    The conventional method for quantification of polyhydroxyalkanoates based on whole-cell methanolysis and gas chromatography (GC) is laborious and time-consuming. In this work, a method based on flow cytometry of Nile red stained bacterial cells was established to quantify poly-3-hydroxybutyrate (PHB) production by the diazotrophic and plant-associated bacteria, Herbaspirillum seropedicae and Azospirillum brasilense. The method consists of three steps: i) cell permeabilization, ii) Nile red staining, and iii) analysis by flow cytometry. The method was optimized step-by-step and can be carried out in less than 5 min. The final results indicated a high correlation coefficient (R2=0.99) compared to a standard method based on methanolysis and GC. This method was successfully applied to the quantification of PHB in epiphytic bacteria isolated from rice roots.

  10. A simple and efficient method for poly-3-hydroxybutyrate quantification in diazotrophic bacteria within 5 minutes using flow cytometry

    PubMed Central

    Alves, L.P.S.; Almeida, A.T.; Cruz, L.M.; Pedrosa, F.O.; de Souza, E.M.; Chubatsu, L.S.; Müller-Santos, M.; Valdameri, G.

    2017-01-01

    The conventional method for quantification of polyhydroxyalkanoates based on whole-cell methanolysis and gas chromatography (GC) is laborious and time-consuming. In this work, a method based on flow cytometry of Nile red stained bacterial cells was established to quantify poly-3-hydroxybutyrate (PHB) production by the diazotrophic and plant-associated bacteria, Herbaspirillum seropedicae and Azospirillum brasilense. The method consists of three steps: i) cell permeabilization, ii) Nile red staining, and iii) analysis by flow cytometry. The method was optimized step-by-step and can be carried out in less than 5 min. The final results indicated a high correlation coefficient (R2=0.99) compared to a standard method based on methanolysis and GC. This method was successfully applied to the quantification of PHB in epiphytic bacteria isolated from rice roots. PMID:28099582

  11. A validated ultra high pressure liquid chromatographic method for qualification and quantification of folic acid in pharmaceutical preparations.

    PubMed

    Deconinck, E; Crevits, S; Baten, P; Courselle, P; De Beer, J

    2011-04-05

    A fully validated UHPLC method for the identification and quantification of folic acid in pharmaceutical preparations was developed. The starting conditions for the development were calculated starting from the HPLC conditions of a validated method. These start conditions were tested on four different UHPLC columns: Grace Vision HT™ C18-P, C18, C18-HL and C18-B (2 mm × 100 mm, 1.5 μm). After selection of the stationary phase, the method was further optimised by testing two aqueous and two organic phases and by adapting to a gradient method. The obtained method was fully validated based on its measurement uncertainty (accuracy profile) and robustness tests. A UHPLC method was obtained for the identification and quantification of folic acid in pharmaceutical preparations, which will cut analysis times and solvent consumption.

  12. Quantification and Statistical Analysis Methods for Vessel Wall Components from Stained Images with Masson's Trichrome

    PubMed Central

    Hernández-Morera, Pablo; Castaño-González, Irene; Travieso-González, Carlos M.; Mompeó-Corredera, Blanca; Ortega-Santana, Francisco

    2016-01-01

    Purpose To develop a digital image processing method to quantify structural components (smooth muscle fibers and extracellular matrix) in the vessel wall stained with Masson’s trichrome, and a statistical method suitable for small sample sizes to analyze the results previously obtained. Methods The quantification method comprises two stages. The pre-processing stage improves tissue image appearance and the vessel wall area is delimited. In the feature extraction stage, the vessel wall components are segmented by grouping pixels with a similar color. The area of each component is calculated by normalizing the number of pixels of each group by the vessel wall area. Statistical analyses are implemented by permutation tests, based on resampling without replacement from the set of the observed data to obtain a sampling distribution of an estimator. The implementation can be parallelized on a multicore machine to reduce execution time. Results The methods have been tested on 48 vessel wall samples of the internal saphenous vein stained with Masson’s trichrome. The results show that the segmented areas are consistent with the perception of a team of doctors and demonstrate good correlation between the expert judgments and the measured parameters for evaluating vessel wall changes. Conclusion The proposed methodology offers a powerful tool to quantify some components of the vessel wall. It is more objective, sensitive and accurate than the biochemical and qualitative methods traditionally used. The permutation tests are suitable statistical techniques to analyze the numerical measurements obtained when the underlying assumptions of the other statistical techniques are not met. PMID:26761643

  13. An Optimized Method for Quantification of Pathogenic Leptospira in Environmental Water Samples

    PubMed Central

    Riediger, Irina N.; Hoffmaster, Alex R.; Biondo, Alexander W.; Ko, Albert I.; Stoddard, Robyn A.

    2016-01-01

    Leptospirosis is a zoonotic disease usually acquired by contact with water contaminated with urine of infected animals. However, few molecular methods have been used to monitor or quantify pathogenic Leptospira in environmental water samples. Here we optimized a DNA extraction method for the quantification of leptospires using a previously described Taqman-based qPCR method targeting lipL32, a gene unique to and highly conserved in pathogenic Leptospira. QIAamp DNA mini, MO BIO PowerWater DNA and PowerSoil DNA Isolation kits were evaluated to extract DNA from sewage, pond, river and ultrapure water samples spiked with leptospires. Performance of each kit varied with sample type. Sample processing methods were further evaluated and optimized using the PowerSoil DNA kit due to its performance on turbid water samples and reproducibility. Centrifugation speeds, water volumes and use of Escherichia coli as a carrier were compared to improve DNA recovery. All matrices showed a strong linearity in a range of concentrations from 106 to 10° leptospires/mL and lower limits of detection ranging from <1 cell /ml for river water to 36 cells/mL for ultrapure water with E. coli as a carrier. In conclusion, we optimized a method to quantify pathogenic Leptospira in environmental waters (river, pond and sewage) which consists of the concentration of 40 mL samples by centrifugation at 15,000×g for 20 minutes at 4°C, followed by DNA extraction with the PowerSoil DNA Isolation kit. Although the method described herein needs to be validated in environmental studies, it potentially provides the opportunity for effective, timely and sensitive assessment of environmental leptospiral burden. PMID:27487084

  14. Absolute configurations of fungal and plant metabolites by chiroptical methods. ORD, ECD, and VCD studies on phyllostin, scytolide, and oxysporone.

    PubMed

    Mazzeo, Giuseppe; Santoro, Ernesto; Andolfi, Anna; Cimmino, Alessio; Troselj, Pavle; Petrovic, Ana G; Superchi, Stefano; Evidente, Antonio; Berova, Nina

    2013-04-26

    The absolute configuration (AC) of the bioactive metabolites phyllostin (1) and scytolide (2), two hexahydro-1,4-benzodioxines produced by Phyllosticta cirsii, and oxysporone (3), a dihydrofuropyranone recently isolated from a strain of Diplodia africana, has been assigned by computational analysis of their optical rotatory dispersion (ORD), electronic circular dichroism (ECD), and vibrational circular dichroism (VCD) spectra. Computational prediction of ORD, ECD, and VCD allowed us to assign (3S,4aR,8S,8aR) AC to naturally occurring (-)-1, while (4aR,8S,8aR) AC was assigned to (-)-2 employing only ECD and VCD, because in this case ORD analysis turned out to be unsuitable for AC assignment. Theoretical prediction of both ORD and ECD spectra of 3 led to assignment of (4S,5R,6R) AC to (+)-3. In this case a satisfactory agreement between experimental and calculated VCD spectra was obtained only after taking into account solvent effects. This study shows that in the case of flexible and complex natural products only a concerted application of more than a single chiroptical technique permits unambiguous assignment of absolute configuration.

  15. A simple and rapid method for optical visualization and quantification of bacteria on textiles

    PubMed Central

    Stiefel, Philipp; Schneider, Jana; Amberg, Caroline; Maniura-Weber, Katharina; Ren, Qun

    2016-01-01

    To prevent bacterial contamination on textiles and the associated undesired effects different biocidal coatings have been investigated and applied. However, due to health and environmental concerns anti-adhesive coatings preventing the binding of bacteria would be favored. To develop such anti-adhesive coatings simple assays for reliable and fast screening are beneficial. Here an easy-to-handle, robust and rapid assay to assess bacteria on textiles utilizing a tetrazolium salt was reported. The assay allowed direct eye visualization of the color change of the textiles containing bacteria, facilitating fast screening. Quantification of the adhered bacteria could be done by generating standard curves which correlate the staining intensity to cell numbers. An additional advantage of the described assay is that with the same detection method anti-adhesive and biocidal effects can be investigated. The method was applied to different coatings, using Pseudomonas aeruginosa and Staphylococcus aureus as model organisms. The detection limit was found to be between 2.5 * 106 and 9.4 * 108 for P. aeruginosa and between 1 * 106 and 3.3 * 108 for S. aureus. The anti-adhesive coating PLUMA was demonstrated to reduce bacterial adhesion without killing them, whereas the biocidal coating TH22-27 caused a clear reduction in the number of viable cells. PMID:28004762

  16. Uncertainty quantification through the Monte Carlo method in a cloud computing setting

    NASA Astrophysics Data System (ADS)

    Cunha, Americo; Nasser, Rafael; Sampaio, Rubens; Lopes, Hélio; Breitman, Karin

    2014-05-01

    The Monte Carlo (MC) method is the most common technique used for uncertainty quantification, due to its simplicity and good statistical results. However, its computational cost is extremely high, and, in many cases, prohibitive. Fortunately, the MC algorithm is easily parallelizable, which allows its use in simulations where the computation of a single realization is very costly. This work presents a methodology for the parallelization of the MC method, in the context of cloud computing. This strategy is based on the MapReduce paradigm, and allows an efficient distribution of tasks in the cloud. This methodology is illustrated on a problem of structural dynamics that is subject to uncertainties. The results show that the technique is capable of producing good results concerning statistical moments of low order. It is shown that even a simple problem may require many realizations for convergence of histograms, which makes the cloud computing strategy very attractive (due to its high scalability capacity and low-cost). Additionally, the results regarding the time of processing and storage space usage allow one to qualify this new methodology as a solution for simulations that require a number of MC realizations beyond the standard.

  17. A comprehensive study of the delay vector variance method for quantification of nonlinearity in dynamical systems

    PubMed Central

    Mandic, D. P.; Ryan, K.; Basu, B.; Pakrashi, V.

    2016-01-01

    Although vibration monitoring is a popular method to monitor and assess dynamic structures, quantification of linearity or nonlinearity of the dynamic responses remains a challenging problem. We investigate the delay vector variance (DVV) method in this regard in a comprehensive manner to establish the degree to which a change in signal nonlinearity can be related to system nonlinearity and how a change in system parameters affects the nonlinearity in the dynamic response of the system. A wide range of theoretical situations are considered in this regard using a single degree of freedom (SDOF) system to obtain numerical benchmarks. A number of experiments are then carried out using a physical SDOF model in the laboratory. Finally, a composite wind turbine blade is tested for different excitations and the dynamic responses are measured at a number of points to extend the investigation to continuum structures. The dynamic responses were measured using accelerometers, strain gauges and a Laser Doppler vibrometer. This comprehensive study creates a numerical and experimental benchmark for structurally dynamical systems where output-only information is typically available, especially in the context of DVV. The study also allows for comparative analysis between different systems driven by the similar input. PMID:26909175

  18. HPLC method for quantification of oxidative stress by salicilate hydroxylation in human plasma.

    PubMed

    Lazalde-Ramos, Blanca P; Lares-Asseff, Ismael; Villanueva-Fierro, Ignacio; Sosa-Macías, Martha; Yahuaca-Mendoza, Patricia; Zamora-Perez, Ana

    2010-09-01

    The aim of the present study was to modify and validate a high-performance liquid chromatographic (HPLC) method for determining 2,3 and 2,5 dihydroxybenzoic acid (2,3-DHBA and 2,5-DHBA) from salicylic acid in human plasma. The mobile phase was a mixture of sodium acetate/citrate (pH 2.5) 30 mM-methanol (93:7, v/v). The injection volume was 10 muL. Retention time for 2,5-DHBA, and 2,3-DHBA was 4.5 +/- 0.10 and 5.8 +/- 0.15 min, respectively. The detection and quantification limits were 10 and 40 nM for 2,3-DHBA and 8 and 20 nM for 2,5-DHBA. Linearity was evaluated in the range of 40-1600 nM for both metabolites. Inter- and intra-analysis variation coefficient was below 10%. Good recoveries of more than 99% were obtained for both metabolites using this method.

  19. Quantification of immunohistochemistry--issues concerning methods, utility and semiquantitative assessment II.

    PubMed

    Taylor, C R; Levenson, R M

    2006-10-01

    Immunohistochemistry is entering its fourth decade of use on formalin-fixed paraffin-embedded tissues. Over this period the method has evolved to become a major part of the practice of diagnostic surgical pathology worldwide. From the beginning immunohistochemistry has been adapted to provide a range of markers of cell lineage and tissue type, with particular application to the diagnosis and classification of tumours. In this modality immunohistochemical methods were employed simply as 'special stains', the results of which were evaluated qualitatively by the pathologist, as for any other stain. More recently, attention has shifted to the demonstration of prognostic markers in tumour cells, driven by the advent of molecular biology and the discovery of numerous regulatory molecules, coupled with manufacture of the corresponding specific antibodies. Immunohistochemistry has rapidly adapted to this new use, but in so doing the demand for quantification has become paramount; it is no longer enough that the 'stain' is there; rather it is a question of 'How much is there?'. This review explores the limitations of immunohistochemistry when employed in a semiquantitative mode, and explores the possibility of fulfilling the full potential of immunohistochemistry, as a true quantitative immunoassay applied in a tissue section environment.

  20. Microplastics in the marine environment: a review of the methods used for identification and quantification.

    PubMed

    Hidalgo-Ruz, Valeria; Gutow, Lars; Thompson, Richard C; Thiel, Martin

    2012-03-20

    This review of 68 studies compares the methodologies used for the identification and quantification of microplastics from the marine environment. Three main sampling strategies were identified: selective, volume-reduced, and bulk sampling. Most sediment samples came from sandy beaches at the high tide line, and most seawater samples were taken at the sea surface using neuston nets. Four steps were distinguished during sample processing: density separation, filtration, sieving, and visual sorting of microplastics. Visual sorting was one of the most commonly used methods for the identification of microplastics (using type, shape, degradation stage, and color as criteria). Chemical and physical characteristics (e.g., specific density) were also used. The most reliable method to identify the chemical composition of microplastics is by infrared spectroscopy. Most studies reported that plastic fragments were polyethylene and polypropylene polymers. Units commonly used for abundance estimates are "items per m(2)" for sediment and sea surface studies and "items per m(3)" for water column studies. Mesh size of sieves and filters used during sampling or sample processing influence abundance estimates. Most studies reported two main size ranges of microplastics: (i) 500 μm-5 mm, which are retained by a 500 μm sieve/net, and (ii) 1-500 μm, or fractions thereof that are retained on filters. We recommend that future programs of monitoring continue to distinguish these size fractions, but we suggest standardized sampling procedures which allow the spatiotemporal comparison of microplastic abundance across marine environments.

  1. Size-exclusion HPLC as a sensitive and calibrationless method for complex peptide mixtures quantification.

    PubMed

    Bodin, Alice; Framboisier, Xavier; Alonso, Dominique; Marc, Ivan; Kapel, Romain

    2015-12-01

    This work describes an original methodology to quantify complex peptide mixtures by size-exclusion high-performance liquid chromatography (SE-HPLC). The methodology was first tested on simulated elutions of peptide mixtures. For this set of experiments, a good estimation of the total peptide concentration was observed (error less than 10 %). Then 30 fractions obtained by ultrafiltration of hydrolysates from two different sources were titrated by Kjeldahl or BCA analysis and analysed by SE-HPLC for an experimental validation of the methodology. Very good matchs between methods were obtained. The linear working range depends on the hydrolysate but is generally between 0.2 and 4gL(-1) (i.e. between 10 and 200μg). Moreover, the presence of organic solvents or salts in samples does not impact the accuracy of the methodology contrary to common quantification methods. Hence, the findings of this study show that total concentration of complex peptide mixture can be efficiently determinate by the proposed methodology using simple SE-HPLC analysis.

  2. Development of a reliable extraction and quantification method for glucosinolates in Moringa oleifera.

    PubMed

    Förster, Nadja; Ulrichs, Christian; Schreiner, Monika; Müller, Carsten T; Mewis, Inga

    2015-01-01

    Glucosinolates are the characteristic secondary metabolites of plants in the order Brassicales. To date the common DIN extraction 'desulfo glucosinolates' method remains the common procedure for determination and quantification of glucosinolates. However, the desulfation step in the extraction of glucosinolates from Moringa oleifera leaves resulted in complete conversion and degradation of the naturally occurring glucosinolates in this plant. Therefore, a method for extraction of intact Moringa glucosinolates was developed and no conversion and degradation of the different rhamnopyranosyloxy-benzyl glucosinolates was found. Buffered eluents (0.1 M ammonium acetate) were necessary to stabilize 4-α-rhamnopyranosyloxy-benzyl glucosinolate (Rhamno-Benzyl-GS) and acetyl-4-α-rhamnopyranosyloxy-benzyl glucosinolate isomers (Ac-Isomers-GS) during HPLC analysis. Due to the instability of intact Moringa glucosinolates at room temperature and during the purification process of single glucosinolates, influences of different storage (room temperature, frozen, thawing and refreezing) and buffer conditions on glucosinolate conversion were analysed. Conversion and degradations processes were especially determined for the Ac-Isomers-GS III.

  3. Methods for the quantification of lysosomal membrane permeabilization: a hallmark of lysosomal cell death.

    PubMed

    Aits, Sonja; Jäättelä, Marja; Nylandsted, Jesper

    2015-01-01

    Lysosomal cell death is triggered by lysosomal membrane permeabilization (LMP) and subsequent release of lysosomal hydrolases from the lysosomal lumen into the cytosol. Once released into the cytosol, the lysosomal cathepsin proteases act as executioner proteases for the subsequent cell death-either autonomously without caspase activation or in concert with the classical apoptotic machinery. Lysosomal cell death usually remains functional in apoptosis-resistant cancer cells and thus holds great potential as a therapeutic strategy for circumventing apoptosis deficiency in cancers. Notably, lysosomal cell death also plays an important role in normal physiology, e.g., during the regression of the mammary gland. Here we present four complementary methods for the quantification and visualization of LMP during the onset of death: (1) enzymatic activity measurements of released lysosomal hydrolases in the cytosol after digitonin extraction, (2) direct visualization of LMP by monitoring the release of fluorescent dextran from lysosomes into the cytosol, (3) immunocytochemistry to detect cathepsins released into the cytosol, and (4) detection of the translocation of galectins to damaged lysosomes. The methods presented here can ideally be combined as needed to provide solid evidence for LMP after a given cytotoxic stimuli.

  4. Testing 3D landform quantification methods with synthetic drumlins in a real digital elevation model

    NASA Astrophysics Data System (ADS)

    Hillier, John K.; Smith, Mike J.

    2012-06-01

    Metrics such as height and volume quantifying the 3D morphology of landforms are important observations that reflect and constrain Earth surface processes. Errors in such measurements are, however, poorly understood. A novel approach, using statistically valid ‘synthetic' landscapes to quantify the errors is presented. The utility of the approach is illustrated using a case study of 184 drumlins observed in Scotland as quantified from a Digital Elevation Model (DEM) by the ‘cookie cutter' extraction method. To create the synthetic DEMs, observed drumlins were removed from the measured DEM and replaced by elongate 3D Gaussian ones of equivalent dimensions positioned randomly with respect to the ‘noise' (e.g. trees) and regional trends (e.g. hills) that cause the errors. Then, errors in the cookie cutter extraction method were investigated by using it to quantify these ‘synthetic' drumlins, whose location and size is known. Thus, the approach determines which key metrics are recovered accurately. For example, mean height of 6.8 m is recovered poorly at 12.5 ± 0.6 (2σ) m, but mean volume is recovered correctly. Additionally, quantification methods can be compared: A variant on the cookie cutter using an un-tensioned spline induced about twice (× 1.79) as much error. Finally, a previously reportedly statistically significant (p = 0.007) difference in mean volume between sub-populations of different ages, which may reflect formational processes, is demonstrated to be only 30-50% likely to exist in reality. Critically, the synthetic DEMs are demonstrated to realistically model parameter recovery, primarily because they are still almost entirely the original landscape. Results are insensitive to the exact method used to create the synthetic DEMs, and the approach could be readily adapted to assess a variety of landforms (e.g. craters, dunes and volcanoes).

  5. Quantification of methane and nitrous oxide emissions from various waste treatment facilities by tracer dilution method

    NASA Astrophysics Data System (ADS)

    Mønster, Jacob; Rella, Chris; Jacobson, Gloria; Kjeldsen, Peter; Scheutz, Charlotte

    2013-04-01

    Urban activities generate solid and liquid waste, and the handling and aftercare of the waste results in the emission of various compounds into the surrounding environment. Some of these compounds are emitted as gasses into the atmosphere, including methane and nitrous oxide. Methane and nitrous oxide are strong greenhouse gases and are considered to have 25 and 298 times the greenhouse gas potential of carbon dioxide on a hundred years term (Solomon et al. 2007). Global observations of both gasses have shown increasing concentrations that significantly contribute to the greenhouse gas effect. Methane and nitrous oxide are emitted from both natural and anthropogenic sources and inventories of source specific fugitive emissions from the anthropogenic sources of methane and nitrous oxide of are often estimated on the basis of modeling and mass balance. Though these methods are well-developed, actual measurements for quantification of the emissions is a very useful tool for verifying the modeling and mass balance as well as for validation initiatives done for lowering the emissions of methane and nitrous oxide. One approach to performing such measurements is the tracer dilution method (Galle et al. 2001, Scheutz et al. 2011), where the exact location of the source is located and a tracer gas is released at this source location at a known flow. The ratio of downwind concentrations of the tracer gas and the methane and nitrous oxide gives the emissions rates of the greenhouse gases. This tracer dilution method can be performed using both stationary and mobile measurements and in both cases, real-time measurements of both tracer and quantified gas are required, placing high demands on the analytical detection method. To perform the methane and nitrous oxide measurements, two robust instruments capable of real-time measurements were used, based on cavity ring-down spectroscopy and operating in the near-infrared spectral region. One instrument measured the methane and

  6. Quantification of camalexin, a phytoalexin from Arabidopsis thaliana: a comparison of five analytical methods.

    PubMed

    Beets, Caryn; Dubery, Ian

    2011-12-15

    Camalexin is a phytoalexin of Arabidopsis thaliana and an important component of inducible defenses. Accurate quantification of low concentrations suffers from interference by structurally related metabolites. A. thaliana plants were induced with silver nitrate and camalexin was extracted using methanol and identified and quantified by (i) TLC as a blue fluorescent band, (ii) microtiter plate-based fluorescence spectroscopy, (iii) GC on a midpolar column coupled to flame ionization detection, (iv) C(18) HPLC coupled to a photodiode detector, and (v) UPLC coupled to a mass spectrometer detector. Standard curves over the range of 0.1-15 μg ml(-1) gave R(2) values from 0.996 to 0.999. The different methods were compared and evaluated for their ability to detect and quantify increasing concentrations (<0.4-8 μgg(-1) FW) of camalexin. Each of the techniques presented advantages and disadvantages with regard to accuracy, precision, interference, analytical sensitivity, and limits of detection. TLC is a good qualitative technique for the identification of camalexin and fluorescence spectroscopy is subject to quenching when performed on crude extracts. Comparable results were obtained with GC-FID, HPLC-PDA, and UPLC-MS, with UPLC-MS having the added advantage of short analysis times and detection based on accurate mass.

  7. Wave reflection quantification based on pressure waveforms alone--methods, comparison, and clinical covariates.

    PubMed

    Hametner, Bernhard; Wassertheurer, Siegfried; Kropf, Johannes; Mayer, Christopher; Holzinger, Andreas; Eber, Bernd; Weber, Thomas

    2013-03-01

    Within the last decade the quantification of pulse wave reflections mainly focused on measures of central aortic systolic pressure and its augmentation through reflections based on pulse wave analysis (PWA). A complementary approach is the wave separation analysis (WSA), which quantifies the total amount of arterial wave reflection considering both aortic pulse and flow waves. The aim of this work is the introduction and comparison of aortic blood flow models for WSA assessment. To evaluate the performance of the proposed modeling approaches (Windkessel, triangular and averaged flow), comparisons against Doppler measurements are made for 148 patients with preserved ejection fraction. Stepwise regression analysis between WSA and PWA parameters are performed to provide determinants of methodological differences. Against Doppler measurement mean difference and standard deviation of the amplitudes of the decomposed forward and backward pressure waves are comparable for Windkessel and averaged flow models. Stepwise regression analysis shows similar determinants between Doppler and Windkessel model only. The results indicate that the Windkessel method provides accurate estimates of wave reflection in subjects with preserved ejection fraction. The comparison with waveforms derived from Doppler ultrasound as well as recently proposed simple triangular and averaged flow waves showed that this approach may reduce variability and provide realistic results.

  8. Relationship between DAPI-fluorescence fading and nuclear DNA content: An alternative method to DNA quantification?

    PubMed

    Gallardo-Escárate, Cristian; Alvarez-Borrego, Josué; Von Brand, Elisabeth; Dupré, Enrique; Del Río-Portilla, Miguel Angel

    2007-01-01

    In observations by confocal or conventional fluorescence microscopy, important factors should be considered in order to obtain accurate images. One of them, such as the fluorescence bleaching from highest intensity to lowest signal of fluorescence is a common problem with several DNA fluorochromes and especially for DAPI stain. The fluorescence of DAPI fades rapidly when it is exposed to UV light, under optimal conditions of observation. Although the fading process can be retarded using a mounting medium with antifading reagents, the photochemical process underlying the fluorescence decay has not yet been fully explained. In addition, no relationship between fluorescence fading and nuclear DNA content has been tested. In order to test this relationship, we measured by means of image analysis the DAPI-fluorescence intensity in several cellular types (spermatozoa, erythrocytes and haemocytes) during their fluorescence bleaching. An algorithm specifically built in MATLAB software was used for this approach. The correlation coefficient between nuclear DNA content and DAPI-fluorescence fading was found equal to 99%. This study demonstrates the feasibility to measure nuclear DNA content by fluorescence fading quantification, as an alternative method concurrently with image analysis procedures.

  9. The Development of Simple Methods for the Maintenance and Quantification of Polymyxa graminis.

    PubMed

    Tyagi, Swati; Sultana, Razia; Ju, Ho-Jong; Lee, Wang-Hyu; Kim, Kangmin; Lee, Bongchoon; Lee, Kui-Jae

    2016-12-01

    Polymyxa graminis, a root endoparasite of several cereal species, is considered to be non-pathogenic but serves as a vector of various plant viruses belonging to the genera Bymovirus, Furovirus, and Pecluvirus. Specifically, it reduces barley productivity by transmitting the Barley Yellow Mosaic Virus (BaYMV). To date, due to its obligate biotrophic property, no artificial culturing of P. graminis was reported and its quantification was also technically challenging. Here, we developed a novel and simple method to infect P. graminis within sterile barley roots in contamination free by preparing nearly pure zoospore inoculum. Such artificial maintenance of P. graminis was verified based on the presence of various developmental stages in infected barley roots under microscope. In addition, the population of resting spores in host tissue was determined by establishing standard curve between manually counted number of spores and Ct values of 18S rDNA amplification using quantitative real-time PCR. Furthermore, it was validated that standard curve generated was also applicable to estimate the abundance of P. graminis in soil environments. In conclusion, the present study would help to generate a system to investigate the etiological causes as well as management of plant diseases caused by P. graminis and BaYMV in tissue and soil.

  10. A convenient method for simultaneous quantification of multiple phytohormones and metabolites: application in study of rice-bacterium interaction

    PubMed Central

    2012-01-01

    Background Simultaneous analysis of multiple functional-related phytohormones and their metabolites will improve our understanding of interactions among different hormones in the same biologic process. Results A method was developed for simultaneous quantification of multiple phytohormones, abscisic acid, indole-3-acetic acid (IAA), jasmonic acid (JA), and salicylic acid, hormone conjugates, IAA-aspartic acid, JA-isoleucine, and methyl JA, and phytoalexins, momilactone A, naringenin, and sakuranetin. This method combines a convenient procedure for preparing filtrated crude extracted samples and a sensitive quantification assay using ultra fast liquid chromatography-electrospray ionization tandem mass spectrometry (UFLC-ESI-MS). With this method, we determined the dynamic profiles of defense-related phytohormones, hormone metabolites, and phytoalexins in the interaction of rice with Xanthomonas oryzae pv. oryzae (Xoo), which causes bacterial blight, one of the most devastating diseases of rice worldwide. Conclusion This UFLC-ESI-MS method is convenient, sensitive, reliable, and inexpensive for quantification of multiple phytohormones and metabolites compared to current methods. The results obtained by application of this method in studying rice-bacterial interaction provide a basis for understanding the molecular mechanisms of rice defense responses. PMID:22243810

  11. Quantification of Methicillin-Resistant Staphylococcus aureus Strains in Marine and Freshwater Samples by the Most-Probable-Number Method

    PubMed Central

    Levin-Edens, Emily; Meschke, John Scott; Roberts, Marilyn C.

    2011-01-01

    Recreational beach environments have been recently identified as a potential reservoir for methicillin-resistant Staphylococcus aureus (MRSA); however, accurate quantification methods are needed for the development of risk assessments. This novel most-probable-number approach for MRSA quantification offers improved sensitivity and specificity by combining broth enrichment with MRSA-specific chromogenic agar. PMID:21441335

  12. PSAQ™ standards for accurate MS-based quantification of proteins: from the concept to biomedical applications.

    PubMed

    Picard, Guillaume; Lebert, Dorothée; Louwagie, Mathilde; Adrait, Annie; Huillet, Céline; Vandenesch, François; Bruley, Christophe; Garin, Jérôme; Jaquinod, Michel; Brun, Virginie

    2012-10-01

    Absolute protein quantification, i.e. determining protein concentrations in biological samples, is essential to our understanding of biological and physiopathological phenomena. Protein quantification methods based on the use of antibodies are very effective and widely used. However, over the last ten years, absolute protein quantification by mass spectrometry has attracted considerable interest, particularly for the study of systems biology and as part of biomarker development. This interest is mainly linked to the high multiplexing capacity of MS analysis, and to the availability of stable-isotope-labelled standards for quantification. This article describes the details of how to produce, control the quality and use a specific type of standard: Protein Standard Absolute Quantification (PSAQ™) standards. These standards are whole isotopically labelled proteins, analogues of the proteins to be assayed. PSAQ standards can be added early during sample treatment, thus they can correct for protein losses during sample prefractionation and for incomplete sample digestion. Because of this, quantification of target proteins is very accurate and precise using these standards. To illustrate the advantages of the PSAQ method, and to contribute to the increase in its use, selected applications in the biomedical field are detailed here.

  13. An ensemble average method to estimate absolute TEC using radio beacon-based differential phase measurements: Applicability to regions of large latitudinal gradients in plasma density

    NASA Astrophysics Data System (ADS)

    Thampi, Smitha V.; Bagiya, Mala S.; Chakrabarty, D.; Acharya, Y. B.; Yamamoto, M.

    2014-12-01

    A GNU Radio Beacon Receiver (GRBR) system for total electron content (TEC) measurements using 150 and 400 MHz transmissions from Low-Earth Orbiting Satellites (LEOS) is fabricated in house and made operational at Ahmedabad (23.04°N, 72.54°E geographic, dip latitude 17°N) since May 2013. This system receives the 150 and 400 MHz transmissions from high-inclination LEOS. The first few days of observations are presented in this work to bring out the efficacy of an ensemble average method to convert the relative TECs to absolute TECs. This method is a modified version of the differential Doppler-based method proposed by de Mendonca (1962) and suitable even for ionospheric regions with large spatial gradients. Comparison of TECs derived from a collocated GPS receiver shows that the absolute TECs estimated by this method are reliable estimates over regions with large spatial gradient. This method is useful even when only one receiving station is available. The differences between these observations are discussed to bring out the importance of the spatial differences between the ionospheric pierce points of these satellites. A few examples of the latitudinal variation of TEC during different local times using GRBR measurements are also presented, which demonstrates the potential of radio beacon measurements in capturing the large-scale plasma transport processes in the low-latitude ionosphere.

  14. Methods of RNA preparation affect mRNA abundance quantification of reference genes in pig maturing oocytes.

    PubMed

    Wang, Y-K; Li, X; Song, Z-Q; Yang, C-X

    2017-04-13

    To ensure accurate normalization and quantification of target RNA transcripts using reverse transcription quantitative polymerase chain reaction (RT-qPCR), most studies focus on the identification of stably expressed gene(s) as internal reference. However, RNA preparation methods could also be an important factor, especially for test samples of limited quantity (e.g. oocytes). In this study, we aimed to select appropriate reference gene(s), and evaluate the effect of RNA preparation methods on gene expression quantification in porcine oocytes and cumulus cells during in vitro maturation. Expression profiles of seven genes (GAPDH, 18S, YWHAG, BACT, RPL4, HPRT1 and PPIA) were examined, on RNA samples extracted from cumulus cells (RNeasy Kit) and oocytes (RNeasy Kit and Lysis Kit) during in vitro maturation, respectively. Interestingly, different RNA preparation methods were found to potentially affect the quantification of reference gene expression in pig oocytes cultured in vitro. After geNorm analyses, the most suitable genes for normalization were identified, GAPDH/18S for cumulus cells and YWHAG/BACT for oocytes, respectively. Thus, our results provide useful data and information on the selection of better reference genes and RNA preparation method for related functional studies.

  15. A rapid Fourier-transform infrared (FTIR) spectroscopic method for direct quantification of paracetamol content in solid pharmaceutical formulations

    NASA Astrophysics Data System (ADS)

    Mallah, Muhammad Ali; Sherazi, Syed Tufail Hussain; Bhanger, Muhammad Iqbal; Mahesar, Sarfaraz Ahmed; Bajeer, Muhammad Ashraf

    2015-04-01

    A transmission FTIR spectroscopic method was developed for direct, inexpensive and fast quantification of paracetamol content in solid pharmaceutical formulations. In this method paracetamol content is directly analyzed without solvent extraction. KBr pellets were formulated for the acquisition of FTIR spectra in transmission mode. Two chemometric models: simple Beer's law and partial least squares employed over the spectral region of 1800-1000 cm-1 for quantification of paracetamol content had a regression coefficient of (R2) of 0.999. The limits of detection and quantification using FTIR spectroscopy were 0.005 mg g-1 and 0.018 mg g-1, respectively. Study for interference was also done to check effect of the excipients. There was no significant interference from the sample matrix. The results obviously showed the sensitivity of transmission FTIR spectroscopic method for pharmaceutical analysis. This method is green in the sense that it does not require large volumes of hazardous solvents or long run times and avoids prior sample preparation.

  16. A high-performance liquid chromatography-electronic circular dichroism online method for assessing the absolute enantiomeric excess and conversion ratio of asymmetric reactions.

    PubMed

    Zhang, Xiang; Wang, Mingchao; Li, Li; Yin, Dali

    2017-03-02

    Asymmetric reactions often need to be evaluated during the synthesis of chiral compounds. However, traditional evaluation methods require the isolation of the individual enantiomer, which is tedious and time-consuming. Thus, it is desirable to develop simple, practical online detection methods. We developed a method based on high-performance liquid chromatography-electronic circular dichroism (HPLC-ECD) that simultaneously analyzes the material conversion ratio and absolute optical purity of each enantiomer. In particular, only a reverse-phase C18 column instead of a chiral column is required in our method because the ECD measurement provides a g-factor that describes the ratio of each enantiomer in the mixtures. We used our method to analyze the asymmetric hydrosilylation of β-enamino esters, and we discussed the advantage, feasibility, and effectiveness of this new methodology.

  17. A high-performance liquid chromatography-electronic circular dichroism online method for assessing the absolute enantiomeric excess and conversion ratio of asymmetric reactions

    PubMed Central

    Zhang, Xiang; Wang, Mingchao; Li, Li; Yin, Dali

    2017-01-01

    Asymmetric reactions often need to be evaluated during the synthesis of chiral compounds. However, traditional evaluation methods require the isolation of the individual enantiomer, which is tedious and time-consuming. Thus, it is desirable to develop simple, practical online detection methods. We developed a method based on high-performance liquid chromatography-electronic circular dichroism (HPLC-ECD) that simultaneously analyzes the material conversion ratio and absolute optical purity of each enantiomer. In particular, only a reverse-phase C18 column instead of a chiral column is required in our method because the ECD measurement provides a g-factor that describes the ratio of each enantiomer in the mixtures. We used our method to analyze the asymmetric hydrosilylation of β-enamino esters, and we discussed the advantage, feasibility, and effectiveness of this new methodology. PMID:28252028

  18. A high-performance liquid chromatography-electronic circular dichroism online method for assessing the absolute enantiomeric excess and conversion ratio of asymmetric reactions

    NASA Astrophysics Data System (ADS)

    Zhang, Xiang; Wang, Mingchao; Li, Li; Yin, Dali

    2017-03-01

    Asymmetric reactions often need to be evaluated during the synthesis of chiral compounds. However, traditional evaluation methods require the isolation of the individual enantiomer, which is tedious and time-consuming. Thus, it is desirable to develop simple, practical online detection methods. We developed a method based on high-performance liquid chromatography-electronic circular dichroism (HPLC-ECD) that simultaneously analyzes the material conversion ratio and absolute optical purity of each enantiomer. In particular, only a reverse-phase C18 column instead of a chiral column is required in our method because the ECD measurement provides a g-factor that describes the ratio of each enantiomer in the mixtures. We used our method to analyze the asymmetric hydrosilylation of β-enamino esters, and we discussed the advantage, feasibility, and effectiveness of this new methodology.

  19. Development and validation of a bioanalytical LC-MS method for the quantification of GHRP-6 in human plasma.

    PubMed

    Gil, Jeovanis; Cabrales, Ania; Reyes, Osvaldo; Morera, Vivian; Betancourt, Lázaro; Sánchez, Aniel; García, Gerardo; Moya, Galina; Padrón, Gabriel; Besada, Vladimir; González, Luis Javier

    2012-02-23

    Growth hormone-releasing peptide 6 (GHRP-6, His-(DTrp)-Ala-Trp-(DPhe)-Lys-NH₂, MW=872.44 Da) is a potent growth hormone secretagogue that exhibits a cytoprotective effect, maintaining tissue viability during acute ischemia/reperfusion episodes in different organs like small bowel, liver and kidneys. In the present work a quantitative method to analyze GHRP-6 in human plasma was developed and fully validated following FDA guidelines. The method uses an internal standard (IS) of GHRP-6 with ¹³C-labeled Alanine for quantification. Sample processing includes a precipitation step with cold acetone to remove the most abundant plasma proteins, recovering the GHRP-6 peptide with a high yield. Quantification was achieved by LC-MS in positive full scan mode in a Q-Tof mass spectrometer. The sensitivity of the method was evaluated, establishing the lower limit of quantification at 5 ng/mL and a range for the calibration curve from 5 ng/mL to 50 ng/mL. A dilution integrity test was performed to analyze samples at higher concentration of GHRP-6. The validation process involved five calibration curves and the analysis of quality control samples to determine accuracy and precision. The calibration curves showed R² higher than 0.988. The stability of the analyte and its internal standard (IS) was demonstrated in all conditions the samples would experience in a real time analyses. This method was applied to the quantification of GHRP-6 in plasma from nine healthy volunteers participating in a phase I clinical trial.

  20. Standardized Method for Quantification of Developing Lymphedema in Patients Treated for Breast Cancer

    SciTech Connect

    Ancukiewicz, Marek; Russell, Tara A.; Otoole, Jean; Specht, Michelle; Singer, Marybeth; Kelada, Alexandra; Murphy, Colleen D.; Pogachar, Jessica; Gioioso, Valeria; Patel, Megha; Skolny, Melissa; Smith, Barbara L.; Taghian, Alphonse G.

    2011-04-01

    Purpose: To develop a simple and practical formula for quantifying breast cancer-related lymphedema, accounting for both the asymmetry of upper extremities' volumes and their temporal changes. Methods and Materials: We analyzed bilateral perometer measurements of the upper extremity in a series of 677 women who prospectively underwent lymphedema screening during treatment for unilateral breast cancer at Massachusetts General Hospital between August 2005 and November 2008. Four sources of variation were analyzed: between repeated measurements on the same arm at the same session; between both arms at baseline (preoperative) visit; in follow-up measurements; and between patients. Effects of hand dominance, time since diagnosis and surgery, age, weight, and body mass index were also analyzed. Results: The statistical distribution of variation of measurements suggests that the ratio of volume ratios is most appropriate for quantification of both asymmetry and temporal changes. Therefore, we present the formula for relative volume change (RVC): RVC = (A{sub 2}U{sub 1})/(U{sub 2}A{sub 1}) - 1, where A{sub 1}, A{sub 2} are arm volumes on the side of the treated breast at two different time points, and U{sub 1}, U{sub 2} are volumes on the contralateral side. Relative volume change is not significantly associated with hand dominance, age, or time since diagnosis. Baseline weight correlates (p = 0.0074) with higher RVC; however, baseline body mass index or weight changes over time do not. Conclusions: We propose the use of the RVC formula to assess the presence and course of breast cancer-related lymphedema in clinical practice and research.

  1. Identification and quantification of aerosol polar oxygenated compounds bearing carboxylic or hydroxyl groups. 1. Method development.

    PubMed

    Jaoui, M; Kleindienst, T E; Lewandowski, M; Edney, E O

    2004-08-15

    In this study, a new analytical technique was developed for the identification and quantification of multifunctional compounds containing simultaneously at least one hydroxyl or one carboxylic group, or both. This technique is based on derivatizing first the carboxylic group(s) of the multifunctional compound using an alcohol (e.g., methanol, 1-butanol) in the presence of a relatively strong Lewis acid (BF3) as a catalyst. This esterification reaction quickly and quantitatively converts carboxylic acids to their ester forms. The second step is based on silylation of the ester compounds using bis(trimethylsilyl) trifluoroacetamide (BSTFA) as the derivatizing agent. For compounds bearing ketone groups in addition to carboxylic and hydroxyl groups, a third step was used based on PFBHA derivatization of the carbonyls. Different parameters including temperature, reaction time, and effect due to artifacts were optimized. A GC/MS in EI and in methane-CI mode was used for the analysis of these compounds. The new approach was tested on a number of multifunctional compounds. The interpretation of their EI (70 eV) and CI mass spectra shows that critical information is gained leading to unambiguous identification of unknown compounds. For example, when derivatized only with BF(3)-methanol, their mass spectra comprise primary ions at m/z M.+ + 1, M.+ + 29, and M.+ - 31 for compounds bearing only carboxylic groups and M.+ + 1, M.+ + 29, M.+ - 31, and M+. - 17 for those bearing hydroxyl and carboxylic groups. However, when a second derivatization (BSTFA) was used, compounds bearing hydroxyl and carboxylic groups simultaneously show, in addition to the ions observed before, ions at m/z M.+ + 73, M.+ - 15, M.+ - 59, M.+ - 75, M.+ - 89, and 73. To the best of our knowledge, this technique describes systematically for the first time a method for identifying multifunctional oxygenated compounds containing simultaneously one or more hydroxyl and carboxylic acid groups.

  2. Quantification of Viral and Prokaryotic Production Rates in Benthic Ecosystems: A Methods Comparison

    PubMed Central

    Rastelli, Eugenio; Dell’Anno, Antonio; Corinaldesi, Cinzia; Middelboe, Mathias; Noble, Rachel T.; Danovaro, Roberto

    2016-01-01

    Viruses profoundly influence benthic marine ecosystems by infecting and subsequently killing their prokaryotic hosts, thereby impacting the cycling of carbon and nutrients. Previously conducted studies, based on different methodologies, have provided widely differing estimates of the relevance of viruses on benthic prokaryotes. There has been no attempt so far to compare these independent approaches, including contextual comparisons among different approaches for sample manipulation (i.e., dilution or not of the sediments during incubations), between methods based on epifluorescence microscopy (EFM) or radiotracers, and between the use of different radiotracers. Therefore, it has been difficult to identify the most suitable methodologies and protocols to be used as standard approaches for the quantification of viral infections of prokaryotes. Here, we compared for the first time different methods for determining viral and prokaryotic production rates in marine sediments collected at two benthic sites, differing in depth and environmental conditions. We used a highly replicated experimental design, testing the potential biases associated to the incubation of sediments as diluted or undiluted. In parallel, we also compared EFM counts with the 3H-thymidine incubations for the determination of viral production rates, and the use of 3H-thymidine versus 3H-leucine radiotracers for the determination of prokaryotic production. We show here that, independent from sediment dilution, EFM-based values of viral production ranged from 1.4 to 4.6 × 107 viruses g-1 h-1, and were similar but overall less variable compared to those obtained by the 3H-thymidine method (0.3 to 9.0 × 107 viruses g-1h-1). In addition, the prokaryotic production rates were not affected by sediment dilution, and the use of different radiotracers provided very consistent estimates (10.3–35.1 and 9.3–34.6 ngC g-1h-1 using the 3H-thymidine or 3H-leucine method, respectively). These results indicated

  3. Absolute cascade-free cross-sections for the 2S to 2P transition in Zn(+) using electron-energy-loss and merged-beams methods

    NASA Technical Reports Server (NTRS)

    Smith, Steven J.; Man, K.-F.; Chutjian, A.; Mawhorter, R. J.; Williams, I. D.

    1991-01-01

    Absolute cascade-free excitation cross-sections in an ion have been measured for the resonance 2S to 2P transition in Zn(+) using electron-energy-loss and merged electron-ion beams methods. Measurements were carried out at electron energies of below threshold to 6 times threshold. Comparisons are made with 2-, 5-, and 15-state close-coupling and distorted-wave theories. There is good agreement between experiment and the 15-state close-coupling cross-sections over the energy range of the calculations.

  4. Validation of a liquid chromatography method for the simultaneous quantification of ochratoxin A and its analogues in red wines.

    PubMed

    Remiro, Rebeca; Ibáñez-Vea, María; González-Peñas, Elena; Lizarraga, Elena

    2010-12-24

    A validated high-performance liquid chromatography (HPLC) method with fluorescence detection for the simultaneous quantification of ochratoxin A (OTA) and its analogues (ochratoxin B (OTB), ochratoxin C (OTC) and methyl ochratoxin A (MeOTA)) in red wine at trace levels is described. Before their analysis by HPLC-FLD, ochratoxins were extracted and purified with immunoaffinity columns from 50 mL of red wine at pH 7.2. Validation of the analytical method was based on the following parameters: selectivity, linearity, robustness, limits of detection and quantification, precision (within-day and between-day variability), recovery and stability. The limits of detection (LOD) in red wine were established at 0.16, 0.32, 0.27 and 0.17 ng L(-1) for OTA, OTB, MeOTA and OTC, respectively. The limit of quantification (LOQ) was established as 0.50 ng L(-1) for all of the ochratoxins. The LOD and LOQ obtained are the lowest found for OTA in the reference literature up to now. Recovery values were 93.5, 81.7, 76.0 and 73.4% for OTA, OTB, MeOTA and OTC, respectively. For the first time, this validated method permits the investigation of the co-occurrence of ochratoxins A, B, C and methyl ochratoxin A in 20 red wine samples from Spain.

  5. A validated liquid chromatography tandem mass spectrometry method for quantification of erlotinib, OSI-420 and didesmethyl erlotinib and semi-quantification of erlotinib metabolites in human plasma.

    PubMed

    Svedberg, Anna; Gréen, Henrik; Vikström, Anders; Lundeberg, Joakim; Vikingsson, Svante

    2015-03-25

    A liquid chromatography tandem mass spectrometry method was developed and validated for quantification of erlotinib and its metabolites in human plasma. The method is suitable for therapeutic drug monitoring and pharmacokinetic studies. The substances were extracted using protein precipitation, separated on a BEH XBridge C18 column (100 ×2.1 mm, 1.7 μm) by gradient elution at 0.7 mL/min of acetonitrile and 5 mM ammonium acetate. The concentration was determined using a Waters Xevo triple quadrupole mass spectrometer in a multi reaction monitoring mode. The total run time was 7 min. Deuterated erlotinib and OSI-597 were used as internal standard for erlotinib and its metabolites, respectively. Erlotinib, OSI-420 and didesmethyl erlotinib were quantified in the concentration range 25-5000 ng/mL, 0.5-500 ng/mL and 0.15-10 ng/mL, respectively. Precision and accuracy was <14% except for OSI-420 at LLOQ (17%). Extraction recovery was above 89%, 99% and 89% for erlotinib, OSI-420 and didesmethyl erlotinib, respectively. The human liver microsomes generated 14 metabolites, three of them not previously reported. Twelve metabolites were measured semi-quantitatively and validated with respect to selectivity, precision and stability.

  6. Absolutely classical spin states

    NASA Astrophysics Data System (ADS)

    Bohnet-Waldraff, F.; Giraud, O.; Braun, D.

    2017-01-01

    We introduce the concept of "absolutely classical" spin states, in analogy to absolutely separable states of bipartite quantum systems. Absolutely classical states are states that remain classical (i.e., a convex sum of projectors on coherent states of a spin j ) under any unitary transformation applied to them. We investigate the maximal size of the ball of absolutely classical states centered on the maximally mixed state and derive a lower bound for its radius as a function of the total spin quantum number. We also obtain a numerical estimate of this maximal radius and compare it to the case of absolutely separable states.

  7. Simultaneous Assessment of Cardiomyocyte DNA Synthesis and Ploidy: A Method to Assist Quantification of Cardiomyocyte Regeneration and Turnover

    PubMed Central

    Richardson, Gavin D.

    2016-01-01

    Although it is accepted that the heart has a limited potential to regenerate cardiomyocytes following injury and that low levels of cardiomyocyte turnover occur during normal ageing, quantification of these events remains challenging. This is in part due to the rarity of the process and the fact that multiple cellular sources contribute to myocardial maintenance. Furthermore, DNA duplication within cardiomyocytes often leads to a polyploid cardiomyocyte and only rarely leads to new cardiomyocytes by cellular division. In order to accurately quantify cardiomyocyte turnover discrimination between these processes is essential. The protocol described here employs long term nucleoside labeling in order to label all nuclei which have arisen as a result of DNA replication and cardiomyocyte nuclei identified by utilizing nuclei isolation and subsequent PCM1 immunolabeling. Together this allows the accurate and sensitive identification of the nucleoside labeling of the cardiomyocyte nuclei population. Furthermore, 4′,6-diamidino-2-phenylindole labeling and analysis of nuclei ploidy, enables the discrimination of neo-cardiomyocyte nuclei from nuclei which have incorporated nucleoside during polyploidization. Although this method cannot control for cardiomyocyte binucleation, it allows a rapid and robust quantification of neo-cardiomyocyte nuclei while accounting for polyploidization. This method has a number of downstream applications including assessing the potential therapeutics to enhance cardiomyocyte regeneration or investigating the effects of cardiac disease on cardiomyocyte turnover and ploidy. This technique is also compatible with additional downstream immunohistological techniques, allowing quantification of nucleoside incorporation in all cardiac cell types. PMID:27285379

  8. Robust optimization of well location to enhance hysteretical trapping of CO2: Assessment of various uncertainty quantification methods and utilization of mixed response surface surrogates

    NASA Astrophysics Data System (ADS)

    Babaei, Masoud; Pan, Indranil; Alkhatib, Ali

    2015-12-01

    The paper aims to solve a robust optimization problem (optimization in presence of uncertainty) for finding the optimal locations of a number of CO2 injection wells for geological sequestration of carbon dioxide in a saline aquifer. The parametric uncertainties are the interfacial tension between CO2 and aquifer brine, the Land's trapping coefficient and the boundary aquifer's absolute permeability. The spatial uncertainties are due to the channelized permeability field which exhibits a binary channel-non-channel system. The objective function of the optimization is the amount of residually trapped CO2 due to the hysteresis of the relative permeability curves. A risk-averse value derived from the cumulative density function of the distribution of the amount of trapped gas is chosen as the objective function value. In order to ensure that the uncertainties are effectively taken into account, Monte Carlo simulation and Polynomial Chaos Expansion (PCE)-based methods are used and compared with each other. For different cases of parametric and spatial uncertainties, the most accurate uncertainty quantification (UQ) method is chosen to be integrated within the optimization algorithm. While for parametric uncertainty cases of up to two uncertain variables, PCE-based methods computationally outperform Monte Carlo simulations, it is shown that for the multimodal distributions of the function of trapped gas occurring for the spatial uncertainty case, Monte Carlo simulations are more reliable than PCE-based UQ methods. For the discrete (integer) optimization problem, various mixed response surface surrogate models are tested and the robust optimization resulted in optimal CO2 injection well locations.

  9. Development and Validation of an LC-MS/MS Method for the Quantification of Agaritine in Mushrooms.

    PubMed

    Merdivan, Simon; Willke, Christoph; Lindequist, Ulrike

    2016-01-01

    Agaritine, an aromatic hydrazine, is found as a secondary metabolite in mushroom species. It is among others suspected to exhibit genotoxic activity. This publication describes the validation of a method for the quantification of agaritine in mushrooms (i.e., extraction and purification by solid phase extraction) and measurement by liquid chromatography with tandem mass spectrometry detection in positive ionization mode. The results show this method to be selective, accurate, and precise. This method could be used for the quality control of pharmaceutical preparations containing mushrooms.

  10. Validation of a P-Glycoprotein (P-gp) Humanized Mouse Model by Integrating Selective Absolute Quantification of Human MDR1, Mouse Mdr1a and Mdr1b Protein Expressions with In Vivo Functional Analysis for Blood-Brain Barrier Transport

    PubMed Central

    Sadiq, Muhammad Waqas; Uchida, Yasuo; Hoshi, Yutaro; Tachikawa, Masanori; Terasaki, Tetsuya; Hammarlund-Udenaes, Margareta

    2015-01-01

    It is essential to establish a useful validation method for newly generated humanized mouse models. The novel approach of combining our established species-specific protein quantification method combined with in vivo functional studies is evaluated to validate a humanized mouse model of P-gp/MDR1 efflux transporter. The P-gp substrates digoxin, verapamil and docetaxel were administered to male FVB Mdr1a/1b(+/+) (FVB WT), FVB Mdr1a/1b(-/-) (Mdr1a/1b(-/-)), C57BL/6 Mdr1a/1b(+/+) (C57BL/6 WT) and humanized C57BL (hMDR1) mice. Brain-to-plasma total concentration ratios (Kp) were measured. Quantitative targeted absolute proteomic (QTAP) analysis was used to selectively quantify the protein expression levels of hMDR1, Mdr1a and Mdr1b in the isolated brain capillaries. The protein expressions of other transporters, receptors and claudin-5 were also quantified. The Kp for digoxin, verapamil, and docetaxel were 20, 30 and 4 times higher in the Mdr1a/1b(-/-) mice than in the FVB WT controls, as expected. The Kp for digoxin, verapamil and docetaxel were 2, 16 and 2-times higher in the hMDR1 compared to the C57BL/6 WT mice. The hMDR1 mice had 63- and 9.1-fold lower expressions of the hMDR1 and Mdr1a proteins than the corresponding expression of Mdr1a in C57BL/6 WT mice, respectively. The protein expression levels of other molecules were almost consistent between C57BL/6 WT and hMDR1 mice. The P-gp function at the BBB in the hMDR1 mice was smaller than that in WT mice due to lower protein expression levels of hMDR1 and Mdr1a. The combination of QTAP and in vivo functional analyses was successfully applied to validate the humanized animal model and evaluates its suitability for further studies. PMID:25932627

  11. Validation of a P-Glycoprotein (P-gp) Humanized Mouse Model by Integrating Selective Absolute Quantification of Human MDR1, Mouse Mdr1a and Mdr1b Protein Expressions with In Vivo Functional Analysis for Blood-Brain Barrier Transport.

    PubMed

    Sadiq, Muhammad Waqas; Uchida, Yasuo; Hoshi, Yutaro; Tachikawa, Masanori; Terasaki, Tetsuya; Hammarlund-Udenaes, Margareta

    2015-01-01

    It is essential to establish a useful validation method for newly generated humanized mouse models. The novel approach of combining our established species-specific protein quantification method combined with in vivo functional studies is evaluated to validate a humanized mouse model of P-gp/MDR1 efflux transporter. The P-gp substrates digoxin, verapamil and docetaxel were administered to male FVB Mdr1a/1b(+/+) (FVB WT), FVB Mdr1a/1b(-/-) (Mdr1a/1b(-/-)), C57BL/6 Mdr1a/1b(+/+) (C57BL/6 WT) and humanized C57BL (hMDR1) mice. Brain-to-plasma total concentration ratios (Kp) were measured. Quantitative targeted absolute proteomic (QTAP) analysis was used to selectively quantify the protein expression levels of hMDR1, Mdr1a and Mdr1b in the isolated brain capillaries. The protein expressions of other transporters, receptors and claudin-5 were also quantified. The Kp for digoxin, verapamil, and docetaxel were 20, 30 and 4 times higher in the Mdr1a/1b(-/-) mice than in the FVB WT controls, as expected. The Kp for digoxin, verapamil and docetaxel were 2, 16 and 2-times higher in the hMDR1 compared to the C57BL/6 WT mice. The hMDR1 mice had 63- and 9.1-fold lower expressions of the hMDR1 and Mdr1a proteins than the corresponding expression of Mdr1a in C57BL/6 WT mice, respectively. The protein expression levels of other molecules were almost consistent between C57BL/6 WT and hMDR1 mice. The P-gp function at the BBB in the hMDR1 mice was smaller than that in WT mice due to lower protein expression levels of hMDR1 and Mdr1a. The combination of QTAP and in vivo functional analyses was successfully applied to validate the humanized animal model and evaluates its suitability for further studies.

  12. Quantification of dental prostheses on cone-beam CT images by the Taguchi method.

    PubMed

    Kuo, Rong-Fu; Fang, Kwang-Ming; Ty, Wong; Hu, Chia Yu

    2016-01-08

    The gray values accuracy of dental cone-beam computed tomography (CBCT) is affected by dental metal prostheses. The distortion of dental CBCT gray values could lead to inaccuracies of orthodontic and implant treatment. The aim of this study was to quantify the effect of scanning parameters and dental metal prostheses on the accuracy of dental cone-beam computed tomography (CBCT) gray values using the Taguchi method. Eight dental model casts of an upper jaw including prostheses, and a ninth prosthesis-free dental model cast, were scanned by two dental CBCT devices. The mean gray value of the selected circular regions of interest (ROIs) were measured using dental CBCT images of eight dental model casts and were compared with those measured from CBCT images of the prosthesis-free dental model cast. For each image set, four consecutive slices of gingiva were selected. The seven factors (CBCTs, occlusal plane canting, implant connection, prosthesis position, coping material, coping thickness, and types of dental restoration) were used to evaluate scanning parameter and dental prostheses effects. Statistical methods of signal to noise ratio (S/N) and analysis of variance (ANOVA) with 95% confidence were applied to quantify the effects of scanning parameters and dental prostheses on dental CBCT gray values accuracy. For ROIs surrounding dental prostheses, the accuracy of CBCT gray values were affected primarily by implant connection (42%), followed by type of restoration (29%), prostheses position (19%), coping material (4%), and coping thickness (4%). For a single crown prosthesis (without support of implants) placed in dental model casts, gray value differences for ROIs 1-9 were below 12% and gray value differences for ROIs 13-18 away from pros-theses were below 10%. We found the gray value differences set to be between 7% and 8% for regions next to a single implant-supported titanium prosthesis, and between 46% and 59% for regions between double implant

  13. A method for in situ absolute DD yield calibration of neutron time-of-flight detectors on OMEGA using CR-39-based proton detectors

    DOE PAGES

    Waugh, C. J.; Rosenberg, M. J.; Zylstra, A. B.; ...

    2015-05-27

    Neutron time of flight (nTOF) detectors are used routinely to measure the absolute DD neutron yield at OMEGA. To check the DD yield calibration of these detectors, originally calibrated using indium activation systems, which in turn were cross-calibrated to NOVA nTOF detectors in the early 1990s, a direct in situ calibration method using CR-39 range filter proton detectors has been successfully developed. By measuring DD neutron and proton yields from a series of exploding pusher implosions at OMEGA, a yield calibration coefficient of 1.09 ± 0.02 (relative to the previous coefficient) was determined for the 3m nTOF detector. In addition,more » comparison of these and other shots indicates that significant reduction in charged particle flux anisotropies is achieved when bang time occurs significantly (on the order of 500 ps) after the trailing edge of the laser pulse. This is an important observation as the main source of the yield calibration error is due to particle anisotropies caused by field effects. The results indicate that the CR-39-nTOF in situ calibration method can serve as a valuable technique for calibrating and reducing the uncertainty in the DD absolute yield calibration of nTOF detector systems on OMEGA, the National Ignition Facility, and laser megajoule.« less

  14. A method for in situ absolute DD yield calibration of neutron time-of-flight detectors on OMEGA using CR-39-based proton detectors

    SciTech Connect

    Waugh, C. J.; Rosenberg, M. J.; Zylstra, A. B.; Frenje, J. A.; Seguin, F. H.; Petrasso, R. D.; Glebov, V. Yu.; Sangster, T. C.; Stoeckl, C.

    2015-05-27

    Neutron time of flight (nTOF) detectors are used routinely to measure the absolute DD neutron yield at OMEGA. To check the DD yield calibration of these detectors, originally calibrated using indium activation systems, which in turn were cross-calibrated to NOVA nTOF detectors in the early 1990s, a direct in situ calibration method using CR-39 range filter proton detectors has been successfully developed. By measuring DD neutron and proton yields from a series of exploding pusher implosions at OMEGA, a yield calibration coefficient of 1.09 ± 0.02 (relative to the previous coefficient) was determined for the 3m nTOF detector. In addition, comparison of these and other shots indicates that significant reduction in charged particle flux anisotropies is achieved when bang time occurs significantly (on the order of 500 ps) after the trailing edge of the laser pulse. This is an important observation as the main source of the yield calibration error is due to particle anisotropies caused by field effects. The results indicate that the CR-39-nTOF in situ calibration method can serve as a valuable technique for calibrating and reducing the uncertainty in the DD absolute yield calibration of nTOF detector systems on OMEGA, the National Ignition Facility, and laser megajoule.

  15. A method for in situ absolute DD yield calibration of neutron time-of-flight detectors on OMEGA using CR-39-based proton detectors

    SciTech Connect

    Waugh, C. J. Zylstra, A. B.; Frenje, J. A.; Séguin, F. H.; Petrasso, R. D.; Rosenberg, M. J.; Glebov, V. Yu.; Sangster, T. C.; Stoeckl, C.

    2015-05-15

    Neutron time of flight (nTOF) detectors are used routinely to measure the absolute DD neutron yield at OMEGA. To check the DD yield calibration of these detectors, originally calibrated using indium activation systems, which in turn were cross-calibrated to NOVA nTOF detectors in the early 1990s, a direct in situ calibration method using CR-39 range filter proton detectors has been successfully developed. By measuring DD neutron and proton yields from a series of exploding pusher implosions at OMEGA, a yield calibration coefficient of 1.09 ± 0.02 (relative to the previous coefficient) was determined for the 3m nTOF detector. In addition, comparison of these and other shots indicates that significant reduction in charged particle flux anisotropies is achieved when bang time occurs significantly (on the order of 500 ps) after the trailing edge of the laser pulse. This is an important observation as the main source of the yield calibration error is due to particle anisotropies caused by field effects. The results indicate that the CR-39-nTOF in situ calibration method can serve as a valuable technique for calibrating and reducing the uncertainty in the DD absolute yield calibration of nTOF detector systems on OMEGA, the National Ignition Facility, and laser megajoule.

  16. A direct qPCR method for residual DNA quantification in monoclonal antibody drugs produced in CHO cells.

    PubMed

    Hussain, Musaddeq

    2015-11-10

    Chinese hamster ovary (CHO) cells are the host cell of choice for manufacturing of monoclonal antibody (mAb) drugs in the biopharmaceutical industry. Host cell DNA is an impurity of such manufacturing process and must be controlled and monitored in order to ensure drug purity and safety. A conventional method for quantification of host residual DNA in drug requires extraction of DNA from the mAb drug substance with subsequent quantification of the extracted DNA using real-time PCR (qPCR). Here we report a method where the DNA extraction step is eliminated prior to qPCR. In this method, which we have named 'direct resDNA qPCR', the mAb drug substance is digested with a protease called KAPA in a 96-well PCR plate, the protease in the digest is then denatured at high temperature, qPCR reagents are added to the resultant reaction wells in the plate along with standards and controls in other wells of the same plate, and the plate subjected to qPCR for analysis of residual host DNA in the samples. This direct resDNA qPCR method for CHO is sensitive to 5.0fg of DNA with high precision and accuracy and has a wide linear range of determination. The method has been successfully tested with four mAbs drug, two IgG1 and two IgG4. Both the purified drug substance as well as a number of process intermediate samples, e.g., bioreactor harvest, Protein A column eluate and ion-exchange column eluates were tested. This method simplifies the residual DNA quantification protocol, reduces time of analysis and leads to increased assay sensitivity and development of automated high-throughput methods.

  17. Comparison of a high-performance liquid chromatography method for quantification of carbamazepine with chemiluminescent microparticle immunoassay.

    PubMed

    Guerrero Garduño, Óscar; González-Esquivel, Dinora F; Escalante-Membrillo, Carmen; Fernández, Ángeles; Rojas-Tomé, Irma Susana; Jung Cook, Helgi; Castro, Nelly

    2016-06-01

    Carbamazepine is an antiepileptic drug widely used for the treatment of epilepsy. In the National Institute of Neurology, monitoring has been performed using the technique chemiluminescent microparticle immunoassay (CMIA) in an automated way during the last five years. The aim of this study was to develop a simple and rapid HPLC analytical method coupled to DAD-UV detection for the determination of plasma concentrations of carbamazepine and compare its feasibility with those used in routine analysis. The developed HPLC method was fully validated and the applicability of the proposed method was verified through the analysis of plasma samples of patients and later compared with the quantification of the same plasma samples with the CMIA method. The limit of quantification obtained was 0.5 μg/mL. The mean value for recovery was 99.05% and the coefficient of variation (CV) was 5.6%. The precision and accuracy of this method were within the acceptable limits; inter- and intraday CV values were <10%. The correlation between the CMIA method and the developed HPLC method was very good (r ≈ 0.999). A Bland-Altman plot showed no significant bias between the results. The HPLC-DAD method may be an alternative to determine and monitoring the carbamazepine levels in human plasma or serum. Copyright © 2015 John Wiley & Sons, Ltd.

  18. Real-time quantification of TEL-AML1 fusion transcripts for MRD detection in relapsed childhood acute lymphoblastic leukaemia. Comparison with antigen receptor-based MRD quantification methods.

    PubMed

    Taube, Tillmann; Eckert, Cornelia; Körner, Gabriele; Henze, Günter; Seeger, Karlheinz

    2004-07-01

    Response to therapy of ALL assessed by molecular methods has been proved to be a predictor of outcome. Alternatively to established but very labour-intensive DNA-based PCR-techniques the TEL-AML1 fusion transcript can serve as a marker for MRD monitoring. MRD quantification using TEL-AML1 is of particular interest if the results are directly comparable to data obtained by established DNA-based assays. Investigation of the potential of MRD monitoring using LightCycler technology for TEL-AML1 real-time quantification and comparison to results from established DNA-based MRD assays revealed corresponding results. Accordingly, TEL-AML1 MRD quantification is a sensitive, specific and rapid method that can supplement clone-specific MRD detection.

  19. A direct droplet digital PCR method for quantification of residual DNA in protein drugs produced in yeast cells.

    PubMed

    Hussain, Musaddeq; Fantuzzo, Rebecca; Mercorelli, Suzanne; Cullen, Constance

    2016-05-10

    Yeast cells, in particular Pichia pastoris, are the host cell of choice for manufacturing several protein therapeutic agents in the biopharmaceutical industry. Host cell DNA is an impurity of such manufacturing process and the residual DNA after the purification process of the drug must be monitored to ensure drug purity and safety. Currently, real-time PCR (qPCR) based methods are widely employed for quantification of host residual DNA. At the same time the digital PCR technology is coming into prominence with promise of higher sensitivity. Here we report a method where the protein drug is directly added to the droplet digital PCR (ddPCR) reaction including yeast-specific primers and fluorescent-tagged probe and nanoliter-sized droplets are generated. The droplets are then subjected to PCR followed by analysis for fluorescence. This Pichia residual DNA direct ddPCR method for yeast can be used to test higher amount of drug compared to the corresponding qPCR method thereby increasing sensitivity, retaining high precision and accuracy and has a wide linear range of determination. The method has been successfully tested with three batches of a recombinant human IgG1-Fc-based drug (RP-1) and with commercially available human insulin, both manufactured in yeast cells. This method simplifies the residual DNA quantification protocol by eliminating DNA extraction or protease digestion and eliminates use of DNA standards in day-to-day running of the method.

  20. Development and validation of a spectrofluorimetric method for the quantification of ceftriaxone in pharmaceutical formulations and plasma.

    PubMed

    Shah, Jasmin; Jan, M Rasul; Shah, Sultan; Inayatullah

    2013-01-01

    We describe the development and validation of a new, simple, sensitive and cost-effective method for the determination of ceftriaxone in commercial formulations and spiked human plasma. The method proposes the conversion of ceftriaxone into a fluorescent product by reacting with ortho-phthalaldehyde (OPA) in the presence of sulfite at room temperature. The reaction medium is buffered to pH 10 using borate buffer. The derivatized reaction product is highly fluorescent and exhibits maximum fluorescence intensity at λ(em) = 386 nm after excitation at λ(ex) = 324 nm. The experimental parameters affecting progress of the derivatization reaction were carefully studied and optimized. Under optimum experimental conditions, the method has an excellent correlation coefficient of 0.9984 with a broad linear range of 0.4-20 µg/mL. The limit of detection (LOD) and limit of quantification (LOQ) were found to be 1.30 × 10(-3) and 3.90 × 10(-3) µg/mL, respectively. The interference effects of common excipients on the quantification of drug were investigated and no interference effect was observed. The proposed method has been successfully applied to the determination of ceftriaxone in pharmaceutical formulations and spiked human plasma samples. The method has been validated statistically through percent recovery studies using standard addition and by comparison with a reference HPLC method. The developed method exhibits excellent inter- and intraday precision.

  1. Alternative method for the quantification by gas chromatography triacylglycerol class analysis of cocoa butter equivalent added to chocolate bars.

    PubMed

    Guyon, François; Destouesse, Sarah; Moustirats, Joëlle; Esclapez, Maryse; Salagoity, Marie-Hélène; Medina, Bernard

    2004-05-19

    Directive 2000/36/EC allows chocolate makers to add up to 5% of only six specific cocoa butter equivalents (CBEs) to cocoa butter (CB). A quantification method based on triacylglycerol (TAG) class analysis by gas chromatography with an unpolar column was set up for routine control purposes of chocolate bars. Mixtures of CBEs/CB were elaborated according to a Placket-Burman experiment design and analyzed by gas chromatography. A matrix was built with the normalized values of TAG classes (C50, C52, C54, and C56) of pure CBs of various origins, homemade CB/CBE mixtures (1 CB type), and mixtures containing CBE with CBs of various origins. A multivariate calibration equation was computed from this matrix using a partial least-squares regression technique. CBE addition can be detected at a minimum level of 2%, and the mathematical model allows its quantification with an uncertainty of 2% with respect to the cocoa butter fats. The model has also been applied for deconvolution and quantification of each CBE of a CBE mixture in chocolate bars.

  2. A method to quantify infectious airborne pathogens at concentrations below the threshold of quantification by culture.

    PubMed

    Cutler, Timothy D; Wang, Chong; Hoff, Steven J; Zimmerman, Jeffrey J

    2013-04-01

    In aerobiology, dose-response studies are used to estimate the risk of infection to a susceptible host presented by exposure to a specific dose of an airborne pathogen. In the research setting, host- and pathogen-specific factors that affect the dose-response continuum can be accounted for by experimental design, but the requirement to precisely determine the dose of infectious pathogen to which the host was exposed is often challenging. By definition, quantification of viable airborne pathogens is based on the culture of micro-organisms, but some airborne pathogens are transmissible at concentrations below the threshold of quantification by culture. In this paper we present an approach to the calculation of exposure dose at microbiologically unquantifiable levels using an application of the "continuous-stirred tank reactor (CSTR) model" and the validation of this approach using rhodamine B dye as a surrogate for aerosolized microbial pathogens in a dynamic aerosol toroid (DAT).

  3. Development and Validation of an Affinity Chromatography-Protein G Method for IgG Quantification

    PubMed Central

    Paradina Fernández, Lesly; Calvo, Loany; Viña, Lisel

    2014-01-01

    Nimotuzumab, an IgG that recognizes the epidermal growth factor receptor (EGF-R) overexpressed in some tumors, is used in the treatment of advanced head and neck cancer. For the quantification of this protein in cell culture supernatants, protein G-HPLC affinity chromatography is used due to its high affinity and specificity for antibodies of this class. The technique relies on the comparison of the area under the curve of the elution peak of the samples to be evaluated versus to a calibration curve of well-known concentrations and was validated by assessment of its robustness, specificity, repeatability, intermediate precision, accuracy, linearity, limit of detection, limit of quantification, and range. According to results of the study all validation parameters fulfilled the preestablished acceptance criteria and demonstrated the feasibility of the assay for the analysis of samples of cell culture supernatant as well as drug product. PMID:27379284

  4. Validated LC-MS/MS Method for the Quantification of Ponatinib in Plasma: Application to Metabolic Stability

    PubMed Central

    Kadi, Adnan A.; Darwish, Hany W.; Attwa, Mohamed W.; Amer, Sawsan M.

    2016-01-01

    In the current work, a rapid, specific, sensitive and validated liquid chromatography tandem mass-spectrometric method was developed for the quantification of ponatinib (PNT) in human plasma and rat liver microsomes (RLMs) with its application to metabolic stability. Chromatographic separation of PNT and vandetanib (IS) were accomplished on Agilent eclipse plus C18 analytical column (50 mm × 2.1 mm, 1.8 μm particle size) maintained at 21±2°C. Flow rate was 0.25 mLmin-1 with run time of 4 min. Mobile phase consisted of solvent A (10 mM ammonium formate, pH adjusted to 4.1 with formic acid) and solvent B (acetonitrile). Ions were generated by electrospray (ESI) and multiple reaction monitoring (MRM) was used as basis for quantification. The results revealed a linear calibration curve in the range of 5–400 ngmL-1 (r2 ≥ 0.9998) with lower limit of quantification (LOQ) and lower limit of detection (LOD) of 4.66 and 1.53 ngmL-1 in plasma, 4.19 and 1.38 ngmL-1 in RLMs. The intra- and inter-day precision and accuracy in plasma ranged from1.06 to 2.54% and -1.48 to -0.17, respectively. Whereas in RLMs ranged from 0.97 to 2.31% and -1.65 to -0.3%. The developed procedure was applied for quantification of PNT in human plasma and RLMs for study metabolic stability of PNT. PNT disappeared rapidly in the 1st 10 minutes of RLM incubation and the disappearance plateaued out for the rest of the incubation. In vitro half-life (t1/2) was 6.26 min and intrinsic clearance (CLin) was 15.182± 0.477. PMID:27764191

  5. A single standardized practical training for surgical scrubbing according to EN1500: Effect Quantification, value of the standardized method and comparison with clinical reference groups

    PubMed Central

    Fichtner, Andreas; Haupt, Elke; Karwath, Tobias; Wullenk, Katharina; Pöhlmann, Christoph; Jatzwauk, Lutz

    2013-01-01

    The standardized training of practical competences in skills labs is relatively new among German Medical Faculties. The broad acceptance and outstanding evaluation results do not provide objective data on the efficiency and cost-efficiency of these trainings. This study aims on the quantification of the teaching effect of the surgical scrubbing technique EN1500 and its comparison with clinical references of OR personnel. Methods: 161 4th year medical students were randomized into intervention and control group. The intervention group received a 45 minute standardized peer-teaching training of practical competences necessary in the OR including the scrubbing according to EN1500. Fluorescence dye was mixed in the disinfectant solution. After hand disinfection, standardized fotographs and semi-automated digital processing resulted in quantification of the insufficiently covered hand area. These results were compared with the control group that received the training after the test. In order to provide information on the achieved clinical competence level, the results were compared with the two clinical reference groups. Results: The intervention group remained with 4,99% (SD 2,34) insufficiently covered hand area after the training compared to the control group 7,33% (SD 3,91), p<0,01. There was no significant difference between control group and reference groups: surgeons 9,32% (SD 4,97), scrub nurses 8,46% (SD 4,66). The student intervention group showed results that were significantly better than the clinical references. The methodic mistake remained negligible. In the sub-group analysis, the students with low or medium experience in surgical scrubbing and hand disinfection derived highest benefit from the training, whereas students with no or high experience did benefit less. All participants showed better results on hand palms compared to back of hand areas. Discussion: A single standardized peer-teaching of surgical scrubbing and hand disinfection according to EN

  6. Absolute entropy and free energy of fluids using the hypothetical scanning method. I. Calculation of transition probabilities from local grand canonical partition functions

    NASA Astrophysics Data System (ADS)

    Szarecka, Agnieszka; White, Ronald P.; Meirovitch, Hagai

    2003-12-01

    The hypothetical scanning (HS) method provides the absolute entropy and free energy from a Boltzmann sample generated by Monte Carlo, molecular dynamics or any other exact simulation procedure. Thus far HS has been applied successfully to magnetic and polymer chain models; in this paper and the following one it is extended to fluid systems by treating a Lennard-Jones model of argon. With HS a probability Pi approximating the Boltzmann probability of system configuration i is calculated with a stepwise reconstruction procedure, based on adding atoms gradually layer-by-layer to an initially empty volume, where they are replaced in their positions at i. At each step a transition probability (TP) is obtained from local grand canonical partition functions calculated over a limited space of the still unvisited (future) volume, the larger this space the better the approximation. Pi is the product of the step TPs, where ln Pi is an upper bound of the absolute entropy, which leads to upper and lower bounds for the free energy. We demonstrate that very good results for the entropy and the free energy can be obtained for a wide range of densities of the argon system by calculating TPs that are based on only a very limited future volume.

  7. Comparison of two enzymatic immunoassays, high resolution mass spectrometry method and radioimmunoassay for the quantification of human plasma histamine.

    PubMed

    Poli, Caroline; Laurichesse, Mathieu; Rostan, Octavie; Rossille, Delphine; Jeannin, Pascale; Drouet, Martine; Renier, Gilles; Chevailler, Alain; Tarte, Karin; Bendavid, Claude; Beauvillain, Céline; Amé-Thomas, Patricia

    2016-01-25

    Histamine (HA) is one of the main immediate mediators involved in allergic reactions. HA plasma concentration is well correlated with the severity of vascular and respiratory signs of anaphylaxis. Consequently, plasma quantification of HA is useful to comfort the diagnosis of anaphylaxis. Currently, radioimmunoassay (RIA) is the gold standard method to quantify HA due to its high sensitivity, but it is time consuming, implicates specific formations and cautions for technicians, and produces hazardous radioactive wastes. The aim of this study was to compare two enzymatic immunoassays (EIA) and one in-house liquid chromatography high-resolution mass spectrometry method (LC-HRMS) with the gold standard method for HA quantification in plasma samples of patients suspected of anaphylaxis reactions. Ninety-two plasma samples were tested with the 4 methods (RIA, 2 EIA and LC-HRMS) for HA quantification. Fifty-eight samples displayed HA concentrations above the positive cut-off of 10nM evaluated by RIA, including 18 highly positive samples (>100 nM). This study shows that Immunotech(®) EIA and LC-HRMS concentrations were highly correlated with RIA values, in particular for samples with a HA concentration around the positive cut-off. In our hands, plasma concentrations obtained with the Demeditec Diagnostics(®) EIA correlated less with results obtained by RIA, and an underestimation of plasma HA levels led to a lack of sensitivity. In conclusion, this study demonstrates that Immunotech(®) EIA and LC-HRMS method could be used instead of RIA to assess plasma HA in human diagnostic use.

  8. Rapid, cost-effective and accurate quantification of Yucca schidigera Roezl. steroidal saponins using HPLC-ELSD method.

    PubMed

    Tenon, Mathieu; Feuillère, Nicolas; Roller, Marc; Birtić, Simona

    2017-04-15

    Yucca GRAS-labelled saponins have been and are increasingly used in food/feed, pharmaceutical or cosmetic industries. Existing techniques presently used for Yucca steroidal saponin quantification remain either inaccurate and misleading or accurate but time consuming and cost prohibitive. The method reported here addresses all of the above challenges. HPLC/ELSD technique is an accurate and reliable method that yields results of appropriate repeatability and reproducibility. This method does not over- or under-estimate levels of steroidal saponins. HPLC/ELSD method does not require each and every pure standard of saponins, to quantify the group of steroidal saponins. The method is a time- and cost-effective technique that is suitable for routine industrial analyses. HPLC/ELSD methods yield a saponin fingerprints specific to the plant species. As the method is capable of distinguishing saponin profiles from taxonomically distant species, it can unravel plant adulteration issues.

  9. Comparison of two methods, UHPLC-UV and UHPLC-MS/MS, for the quantification of polyphenols in cider apple juices.

    PubMed

    Verdu, Cindy F; Gatto, Julia; Freuze, Ingrid; Richomme, Pascal; Laurens, François; Guilet, David

    2013-08-22

    The aim of this study was to develop faster and more efficient phenotyping methods for in-depth genetic studies on cider apple progeny. The UHPLC chromatographic system was chosen to separate polyphenolic compounds, and quantifications were then simultaneously performed with a UV-PDA detector and an ESI-triple quadrupole mass analyzer (SRM mode). Both quantification methods were validated for 15 major compounds using two apple juice samples, on the basis of linearity, limits of detection and quantification, recovery and precision tests. The comparison between UV and SRM quantifications in 120 different samples of a cider apple progeny showed an excellent correlation for major compounds quantified with both methods. However, an overestimation was revealed for five compounds with the UV detector and the mass analyzer. Co-elution and matrix effects are discussed to explain this phenomenon. SRM methods should therefore be considered with restrictions in some cases for quantification measurements when several phenolic compounds are simultaneously quantified in complex matrices such as apple juices. For both methods, analyses were carried out over short periods of time while maintaining a high quality for the simultaneous quantification of phenolic compounds in apple juice. Each method is relevant for more in-depth genetic studies of the polyphenol content of apple juice.

  10. Quantification methods for human and large animal leukocytes using DNA dyes by flow cytometry.

    PubMed

    Pieper, Ina Laura; Radley, Gemma; Chan, Chris H H; Friedmann, Yasmin; Foster, Graham; Thornton, Catherine A

    2016-06-01

    Ovine and bovine blood is used heavily within the development of blood-handling medical devices, such as heart pumps (left ventricular assist devices, LVADs), for which blood cell damage needs to be monitored during in vitro testing. Hematology analyzers provide cell counts but no information about cell viability. The anthraquinone DNA dyes CyTRAK Orange™ and DRAQ7™ have practical and spectral properties rendering them suitable for multicolor assays. Compared to other DNA dyes such as Vybrant Dyecycle, CyTRAK Orange enables a faster staining protocol and does not require incubation at +37°C. Compared to traditional viability dyes such as propidium iodide and 7AAD, DRAQ7's unique spectral profile of excitation in both blue and red lasers and far-red emission enables identification of dual positive dead cell events and frees up detectors for use with other reagents. CyTRAK Orange and DRAQ7 could be used in combination with absolute counting bead standards to provide cell counts and viability but the combination of these dyes has previously only been used for microscopy on rodent cells. The purpose of this study was to evaluate the use of these dyes in combination in large animal blood samples for flow cytometry. A viability and cell counting protocol for bovine, ovine, and human leukocytes using CyTRAK Orange and DRAQ7 was prepared. Four different counting bead standards were evaluated using the Navios and FACSAria cytometers and compared to counts obtained from hematology analyzers. CyTRAK Orange successfully detected CD45(+) leukocytes in all species. The DRAQ7 single-stained dead cell counts correlated well with the CyTRAK Orange/DRAQ7 double-stained dead cell counts in human and bovine blood, but not in ovine blood, which could be related to the blood source. In conclusion, for human and bovine blood, this method works well for viability counts using different flow cytometers and bead standards. © 2016 International Society for Advancement of Cytometry.

  11. Methane fugitive emissions quantification using the novel 'plume camera' (spatial correlation) method

    NASA Astrophysics Data System (ADS)

    Crosson, E.; Rella, C.

    2012-12-01

    Fugitive emissions of methane into the atmosphere are a major concern facing the natural gas production industry. Given that the global warming potential of methane is many times greater than that of carbon dioxide, the importance of quantifying methane emissions becomes clear. The rapidly increasing reliance on shale gas (or other unconventional sources) is only intensifying the interest in fugitive methane releases. Natural gas (which is predominantly methane) is an attractive energy source, as it emits 40% less carbon dioxide per Joule of energy generated than coal. However, if just a small percentage of the natural gas consumed is lost due to fugitive emissions during production, processing, or transport, this global warming benefit is lost (Howarth et al. 2012). It is therefore imperative, as production of natural gas increases, that the fugitive emissions of methane are quantified accurately. Traditional direct measurement techniques often involve physical access of the leak itself to quantify the emissions rate, and are generally require painstaking effort to first find the leak and then quantify the emissions rate. With over half a million natural gas producing wells in the U.S. (U.S. Energy Information Administration), not including the associated processing, storage, and transport facilities, and with each facility having hundreds or even thousands of fittings that can potentially leak, the need is clear to develop methodologies that can provide a rapid and accurate assessment of the total emissions rate on a per-well head basis. In this paper we present a novel method for emissions quantification which uses a 'plume camera' with three 'pixels' to quantify emissions using direct measurements of methane concentration in the downwind plume. By analyzing the spatial correlation between the pixels, the spatial extent of the instantaneous plume can be inferred. This information, when combined with the wind speed through the measurement plane, provides a direct

  12. Standardless Quantification of Heavy Elements by Electron Probe Microanalysis.

    PubMed

    Moy, Aurélien; Merlet, Claude; Dugne, Olivier

    2015-08-04

    Absolute Mα and Mβ X-ray intensities were measured for the elements Pt, Au, Pb, U, and Th by electron impact for energies ranging from 6 to 38 keV. Experimental data were obtained by measuring the X-ray intensity emitted from bulk samples with an electron microprobe using high-resolution wavelength-dispersive spectrometers. Recorded X-ray intensities were converted into absolute X-ray yields by evaluation of the detector efficiency and then compared with X-ray intensities calculated by means of Monte Carlo simulations. Simulated Mα and Mβ X-ray intensities were found to be in good agreement with the measurements, allowing their use in standardless quantification methods. A procedure and a software program were developed to accurately obtain virtual standard values. Standardless quantifications of Pb and U were tested on standards of PbS, PbTe, PbCl2, vanadinite, and UO2.

  13. Rapid and Reliable Method for Quantification of Mycobacterium paratuberculosis by Use of the BACTEC MGIT 960 System▿

    PubMed Central

    Shin, Sung Jae; Han, Jun Hee; Manning, Elizabeth J. B.; Collins, Michael T.

    2007-01-01

    A simple method for the enumeration of viable Mycobacterium paratuberculosis cells was developed and evaluated using the MGIT 960 culture system. For each of 12 M. paratuberculosis strains isolated from either cattle or humans, single-cell suspensions of M. paratuberculosis cells were adjusted to an optical density at 600 nm of 1.00 (107.6 to 108.2 cells/ml), and serial dilutions were prepared. Standard curves were established by relating the MGIT time-to-detection data to the log10 CFU for these suspensions using standard plate counting and BACTEC 460 results as reference methods. Universal and strain-specific standard quantification curves were generated. A one-phase exponential decay equation best fit the universal standard curve and strain-specific curves (R2 of 0.96 and >0.99, respectively). Two subgroups within the universal curves were distinguished: one for laboratory-adapted strains and the other for recently isolated low-passage bovine strains. The predictive errors for log10 estimations using the universal standard curve, each subgroup's standard curve, and strain-specific curves were ±0.87, ±0.45, and ±0.31 log10 units, respectively. CFU estimations by all three standard curves were highly reproducible, regardless of the M. paratuberculosis strain or inoculum volume. In comparison with the previously described BACTEC 460 M. paratuberculosis counting method, quantification with MGIT 960 was less expensive, more rapid, more accurate, and more sensitive (<10 CFU). This MGIT counting method has broad applications for studies requiring the quantification of viable M. paratuberculosis cells, such as drug susceptibility testing or environmental survival studies. PMID:17428943

  14. Simultaneous Quantification of Gymnemic Acid as Gymnemagenin and Charantin as β-Sitosterol Using Validated HPTLC Densitometric Method.

    PubMed

    Ahamad, Javed; Amin, Saima; Mir, Showkat R

    2015-08-01

    Gymnemic acid and charantin are well-established antidiabetic phytosterols found in Gymnema sylvestre and Momordica charantia, respectively. The fact that these plants are often used together in antidiabetic poly-herbal formulations lured us to develop an HPTLC densitometric method for the simultaneous quantification of their bioactive compounds. Indirect estimation of gymnemic acid as gymnemagenin and charantin as β-sitosterol after hydrolysis has been proposed. Aluminum-backed silica gel 60 F254 plates (20 × 10 cm) were used as stationary phase and toluene-ethyl acetate-methanol-formic acid (60 : 20 : 15 : 5, v/v) as mobile phase. Developed chromatogram was scanned at 550 nm after derivatization with modified vanillin-sulfuric acid reagent. Regression analysis of the calibration data showed an excellent linear relationship between peak area versus concentration of the analytes. Linearity was found to be in the range of 500-2,500 and 100-500 ng/band for gymnemagenin and β-sitosterol, respectively. The suitability of the developed HPTLC method for simultaneous estimation of analytes was established by validating it as per the ICH guidelines. The limits of detection and quantification for gymnemagenin were found to be ≈60 and ≈190 ng/band, and those for β-sitosterol ≈30 and ≈90 ng/band, respectively. The developed method was found to be linear (r(2) = 0.9987 and 0.9943), precise (relative standard deviation <1.5 and <2% for intra- and interday precision) and accurate (mean recovery ranged between 98.43-101.44 and 98.68-100.20%) for gymnemagenin and β-sitosterol, respectively. The proposed method was also found specific and robust for quantification of both the analytes and was successfully applied to herbal drugs and in-house herbal formulation without any interference.

  15. Evaluation of Molecular Methods for the Detection and Quantification of Pathogen-Derived Nucleic Acids in Sediment

    PubMed Central

    Farkas, Kata; Hassard, Francis; McDonald, James E.; Malham, Shelagh K.; Jones, Davey L.

    2017-01-01

    The accurate detection of pathogens in environmental matrices, such as sediment, is critical in understanding pathogen fate and behavior in the environment. In this study, we assessed the usefulness of methods for the detection and quantification of Vibrio spp. and norovirus (NoV) nucleic acids in sediment. For bacteria, a commonly used direct method using hexadecyltrimethylammonium bromide (CTAB) and phenol-chloroform-isoamyl alcohol (PCI) extraction was optimized, whereas for NoV, direct and indirect (virus elution—concentration) methods were evaluated. For quantification, commercially available quantitative PCR (qPCR) and reverse transcription qPCR (RT-qPCR) kits were tested alongside a digital PCR (dPCR) approach. CTAB-based extraction combined with 16 h polyethylene glycol 6000 (PEG6000) precipitation was found to be suitable for the direct extraction of high abundance bacterial and viral nucleic acids. For the indirect extraction of viral RNA, beef extract-based elution followed by PEG6000 precipitation and extraction using the NucliSENS® MiniMag® Nucleic Acid Purification System and the PowerViral® Environmental RNA/DNA Isolation Kit and qRT-PCR resulted in 83–112 and 63–69% recoveries of NoV, respectively. dPCR resulted in lower viral recoveries (47 and 9%) and ~4 orders of magnitude lower Vibrio concentrations (3.6–4.6 log10 gc/100 g sediment) than was observed using qPCR. The use of internal controls during viral quantification revealed that the RT step was more affected by inhibitors than the amplification. The methods described here are suitable for the enumeration of viral and/or bacterial pathogens in sediment, however the use of internal controls to assess efficiency is recommended. PMID:28174565

  16. Comparison of derivative preprocessing and automated polynomial baseline correction method for classification and quantification of narcotics in solid mixtures.

    PubMed

    Leger, Marc N; Ryder, Alan G

    2006-02-01

    This work offers a real-world comparison of derivative preprocessing and a new polynomial method described by Lieber and Mahadevan-Jansen (LMJ) for baseline correction of Raman spectra with widely varying backgrounds. This comparison is based on their outcomes in factor analysis, analyte discrimination, and quantification. Both correction methods are applied to a Raman spectra data set taken from 85 solid samples of illegal narcotics diluted with various materials. It is found that neither approach outperforms the other, as they give similar principal component analysis (PCA) models and quantification errors: cocaine and heroin show cross-validation errors of approximately 8%, while MDMA is quantified to a cross-validation error of approximately 3-4%. The LMJ method does offer several other advantages, the most significant being the retention of original peak shapes after the correction, which simplifies the interpretation of the preprocessed spectra. The LMJ method is therefore recommended for use as a baseline correction method in future research with Raman spectroscopy.

  17. Simultaneous Quantification of Ten Active Components in Traditional Chinese Formula Sijunzi Decoction Using a UPLC-PDA Method.

    PubMed

    An, Kang; Jin-Rui, Guo; Zhen, Zhang; Xiao-Long, Wang

    2014-01-01

    Sijunzi decoction (SJZT), a traditional Chinese formula (TCMF) consisting of four herbs, has been widely used for the treatment of various gastrointestinal symptoms. However, its modernization process is hindered by the lack of a powerful quality control method that covers the major active components in the formula. The aim of this study was to establish a UPLC method for the quantitative determination of ten active components in Sijunzi decoction including ginsenoside Rg1, Re, Rb1, liquiritin, liquiritigenin, glycyrrhizic acid, atractylenolide I, atractylenolide II, atractylenolide III, and pachymic acid. Separation was achieved using an ACQUITY UPLC BEHC18 column (2.1 mm × 100 mm, 1.7  μ m) with a gradient elution program consisting of acetonitrile and 0.1% phosphoric acid solution. The detection wavelengths were set at 203, 254, 222, and 267 nm. The method was validated for linearity, accuracy, precision, limit of detection, and limit of quantification. The validated method was successfully applied to the simultaneous quantification of ten active compounds from several finished batches of SJZT. This validated that UPLC method is expected to provide a new basis for the quality control of SJZT.

  18. A validated GC-MS method for the determination and quantification of residual solvents in counterfeit tablets and capsules.

    PubMed

    Deconinck, E; Canfyn, M; Sacré, P-Y; Baudewyns, S; Courselle, P; De Beer, J O

    2012-11-01

    A fast headspace GC-MS method was developed and validated for the detection and quantification of residual solvents of all three ICH-classes in counterfeit tablets and capsules. The method was validated for 10 solvents, selected based on an initial screening of counterfeit medicinal products. The considered solvents were ethanol, 2-propanol, acetone, ethylacetate, chloroform, carbon tetrachloride, benzene, toluene, dichloromethane and ethylbenzene. The proposed method uses a Phenomenex 624 capillary column (60 m × 0.32 mm; 1.8 μm film thickness) (Phenomenex, Torrance, USA) with an oven temperature program from 60 °C (held for 5 min) to 270 °C at 25 °C/min. 270 °C is held for 10 min. The total run time is 23.4 min. The obtained method was fully validated by applying the "total error" profile. Calibration lines for all components were linear within the studied ranges. The relative bias and the relative standard deviations for all components were smaller than 5%, the β-expectation tolerance limits did not exceed the acceptance limits of ±10% and the relative expanded uncertainties were acceptable for all of the considered components. A method was obtained for the screening and quantification of residual solvents in counterfeit tablets and capsules, which will allow a fast screening of these products for the presence of residual solvents.

  19. Dystrophin quantification

    PubMed Central

    Anthony, Karen; Arechavala-Gomeza, Virginia; Taylor, Laura E.; Vulin, Adeline; Kaminoh, Yuuki; Torelli, Silvia; Feng, Lucy; Janghra, Narinder; Bonne, Gisèle; Beuvin, Maud; Barresi, Rita; Henderson, Matt; Laval, Steven; Lourbakos, Afrodite; Campion, Giles; Straub, Volker; Voit, Thomas; Sewry, Caroline A.; Morgan, Jennifer E.; Flanigan, Kevin M.

    2014-01-01

    Objective: We formed a multi-institution collaboration in order to compare dystrophin quantification methods, reach a consensus on the most reliable method, and report its biological significance in the context of clinical trials. Methods: Five laboratories with expertise in dystrophin quantification performed a data-driven comparative analysis of a single reference set of normal and dystrophinopathy muscle biopsies using quantitative immunohistochemistry and Western blotting. We developed standardized protocols and assessed inter- and intralaboratory variability over a wide range of dystrophin expression levels. Results: Results from the different laboratories were highly concordant with minimal inter- and intralaboratory variability, particularly with quantitative immunohistochemistry. There was a good level of agreement between data generated by immunohistochemistry and Western blotting, although immunohistochemistry was more sensitive. Furthermore, mean dystrophin levels determined by alternative quantitative immunohistochemistry methods were highly comparable. Conclusions: Considering the biological function of dystrophin at the sarcolemma, our data indicate that the combined use of quantitative immunohistochemistry and Western blotting are reliable biochemical outcome measures for Duchenne muscular dystrophy clinical trials, and that standardized protocols can be comparable between competent laboratories. The methodology validated in our study will facilitate the development of experimental therapies focused on dystrophin production and their regulatory approval. PMID:25355828

  20. Absolute method for the assay of oleuropein in olive oils by atmospheric pressure chemical ionization tandem mass spectrometry.

    PubMed

    De Nino, Antonio; Di Donna, Leonardo; Mazzotti, Fabio; Muzzalupo, Enzo; Perri, Enzo; Sindona, Giovanni; Tagarelli, Antonio

    2005-09-15

    Oleuropein (OLP, 1), the active ingredient present (i) in food integrators extracted from olive leaves, (ii) in table olives, and (iii) in extra virgin olive oils is a nutraceutical whose health benefits have been widely documented. A new analytical method for its assay, which is based on the utilization of atmospheric pressure chemical ionization tandem mass spectrometry and on the use of a synthetic labeled analogue, the 4-trideuteriocarboxyoleuropein (2), as an internal standard, is presented. The results obtained with extra virgin olive oils from different cultivars and different Italian regions are discussed.

  1. High-performance capillary electrophoretic method for the quantification of 5-methyl 2'-deoxycytidine in genomic DNA: application to plant, animal and human cancer tissues.

    PubMed

    Fraga, Mario F; Uriol, Esther; Borja Diego, L; Berdasco, María; Esteller, Manel; Cañal, María Jesús; Rodríguez, Roberto

    2002-06-01

    A new approach to the evaluation of the relative degree of genomic DNA methylation through the quantification of 2'-deoxynucleosides is proposed. Detection and quantification of 5-methyl 2'-deoxycytidine in genomic DNA has been performed using micellar high-performance capillary electrophoresis (HPCE) with UV-Vis detection. This approach has been demonstrated to be more sensitive and specific than other HPCE methods for the quantification of DNA methylation degree and also to be faster than other HPLC-based methods. The detection and quantification of nucleosides through enzymatic hydrolyses notably increases the specificity of the technique and allows its exploitation in the analysis of poorly purified and/or concentrated DNA samples such as those obtained from meristematic plant regions and paraffin-embedded tissues.

  2. Utilizing a reference material for assessing absolute tumor mechanical properties in modality independent elastography

    NASA Astrophysics Data System (ADS)

    Kim, Dong Kyu; Weis, Jared A.; Yankeelov, Thomas E.; Miga, Michael I.

    2014-03-01

    There is currently no reliable method for early characterization of breast cancer response to neoadjuvant chemotherapy (NAC) [1,2]. Given that disruption of normal structural architecture occurs in cancer-bearing tissue, we hypothesize that further structural changes occur in response to NAC. Consequently, we are investigating the use of modalityindependent elastography (MIE) [3-8] as a method for monitoring mechanical integrity to predict long term outcomes in NAC. Recently, we have utilized a Demons non-rigid image registration method that allows 3D elasticity reconstruction in abnormal tissue geometries, making it particularly amenable to the evaluation of breast cancer mechanical properties. While past work has reflected relative elasticity contrast ratios [3], this study improves upon that work by utilizing a known stiffness reference material within the reconstruction framework such that a stiffness map becomes an absolute measure. To test, a polyvinyl alcohol (PVA) cryogel phantom and a silicone rubber mock mouse tumor phantom were constructed with varying mechanical stiffness. Results showed that an absolute measure of stiffness could be obtained based on a reference value. This reference technique demonstrates the ability to generate accurate measurements of absolute stiffness to characterize response to NAC. These results support that `referenced MIE' has the potential to reliably differentiate absolute tumor stiffness with significant contrast from that of surrounding tissue. The use of referenced MIE to obtain absolute quantification of biomarkers is also translatable across length scales such that the characterization method is mechanics-consistent at the small animal and human application.

  3. A liquid biopsy-based method for the detection and quantification of circulating tumor cells in surgical osteosarcoma patients

    PubMed Central

    Zhang, Haoqiang; Gao, Peng; Xiao, Xin; Heger, Michal; Geng, Lei; Fan, Bo; Yuan, Yulin; Huang, Chen; Chen, Guojing; Liu, Yao; Hu, Yongchen; Yu, Xiuchun; Wu, Sujia; Wang, Ling; Wang, Zhen

    2017-01-01

    A method for the enumeration and quantification of osteosarcoma (OS) circulating tumor cells (CTCs) is currently not available. A correlation between the number of CTCs and progression-free survival (PFS) has been established for other cancers, but not for OS CTCs. A method was therefore developed for CTC quantification in OS and validated in a prospective cohort of surgical patients with primary and recurrent/metastatic OS (N=23). Human OS cells, acting as CTCs, were enumerated from spiked human peripheral blood (PB) following erythrocyte and leukocyte depletion. The OS cells were quantified microscopically based on aneuploidy and a CK18−/CD45− phenotype. Aneuploidy was assayed by fluorescence in situ hybridization (FISH) using fluorescence-labeled alpha-satellite probes for the centromeres of chromosome (CEP 8). CK18 and CD45 phenotyping was performed with immunocytochemistry. HOS cells in spiked PB could be effectively retrieved with the FISH-based enumeration method, which was subsequently employed in an OS patient cohort. PB of recurrent/metastatic OS patients contained more CTCs than the PB of primary OS patients. OS patients with ≥2 CTCs per 7.5 ml of PB had worse PFS than patients whose PB contained <2 CTCs. In 2 cases, CTCs were present in PB of OS patients with negative X-ray and chest CT scans. In conclusion, our method was able to quantitate CTCs in liquid biopsies of OS patients. The number of CTCs has diagnostic and prognostic value. PMID:28350107

  4. A liquid biopsy-based method for the detection and quantification of circulating tumor cells in surgical osteosarcoma patients.

    PubMed

    Zhang, Haoqiang; Gao, Peng; Xiao, Xin; Heger, Michal; Geng, Lei; Fan, Bo; Yuan, Yulin; Huang, Chen; Chen, Guojing; Liu, Yao; Hu, Yongchen; Yu, Xiuchun; Wu, Sujia; Wang, Ling; Wang, Zhen

    2017-03-08

    A method for the enumeration and quantification of osteosarcoma (OS) circulating tumor cells (CTCs) is currently not available. A correlation between the number of CTCs and progression-free survival (PFS) has been established for other cancers, but not for OS CTCs. A method was therefore developed for CTC quantification in OS and validated in a prospective cohort of surgical patients with primary and recurrent/metastatic OS (N=23). Human OS cells, acting as CTCs, were enumerated from spiked human peripheral blood (PB) following erythrocyte and leukocyte depletion. The OS cells were quantified microscopically based on aneuploidy and a CK18-/CD45- phenotype. Aneuploidy was assayed by fluorescence in situ hybridization (FISH) using fluorescence-labeled alpha-satellite probes for the centromeres of chromosome (CEP 8). CK18 and CD45 phenotyping was performed with immunocytochemistry. HOS cells in spiked PB could be effectively retrieved with the FISH-based enumeration method, which was subsequently employed in an OS patient cohort. PB of recurrent/metastatic OS patients contained more CTCs than the PB of primary OS patients. OS patients with ≥2 CTCs per 7.5 ml of PB had worse PFS than patients whose PB contained <2 CTCs. In 2 cases, CTCs were present in PB of OS patients with negative X-ray and chest CT scans. In conclusion, our method was able to quantitate CTCs in liquid biopsies of OS patients. The number of CTCs has diagnostic and prognostic value.

  5. Multiclass method for pesticides quantification in honey by means of modified QuEChERS and UHPLC-MS/MS.

    PubMed

    Tette, Patrícia Amaral Souza; da Silva Oliveira, Fabiano Aurélio; Pereira, Elba Nathália Corrêa; Silva, Gilsara; de Abreu Glória, Maria Beatriz; Fernandes, Christian

    2016-11-15

    Bee products can be produced in an environment contaminated by pesticides that can be transported by honey bees to the hive and incorporated into the honey. Therefore, rapid and modern methods to determine pesticide residues in honey samples are essential to guarantee consumers' health. In this study, a simple multiresidue method for the quantification of 116 pesticides in honey is proposed. It involves the use of a modified QuEChERS procedure followed by UHPLC-MS/MS analysis. The method was validated according to the European Union SANCO/12571/2013 guidelines. Acceptable values were obtained for the following parameters: linearity, limit of detection (0.005mg/kg) and limit of quantification (0.010 and 0.025mg/kg), trueness (for the four tested levels the recovery assays values were between 70 and 120%), intermediate precision (RSD<20.0%) and measurement uncertainty tests (<50.0%). The validated method was applied for determination of 100 honey samples from five states of Brazil.

  6. Review of methods for the detection and quantification of adulteration of rice: Basmati as a case study.

    PubMed

    Vemireddy, Lakshminarayana R; Satyavathi, V V; Siddiq, E A; Nagaraju, J

    2015-06-01

    Rice is a staple and widely grown crop endowed with rich genetic diversity. As it is difficult to differentiate seeds of various rice varieties based on visual observation accurately, the harvested seeds and subsequent processed products are highly prone to adulteration with look-alike and low quality seeds by the dishonest traders. To protect the interests of importing countries and consumers, several methods have been employed over the last few decades for unambiguous discrimination of cultivars, accurate quantification of the adulterants, and for determination of cultivated geographical area. With recent advances in biotechnology, DNA based techniques evolved rapidly and proved successful over conventional non-DNA based methods to purge the problem of adulteration at commercial level. In the current review, we made an attempt to summarize the existing methods of adulteration detection and quantification in a comprehensive manner by providing Basmati as a case study to enable the traders to arrive at a quick resolution in choosing the apt method to eliminate the adulteration practice in the global rice industry.

  7. Development of a fast and reliable method for the assessment of microbial colonization and growth on textiles by DNA quantification.

    PubMed

    Teufel, Linda; Schuster, K Christian; Merschak, Petra; Bechtold, Thomas; Redl, Bernhard

    2008-01-01

    There is a lack of relevant methods to assess the colonization of textiles by skin bacteria because present methods are mainly culture-based procedures. Therefore, the goal of this study was to develop a fast and sensitive culture-independent procedure for the quantification of microbial colonization and growth on textiles. We have established a suitable protocol to use DNA quantification as a reliable method for in vitroand in vivoinvestigations of textiles. For DNA extraction, a two-step procedure comprising treatment of the textile with a solution containing Triton X-100 and lysozyme for 1 h and a successive treatment by SDS and proteinase K for 2 h turned out to be most efficient. DNA extracted from textiles and fabrics was than quantified with the highly sensitive PicoGreen fluorescent dye. In vitrochallenge tests demonstrated a strong correlation between numbers of bacteria on textiles and amount of DNA extracted from textiles. Therefore, this method was used to compare different materials after in vivotrials for assessment of their susceptibility for microbial colonization and growth.

  8. Development of a Gas Chromatography-Mass Spectrometry Method for the Quantification of Glucaric Acid Derivatives in Beverage Substrates

    PubMed Central

    Craig, Ana Paula; Fields, Christine C.; Simpson, John V.

    2014-01-01

    A gas chromatography-mass spectrometry (GC-MS) method using the standard addition methodology was developed for the determination of glucuronolactone (GL) and glucuronic acid (DGuA) in four beverages categorized as detoxification, recovery, or energy drinks. The method features a precolumn derivatization step with a combination of BSTFA (N,O-bis(trimethylsilyl)trifluoroacetamide) and TMCS (trimethylchlorosilane) to silylate the analytes. The sample pretreatment required no extraction, filtration, or reduction step prior to the injection. The quantification of the analytes was performed using a five-point standard addition protocol. The proposed method presented excellent intraday precision (%RSD < 10) and linearity for GL calibration curves (correlation coefficients > 0.995) and acceptable linearity for DGuA calibration curves (correlation coefficients > 0.97). The estimated limits of detection (LOD) and quantification (LOQ) for GL ranged from 0.006 ppm to 0.14 ppm, and 0.02 ppm to 0.47 ppm, respectively. The estimated LOD and LOQ for DGuA determination ranged, respectively, from 0.06 ppm to 1.1 ppm and 0.2 ppm to 3.8 ppm. The results demonstrated that the method should be regarded as a reliable alternative to the simultaneous determination of GL and DGuA. PMID:25024704

  9. Eddy current modeling by finite element method for evaluation of mechanical properties of the structure cracked in absolute probe

    NASA Astrophysics Data System (ADS)

    Harzallah, Salaheddine; Chabaat, Mohamed; Belgacem, Fethi Bin Muhammad

    2014-12-01

    In this paper, a nondestructive evaluation by sensor Eddy current is used as a tool to control cracks and micro-cracks in materials. A simulation by a numerical approach based on the finite element method is employed to detect cracks in materials and eventually to study their propagation using a crucial parameter such as a Stress Intensity Factor (SIF). This method has emerged as one of the most efficient techniques for prospecting cracks in materials, evaluating SIFs and analyzing crack's growth in the context of linear elastic fracture mechanics (LEFM). This technique uses extrapolation of displacements from results compared with those obtained by the integral interaction. On the other hand, crack's growth is analyzed as a model by combining the maximum circumferential stress criteria with the critical plane for predicting the direction of crack growth. Moreover, a constant crack growth increment is determined using the modified Paris's model. Furthermore, stress intensity factors needed for these models are calculated using the domain form of the J-integral interactions.

  10. Aplisulfamines, new sulfoxide-containing metabolites from an aplidium tunicate: absolute stereochemistry at chiral sulfur and carbon atoms assigned through an original combination of spectroscopic and computational methods.

    PubMed

    Aiello, Anna; Fattorusso, Ernesto; Imperatore, Concetta; Luciano, Paolo; Menna, Marialuisa; Vitalone, Rocco

    2012-01-01

    Two new sulfoxide-containing metabolites, aplisulfamines A and B, have been isolated from an Aplidium sp. collected in the Bay of Naples. Their planar structure and geometry of a double bond were readily determined by using standard methods, mainly NMR spectroscopy. An original approach was used to assign the absolute configuration at the three contiguous chiral centers present in the structures of both aplisulfamines, two at carbon and one at sulfur. This involved Electronic Circular Dichroism (ECD) studies, J-based configuration analysis and Density Functional Theory (DFT) calculations and represents an interesting integration of modern techniques in stereoanalysis, which could contribute to the enhancement of theoretical protocols recently applied to solve stereochemical aspects in structure elucidation.

  11. Quantification of overlapping polygonal-shaped particles based on a new segmentation method of in situ images during crystallization

    NASA Astrophysics Data System (ADS)

    Ahmad, Ola Suleiman; Debayle, Johan; Gherras, Nesrine; Presles, Benoît; Févotte, Gilles; Pinoli, Jean-Charles

    2012-04-01

    Quantification of the overlapping particles in crystallization processes is very important for the quality control of chemical products or drugs. We present a method of segmentation of polygonal-shaped (i.e., rectangles, regular/irregular prisms) and overlapping particles from in situ images during a crystallization process for measuring their size distributions. The method is first based on detecting the geometric features of the particles identified by their salient corners. A clustering technique is then applied by grouping three correspondent salient corners belonging to the same particle. The proposed method is applied on particles of ammonium oxalate during batch crystallization in pure water. The particle size distributions are calculated, and a quantitative comparison between the proposed method and a manual sizing is performed. The method showed that it is valid for analyzing the crystal growth, and the results are promising for monitoring the particle size distribution.

  12. The 1H NMR method for the determination of the absolute configuration of 1,2,3-prim,sec,sec-triols.

    PubMed

    Lallana, Enrique; Freire, Félix; Seco, José Manuel; Quiñoa, Emilio; Riguera, Ricardo

    2006-09-28

    The absolute configuration of 1,2,3-prim,sec,sec-triols can be assigned by comparison of the 1H NMR spectra of the tris-(R)- and the tris-(S)-MPA ester derivatives. An experimental demonstration of this correlation with 24 triols of known absolute configuration and a protocol using two parameters-Deltadelta(RS)(H3) and the difference between Deltadelta RS (H2) and Deltadelta RS (H3) = absolute value (Delta(Deltadelta RS))-for its application to the determination of the absolute configuration of other triols are presented.

  13. Pseudo-absolute quantitative analysis using gas chromatography - Vacuum ultraviolet spectroscopy - A tutorial.

    PubMed

    Bai, Ling; Smuts, Jonathan; Walsh, Phillip; Qiu, Changling; McNair, Harold M; Schug, Kevin A

    2017-02-08

    The vacuum ultraviolet detector (VUV) is a new non-destructive mass sensitive detector for gas chromatography that continuously and rapidly collects full wavelength range absorption between 120 and 240 nm. In addition to conventional methods of quantification (internal and external standard), gas chromatography - vacuum ultraviolet spectroscopy has the potential for pseudo-absolute quantification of analytes based on pre-recorded cross sections (well-defined absorptivity across the 120-240 nm wavelength range recorded by the detector) without the need for traditional calibration. The pseudo-absolute method was used in this research to experimentally evaluate the sources of sample loss and gain associated with sample introduction into a typical gas chromatograph. Standard samples of benzene and natural gas were used to assess precision and accuracy for the analysis of liquid and gaseous samples, respectively, based on the amount of analyte loaded on-column. Results indicate that injection volume, split ratio, and sampling times for splitless analysis can all contribute to inaccurate, yet precise sample introduction. For instance, an autosampler can very reproducibly inject a designated volume, but there are significant systematic errors (here, a consistently larger volume than that designated) in the actual volume introduced. The pseudo-absolute quantification capability of the vacuum ultraviolet detector provides a new means for carrying out system performance checks and potentially for solving challenging quantitative analytical problems. For practical purposes, an internal standardized approach to normalize systematic errors can be used to perform quantitative analysis with the pseudo-absolute method.

  14. Simultaneous Quantification of Dexpanthenol and Resorcinol from Hair Care Formulation Using Liquid Chromatography: Method Development and Validation

    PubMed Central

    De, Amit Kumar; Chowdhury, Partha Pratim; Chattapadhyay, Shyamaprasad

    2016-01-01

    The current study presents the simultaneous quantification of dexpanthenol and resorcinol from marketed hair care formulation. Dexpanthenol is often present as an active ingredient in personal care products for its beautifying and invigorating properties and restorative and smoothing properties. On the other hand resorcinol is mainly prescribed for the treatment of seborrheic dermatitis of scalp. The toxic side effects of resorcinol limit its use in dermatological preparations. Therefore an accurate quantification technique for the simultaneous estimation of these two components can be helpful for the formulation industries for the accurate analysis of their product quality. In the current study a high performance liquid chromatographic technique has been developed using a C18 column and a mobile phase consisting of phosphate buffer of pH = 2.8 following a gradient elution. The mobile phase flow rate was 0.6 mL per minute and the detection wavelength was 210 nm for dexpanthenol and 280 nm for resorcinol. The linearity study was carried out using five solutions having concentrations ranging between 10.34 μg·mL−1 and 82.69 μg·mL−1 (r2 = 0.999) for resorcinol and 10.44 μg·mL−1 and 83.50 μg·mL−1 (r2 = 0.998) for dexpanthenol. The method has been validated as per ICH Q2(R1) guidelines. The ease of single step sample preparation, accuracy, and precision (intraday and interday) study presents the method suitable for the simultaneous quantification of dexpanthenol and resorcinol from any personal care product and dermatological preparations containing these two ingredients. PMID:27042377

  15. Simultaneous Quantification of Dexpanthenol and Resorcinol from Hair Care Formulation Using Liquid Chromatography: Method Development and Validation.

    PubMed

    De, Amit Kumar; Chowdhury, Partha Pratim; Chattapadhyay, Shyamaprasad

    2016-01-01

    The current study presents the simultaneous quantification of dexpanthenol and resorcinol from marketed hair care formulation. Dexpanthenol is often present as an active ingredient in personal care products for its beautifying and invigorating properties and restorative and smoothing properties. On the other hand resorcinol is mainly prescribed for the treatment of seborrheic dermatitis of scalp. The toxic side effects of resorcinol limit its use in dermatological preparations. Therefore an accurate quantification technique for the simultaneous estimation of these two components can be helpful for the formulation industries for the accurate analysis of their product quality. In the current study a high performance liquid chromatographic technique has been developed using a C18 column and a mobile phase consisting of phosphate buffer of pH = 2.8 following a gradient elution. The mobile phase flow rate was 0.6 mL per minute and the detection wavelength was 210 nm for dexpanthenol and 280 nm for resorcinol. The linearity study was carried out using five solutions having concentrations ranging between 10.34 μg·mL(-1) and 82.69 μg·mL(-1) (r (2) = 0.999) for resorcinol and 10.44 μg·mL(-1) and 83.50 μg·mL(-1) (r (2) = 0.998) for dexpanthenol. The method has been validated as per ICH Q2(R1) guidelines. The ease of single step sample preparation, accuracy, and precision (intraday and interday) study presents the method suitable for the simultaneous quantification of dexpanthenol and resorcinol from any personal care product and dermatological preparations containing these two ingredients.

  16. Separation, identification, quantification, and method validation of anthocyanins in botanical supplement raw materials by HPLC and HPLC-MS.

    PubMed

    Chandra, A; Rana, J; Li, Y

    2001-08-01

    A method has been established and validated for identification and quantification of individual, as well as total, anthocyanins by HPLC and LC/ES-MS in botanical raw materials used in the herbal supplement industry. The anthocyanins were separated and identified on the basis of their respective M(+) (cation) using LC/ES-MS. Separated anthocyanins were individually calculated against one commercially available anthocyanin external standard (cyanidin-3-glucoside chloride) and expressed as its equivalents. Amounts of each anthocyanin calculated as external standard equivalent were then multiplied by a molecular-weight correction factor to afford their specific quantities. Experimental procedures and use of a molecular-weight correction factors are substantiated and validated using Balaton tart cherry and elderberry as templates. Cyanidin-3-glucoside chloride has been widely used in the botanical industry to calculate total anthocyanins. In our studies on tart cherry and elderberry, its use as external standard followed by use of molecular-weight correction factors should provide relatively accurate results for total anthocyanins, because of the presence of cyanidin as their major anthocyanidin backbone. The method proposed here is simple and has a direct sample preparation procedure without any solid-phase extraction. It enables selection and use of commercially available anthocyanins as external standards for quantification of specific anthocyanins in the sample matrix irrespective of their commercial availability as analytical standards. It can be used as a template and applied for similar quantification in several anthocyanin-containing raw materials for routine quality control procedures, thus providing consistency in analytical testing of botanical raw materials used for manufacturing efficacious and true-to-the-label nutritional supplements.

  17. Spectrophotometric method for fast quantification of ascorbic acid and dehydroascorbic acid in simple matrix for kinetics measurements.

    PubMed

    Gómez Ruiz, Braulio; Roux, Stéphanie; Courtois, Francis; Bonazzi, Catherine

    2016-11-15

    A simple, rapid and reliable method was developed for quantifying ascorbic (AA) and dehydroascorbic (DHAA) acids and validated in 20mM malate buffer (pH 3.8). It consists in a spectrophotometric measurement of AA, either directly on the solution added with metaphosphoric acid or after reduction of DHAA into AA by dithiothreitol. This method was developed with real time measurement of reactions kinetics in bulk reactors in mind, and was checked in terms of linearity, limits of detection and quantification, fidelity and accuracy. The linearity was found satisfactory on the range of 0-6.95mM with limits of detection and quantification of 0.236mM and 0.467mM, respectively. The method was found acceptable in terms of fidelity and accuracy with a coefficient of variation for repeatability and reproducibility below 6% for AA and below 15% for DHAA, and with a recovery range of 97-102% for AA and 88-112% for DHAA.

  18. Development and validation of a reversed-phase HPLC method for the quantification of paclitaxel in different PLGA nanocarriers.

    PubMed

    Furman, Christophe; Carpentier, Rodolphe; Barczyk, Amélie; Chavatte, Philippe; Betbeder, Didier; Lipka, Emmanuelle

    2017-03-31

    A reversed-phase high-performance liquid chromatography (RP-HPLC) method has been developed and validated for the quantification of paclitaxel encapsulated in biodegradable poly(lactic-co-glycolic) (PLGA) copolymer nanoparticles. This simple (isocratic mode, without additive) and rapid (retention time of the paclitaxel under 4 minutes) methodology permits the detection of low quantities of paclitaxel in nanoparticulate formulations and the determination of the encapsulation efficiency (EE). Analysis was achieved on an octadecyl stationary phase. The isocratic mobile phase consisted of acetonitrile:water 80:20 (v/v) (flow rate = 0.8 mL/min). Stability of free paclitaxel was preliminary studied in those chromatographic conditions. The calibration curve was linear in the concentration range of 2 to 10 μg/mL (R(2) = 0.9994). The method was specific with valuable trueness, repeatability (intra-day precision) and intermediate precision (inter-day precision) based on relative standard deviation (RSD) values (less than 2%). The limits of detection (LOD) and quantification (LOQ) were 0.56 and 1.85 ng/mL respectively. This developed method was successfully employed for quantifying paclitaxel in PLGA 50:50 co-polymer nanoparticles. The accurate knowledge of the encapsulated paclitaxel concentration is essential to define the quantities of PLGA nanoparticles necessary to achieve the in vitro cell viability study. This article is protected by copyright. All rights reserved.

  19. A quantification method for determination of racemate praziquantel and R-enantiomer in rat plasma for comparison of their pharmacokinetics.

    PubMed

    Zhang, Danlu; Wang, Haina; Ji, Jianbo; Nie, Lei; Sun, Dequn

    2017-03-24

    Praziquantel is the drug of first choice for the control and treatment of all forms of schistosomiasis. Praziquantel is administered as a racemate, including R-enantiomer and S-enantiomer. Among them, R-enantiomer has main contribution to schistosomicidal activity. In this study, a sensitive and rapid liquid chromatography with tandem mass spectrometry was established and validated to determine the concentration of racemate praziquantel and R-enantiomer in rat plasma after oral administration. Chromatographic separation was performed on an Agilent Zorbax SB-C18 column. An entire run time for chromatographic separation was no more than 5min. The present method for analytes manifested that high sensitivity (the lower limit of quantification was 3.0ng/mL), satisfactory accuracy (relative error ≤±15%) and precision (relative standard deviation ≤15%) were achieved. There was no obvious matrix effect found. The average recoveries of racemate praziquantel and R-enantiomer were both above 85%. Then, the developed method had a successful application to comparative pharmacokinetic study of racemate praziquantel and R-enantiomer. Meanwhile, the differences in their pharmacokinetic parameters were compared and analyzed. The present quantification method and comparative pharmacokinetic study would provide a useful reference for the drug development of enantiopure schistosomicidal R-enantiomer as a replacement of racemate praziquantel for treatment of schistosomiasis.

  20. A novel quantification method of pantaprazole sodium monohydrate in sesquihydrate by thermogravimetric analyzer.

    PubMed

    Reddy, V Ranga; Rajmohan, M Anantha; Shilpa, R Laxmi; Raut, Dilip M; Naveenkumar, Kolla; Suryanarayana, M V; Mathad, Vijayavitthal T

    2007-04-11

    To demonstrate the applicability of thermogravimetric analyzer as a tool for the quantification of pantaprazole sodium monohydrate in sesquihydrate, studies have been conducted. Thermal analysis (DSC, TGA) crystallographic (PXRD) and spectroscopic techniques (FT-IR) were used for the characterization of the polymorphs. Thermogravimetric analysis (TGA) analysis was explored by high-resolution dynamic (Hi-Res-dynamic) and high-resolution modulated (Hi-Res-modulated) test procedures to quantify the hydrate polymorphic mixtures. The two polymorphic forms exhibited significant differences and good resolution in the second derivative thermogram generated by Hi-Res-modulated test procedure. Thus, the TGA with Hi-Res-modulated test procedure was considered for the quantification of monohydrate in sesquihydrate. The calibration plot was constructed from the known mixtures of two polymorphs by plotting the peak area of the second derivative thermogram against the weight percent of monohydrate. Using this novel approach, 1 wt% limit of detection (LOD) was achieved. The polymorphic purity results, obtained by TGA in Hi-Res-modulated test procedure were found to be in good agreement with the results predicted by FT-IR and was comparable with the actual values of the known polymorphic mixtures. The Hi-Res-modulated TGA technique is very simple and easy to perform the analysis.

  1. Method validation for detection and quantification of cocoa butter equivalents in cocoa butter and plain chocolate.

    PubMed

    Buchgraber, Manuela; Ulberth, Franz; Anklam, Elke

    2004-01-01

    A European interlaboratory study was conducted to validate an analytical procedure for the detection and quantification of cocoa butter equivalents in cocoa butter and plain chocolate. In principle, the fat obtained from plain chocolate according to the Soxhlet principle is separated by high-resolution capillary gas chromatography into triacylglycerol fractions according to their acyl-C-numbers, and within a given number, also according to unsaturation. The presence of cocoa butter equivalents is detected by linear regression analysis applied to the relative proportions of the 3 main triacylglycerol fractions of the fat analyzed. The amount of the cocoa butter equivalent admixture is estimated by partial least-squares regression analysis applied to the relative proportions of the 5 main triacylglycerols. Cocoa butter equivalent admixtures were detected down to a level of 2% related to the fat phase, corresponding to 0.6% in chocolate (assumed fat content of chocolate, 30%), without false-positive or -negative results. By using a quantification model based on partial least-squares regression analysis, the predicted cocoa butter equivalent amounts were in close agreement with the actual values. The applied model performed well at the level of the statutory limit of 5% cocoa butter equivalent addition to chocolate with a prediction error of 0.6%, assuming a chocolate fat content of 30%.

  2. Development and validation of a liquid chromatography-tandem mass spectrometry method for the quantification of opiorphin in human saliva.

    PubMed

    Brkljačić, Lidija; Sabalić, Maja; Salarić, Ivan; Jerić, Ivanka; Alajbeg, Ivan; Nemet, Ina

    2011-12-15

    Opiorphin, QRFSR-peptide, is a mature product of the PROL1 (proline rich, lacrimal 1) protein that showed beneficial effects in pain management, antidepressant-like actions as well as involvement in colonic motility and erectile physiology. Using opiorphin as a potential biomarker of different pathological states requires the development of robust and sensitive methods. We report a highly sensitive and specific liquid chromatography with tandem mass spectrometric detection (LC-MS/MS) analytical method for the analysis of opiorphin in human saliva. Quantification was based on multiple reaction monitoring using characteristic transitions (m/z 347/120 - as quantifying ion; 347/175 and 347/268 as qualifying ions). The assay was linear in the range of 0-110 ng/ml and the lower limit of quantification reached was 1.0 ng/ml. The intra-day precision and accuracy were between 2.7-5.6% and -2.3 to 3.2%, respectively. The inter-day precision and accuracy were between 10.8-13.7% and -11.0 to 52%, respectively. Mean recovery was 106% and mean matrix effect was 0.97. Opiorphin in TFA treated saliva samples was stable for at least 12h at room temperature and up to 30 days at -20°C. Opiorphin levels in human saliva samples collected from young healthy individuals ranged from 2.8 to 25.9 ng/ml.

  3. Use of the Relaxometry Technique for Quantification of Paramagnetic Ions in Aqueous Solutions and a Comparison with Other Analytical Methods

    PubMed Central

    Burato, Juliana Soares da Silva; Silva Lobo, Carlos Manuel; Colnago, Luiz Alberto

    2016-01-01

    We have demonstrated that the relaxometry technique is very efficient to quantify paramagnetic ions during in situ electrolysis measurements. Therefore, the goal of this work was to validate the relaxometry technique in the determination of the concentration of the ions contained in electrolytic solutions, Cu2+, Ni2+, Cr3+, and Mn2+, and compare it with other analytical methods. Two different NMR spectrometers were used: a commercial spectrometer with a homogeneous magnetic field and a home-built unilateral sensor with an inhomogeneous magnetic field. Without pretreatment, manganese ions do not have absorption bands in the UV-Visible region, but it is possible to quantify them using relaxometry (the limit of quantification is close to 10−5 mol L−1). Therefore, since the technique does not require chemical indicators and is a cheap and robust method, it can be used as a replacement for some conventional quantification techniques. The relaxometry technique could be applied to evaluate the corrosion of metallic surfaces. PMID:27293437

  4. Bioanalytical method development, validation and quantification of flupirtine maleate in rat plasma by liquid chromatography-tandem mass spectrometry.

    PubMed

    Kandasamy, Karthikeyan; Gowdra, Vasantharaju Surenahalli; Nammalvar, Hariprabhu; Govindarajan, Arul Kumaran Kottur S

    2011-01-01

    A simple, highly sensitive, precise and accurate high-performance liquid chromatographic (LCMSMS) method with mass detection was developed and validated for the rapid quantification of flupirtine (CAS 75507-68-5) in rat plasma samples. The chromatographic separation was achieved with a reverse phase column (4.6 x 50 mm, 5 microm) and the mobile phase consisted of cyanomethane and 5 mM ammonium formate buffer pH 4.5 (70:30 v/v) as eluent, at a flow rate of 0.6 mL/min. Labetalol (CAS 36894-69-6) was used as an internal standard. The effluence was ionized by positive electrospray ionization and measured by mass spectrometry. The retention times of flupirtine and labetalol were found to be 2.16 and 1.66 min respectively. The calibration curve was linear (r2 > or = 0.99) ranging from 0.98 to 1000 ng/ml and the lower limit of quantification was 0.98 ng/ mL. Inter-day and Intra-day precision were lower than 5% (CV) and accuracy ranged from 90 to 110% in terms of percent accuracy. Mean extraction recovery was found to be above 86.5%. The method was successfully applied for evaluation of the pharmacokinetic profile of flupirtine in male Sprague-Dawley rats and validated for excellent selectivity, accuracy, precision, recovery and stability.

  5. LV wall segmentation using the variational level set method (LSM) with additional shape constraint for oedema quantification

    NASA Astrophysics Data System (ADS)

    Kadir, K.; Gao, H.; Payne, A.; Soraghan, J.; Berry, C.

    2012-10-01

    In this paper an automatic algorithm for the left ventricle (LV) wall segmentation and oedema quantification from T2-weighted cardiac magnetic resonance (CMR) images is presented. The extent of myocardial oedema delineates the ischaemic area-at-risk (AAR) after myocardial infarction (MI). Since AAR can be used to estimate the amount of salvageable myocardial post-MI, oedema imaging has potential clinical utility in the management of acute MI patients. This paper presents a new scheme based on the variational level set method (LSM) with additional shape constraint for the segmentation of T2-weighted CMR image. In our approach, shape information of the myocardial wall is utilized to introduce a shape feature of the myocardial wall into the variational level set formulation. The performance of the method is tested using real CMR images (12 patients) and the results of the automatic system are compared to manual segmentation. The mean perpendicular distances between the automatic and manual LV wall boundaries are in the range of 1-2 mm. Bland-Altman analysis on LV wall area indicates there is no consistent bias as a function of LV wall area, with a mean bias of -121 mm2 between individual investigator one (IV1) and LSM, and -122 mm2 between individual investigator two (IV2) and LSM when compared to two investigators. Furthermore, the oedema quantification demonstrates good correlation when compared to an expert with an average error of 9.3% for 69 slices of short axis CMR image from 12 patients.

  6. Photometric method for the quantification of chlorophylls and their derivatives in complex mixtures: fitting with Gauss-peak spectra.

    PubMed

    Küpper, H; Spiller, M; Küpper, F C

    2000-11-15

    Accurate quantification of pigments in mixtures is essential in all cases in which separation of pigments by chromatography is impracticable for one reason or another. An example is the analysis of in vivo formation of heavy metal-substituted chlorophylls in heavy metal-stressed plants. We describe here a novel, accurate UV/VIS spectrophotometric method for the quantification of individual chlorophyll derivatives in complex mixtures, which has the potential for universal applicability for mixtures difficult to separate. The method is based on the description of each pigment spectrum by a series of Gaussian peaks. A sample spectrum is then fitted by a linear combination of these "Gauss-peak spectra" including an automatic correction of wavelength inaccuracy and baseline instability of the spectrometer as well as a correction of the widening of absorbance peaks in more concentrated pigment solutions. The automatic correction of peak shifts can also partially correct shifts caused by processes like allomerization. In this paper, we present the Gauss-peak spectra for Mg-chlorophyll a, b, c, pheophytin a, b, c, Cu-chlorophyll a, b, c, and Zn-chlorophyll a in acetone; Mg-chlorophyll a, b, pheophytin a, b, Cu-chlorophyll a, b, allomerized Cu-chlorophyll a, b, and Zn-chlorophyll a, b in cyclohexane; Mg-chlorophyll a, b, pheophytin a, b, and Cu-chlorophyll a, b in diethyl ether.

  7. Quantification of fibronectin as a method to assess ex vivo extracellular matrix remodeling.

    PubMed

    Bager, C L; Gudmann, N; Willumsen, N; Leeming, D J; Karsdal, M A; Bay-Jensen, A C; Høgdall, E; Balslev, I; He, Y

    2016-09-16

    Altered architecture, composition and quality of the extracellular matrix (ECM) are pathological hallmarks of several inflammatory and fibro-proliferative pathological processes such as osteoarthritis (OA), rheumatoid arthritis (RA), fibrosis and cancer. One of the most important components of the ECM is fibronectin. Fibronectin serves as an adhesion molecule anchoring cells to the underlying basement membrane through direct interaction with integrin receptors. Fibronectin hereby modulates the properties of the ECM and affects cellular processes. Quantification of fibronectin remodeling could therefore be used to assess the changes in the ECM that occur during progression of fibro-proliferative pathologies. Ex vivo models are becoming state-of-the-art tools to study ECM remodeling as the cellular composition and the organization of the ECM are preserved. Ex vivo models may therefore be a valuable tool to study the ECM remodeling that occurs during progression of fibro-proliferative pathologies. The aim of this study was to quantify fibronectin remodeling in ex vivo models of cartilage and cancer. A competitive The enzyme-linked immunosorbent assay (ELISA) against the C-terminus of fibronectin was developed (FBN-C). The assay was evaluated in relation to specificity, technical performance and as a marker for quantification of fibronectin in cartilage and cancer ex vivo models. The ELISA was specific and technically stable. Cleavage of tumor tissue with MMP-2 released significantly higher levels of FBN-C compared to tissue with buffer only and western blot analysis revealed that FBN-C recognizes both full length and degraded fibronectin. When ex vivo cartilage cultures were stimulated with the anabolic factor TGFβ and catabolic factors TNF-α and OSM, significantly higher levels of FBN-C were found in the conditioned media. Lastly, FBN-C was released from a cancer ex vivo model. In conclusion, we were able to quantify fibronectin remodeling in ex vivo models

  8. P197-M Automated Ion Selection and Method Building For MRM-Based Protein Validation And Quantification

    PubMed Central

    Duchoslav, E.; Cox, D.; Tate, S. A.

    2007-01-01

    A number of successful multiple reaction monitoring (MRM)-directed MS/MS methods for protein characterization and quantification have been published over the past couple of years. These methods have been created either by the generation of in silico MRM transitions or from taking discovery data generated by automated MS/MS analysis and formulating MRM transitions from this. However, such a workflow, where a vast amount of discovery data needs to be searched for idealized MS/MS to target peptides, is difficult and time consuming. Automation of this would simplify the whole workflow, and allowing methods to be created smarter by utilizing the data of peptides that have been previously identified, provides a better chance of success. In this poster we highlight one possible workflow for the processing of a number of database search results that were generated during proteomic analysis of a complex sample. Proteins identified as of potential interest in these results were then added to a MRM method-building list, and all MS/MS associated with those proteins were extracted from the general proteomic search results. These MS/MS are then automatically processed to create instrument methods for MRM-directed MS/MS quantification. After analysis of the sample by these methods, the data are processed, both from a sequence identification and a chromatographic LC-MRM peak signal-to-noise perspective. These data are associated with the original proteomic workflow data, allowing visualization of the results and construction of a method that can then undergo validation.

  9. A quantification method for heat-decomposable methylglyoxal oligomers and its application on 1,3,5-trimethylbenzene SOA

    NASA Astrophysics Data System (ADS)

    Rodigast, Maria; Mutzel, Anke; Herrmann, Hartmut

    2017-03-01

    Methylglyoxal forms oligomeric compounds in the atmospheric aqueous particle phase, which could establish a significant contribution to the formation of aqueous secondary organic aerosol (aqSOA). Thus far, no suitable method for the quantification of methylglyoxal oligomers is available despite the great effort spent for structure elucidation. In the present study a simplified method was developed to quantify heat-decomposable methylglyoxal oligomers as a sum parameter. The method is based on the thermal decomposition of oligomers into methylglyoxal monomers. Formed methylglyoxal monomers were detected using PFBHA (o-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine hydrochloride) derivatisation and gas chromatography-mass spectrometry (GC/MS) analysis. The method development was focused on the heating time (varied between 15 and 48 h), pH during the heating process (pH = 1-7), and heating temperature (50, 100 °C). The optimised values of these method parameters are presented. The developed method was applied to quantify heat-decomposable methylglyoxal oligomers formed during the OH-radical oxidation of 1,3,5-trimethylbenzene (TMB) in the Leipzig aerosol chamber (LEipziger AerosolKammer, LEAK). Oligomer formation was investigated as a function of seed particle acidity and relative humidity. A fraction of heat-decomposable methylglyoxal oligomers of up to 8 % in the produced organic particle mass was found, highlighting the importance of those oligomers formed solely by methylglyoxal for SOA formation. Overall, the present study provides a new and suitable method for quantification of heat-decomposable methylglyoxal oligomers in the aqueous particle phase.

  10. Comparing paired vs non-paired statistical methods of analyses when making inferences about absolute risk reductions in propensity-score matched samples.

    PubMed

    Austin, Peter C

    2011-05-20

    Propensity-score matching allows one to reduce the effects of treatment-selection bias or confounding when estimating the effects of treatments when using observational data. Some authors have suggested that methods of inference appropriate for independent samples can be used for assessing the statistical significance of treatment effects when using propensity-score matching. Indeed, many authors in the applied medical literature use methods for independent samples when making inferences about treatment effects using propensity-score matched samples. Dichotomous outcomes are common in healthcare research. In this study, we used Monte Carlo simulations to examine the effect on inferences about risk differences (or absolute risk reductions) when statistical methods for independent samples are used compared with when statistical methods for paired samples are used in propensity-score matched samples. We found that compared with using methods for independent samples, the use of methods for paired samples resulted in: (i) empirical type I error rates that were closer to the advertised rate; (ii) empirical coverage rates of 95 per cent confidence intervals that were closer to the advertised rate; (iii) narrower 95 per cent confidence intervals; and (iv) estimated standard errors that more closely reflected the sampling variability of the estimated risk difference. Differences between the empirical and advertised performance of methods for independent samples were greater when the treatment-selection process was stronger compared with when treatment-selection process was weaker. We recommend using statistical methods for paired samples when using propensity-score matched samples for making inferences on the effect of treatment on the reduction in the probability of an event occurring.

  11. Comprehensive identification and quantification of microbial transcriptomes by genome-wide unbiased methods.

    PubMed

    Mäder, Ulrike; Nicolas, Pierre; Richard, Hugues; Bessières, Philippe; Aymerich, Stéphane

    2011-02-01

    Genomic tiling array transcriptomics and RNA-seq are two powerful and rapidly developing approaches for unbiased transcriptome analysis. Providing comprehensive identification and quantification of transcripts with an unprecedented resolution, they are leading to major breakthroughs in systems biology. Here we review each step of the analysis from library preparation to the interpretation of the data, with particular attention paid to the possible sources of artifacts. Methodological requirements and statistical frameworks are often similar in both the approaches despite differences in the nature of the data. Tiling array analysis does not require rRNA depletion and benefits from a more mature computational workflow, whereas RNA-Seq has a clear lead in terms of background noise and dynamic range with a considerable potential for evolution with the improvements of sequencing technologies. Being independent of prior sequence knowledge, RNA-seq will boost metatranscriptomics and evolutionary transcriptomics applications.

  12. Development and validation of high-throughput liquid chromatography-tandem mass spectrometric method for simultaneous quantification of loratadine and desloratadine in human plasma.

    PubMed

    Srinubabu, G; Patel, Rajaram S; Shedbalkar, Vinay P; Rao, Allam Appa; Rao, M Narasimha; Bandaru, Veera Venkata Ratnam

    2007-12-15

    As a continuation of effort to improve our high flow on-line bioanalytical approach for high-throughput quantification of drugs and metabolites in plasma by high-throughput liquid chromatography tandem mass spectrometry (HTLC-MS/MS), we have developed a simple, sensitive and reliable method for simultaneous quantification of loratadine and desloratadine in human plasma. We have performed on-line coupling of extraction with Cyclone P 50 mm x 0.5 mm 50 microm HTLC column and chromatographic separation is performed with Zorbax XDB C18 50 mm x 2.1 mm 5 microm, followed by quantification with mass detector. The method is validated and showed good performances in terms of linearity, sensitivity, precision, accuracy and stability. A marked improvement in sample throughput efficiency is realized with this method and the proposed method will be useful for pharmacokinetic and/or bioequivalence studies.

  13. An Improved Method for Discriminating ECG Signals using Typical Nonlinear Dynamic Parameters and Recurrence Quantification Analysis in Cardiac Disease Therapy.

    PubMed

    Tang, M; Chang, C Q; Fung, P C W; Chau, K T; Chan, F H Y

    2005-01-01

    The discrimination of ECG signals using nonlinear dynamic parameters is of crucial importance in the cardiac disease therapy and chaos control for arrhythmia defibrillation in the cardiac system. However, the discrimination results of previous studies using features such as maximal Lyapunov exponent (λmax) and correlation dimension (D2) alone are somewhat limited in recognition rate. In this paper, improved methods for computing λmaxand D2are purposed. Another parameter from recurrence quantification analysis is incorporated to the new multi-feature Bayesian classifier with λmaxand D2so as to improve the discrimination power. Experimental results have verified the prediction using Fisher discriminant that the maximal vertical line length (Vmax) from recurrence quantification analysis is the best to distinguish different ECG classes. Experimental results using the MIT-BIH Arrhythmia Database show improved and excellent overall accuracy (96.3%), average sensitivity (96.3%) and average specificity (98.15%) for discriminating sinus, premature ventricular contraction and ventricular flutter signals.

  14. High-performance liquid chromatographic method for the quantification of Mitragyna inermis alkaloids in order to perform pharmacokinetic studies.

    PubMed

    Sinou, Veronique; Fiot, Julien; Taudon, Nicolas; Mosnier, Joël; Martelloni, Maryse; Bun, Sok S; Parzy, Daniel; Ollivier, Evelyne

    2010-06-01

    In Africa, Mitragyna inermis (Willd.) O. Kuntze (Rubiaceae) is commonly used in traditional medicine to treat malaria. Antimalarial activity is mostly due to the hydromethanolic extract of M. inermis leaves and especially to the main alkaloids, uncarine D and isorhynchophilline. In the present study, we describe for the first time an HPLC method for the simultaneous quantification of uncarine D and isorhynchophylline in biological matrices. SPE was used to extract the components and the internal standard naphthalene from human and pig plasma samples. Chromatographic separation was performed on a C-18 reversed column at a flow rate of 1 mL/min, using methanol-phosphate buffer (10:90, pH 7), as a mobile phase. Good linearity was observed over the concentration ranges of 0.0662-3.31 microg/mL for uncarine D and 0.0476-2.38 microg/mL for isorynchophylline. The precision was less than 12% and the accuracy was from 86 to 107% without any discrepancy between the two species. Uncarine D and isorhynchophylline recoveries were over 80%. These results allowed the quantification of both uncarine D and isorhynchophylline in pig plasma after intravenous administration of M. inermis extract.

  15. HPLC method for the quantification of procyanidins in cocoa and chocolate samples and correlation to total antioxidant capacity.

    PubMed

    Adamson, G E; Lazarus, S A; Mitchell, A E; Prior, R L; Cao, G; Jacobs, P H; Kremers, B G; Hammerstone, J F; Rucker, R B; Ritter, K A; Schmitz, H H

    1999-10-01

    Monomeric and oligomeric procyanidins present in cocoa liquors and chocolates were separated and quantified in four different laboratories using a normal-phase high-performance liquid chromatography (HPLC) method with fluorescence detection. Procyanidin standards through decamers were obtained by extraction from cocoa beans, enrichment by Sephadex LH-20 gel permeation chromatography, and final purification by preparative normal-phase HPLC. The purity of each oligomeric fraction was assessed using HPLC coupled to mass spectrometry. A composite standard was then prepared, and calibration curves were generated for each oligomeric class using a quadratic fit of area sum versus concentration. Results obtained by each of the laboratories were in close agreement, which suggests this method is reliable and reproducible for quantification of procyanidins. Furthermore, the procyanidin content of the samples was correlated to the antioxidant capacity measured using the ORAC assay as an indicator for potential biological activity.

  16. Quantification Bias Caused by Plasmid DNA Conformation in Quantitative Real-Time PCR Assay

    PubMed Central

    Lin, Chih-Hui; Chen, Yu-Chieh; Pan, Tzu-Ming

    2011-01-01

    Quantitative real-time PCR (qPCR) is the gold standard for the quantification of specific nucleic acid sequences. However, a serious concern has been revealed in a recent report: supercoiled plasmid standards cause significant over-estimation in qPCR quantification. In this study, we investigated the effect of plasmid DNA conformation on the quantification of DNA and the efficiency of qPCR. Our results suggest that plasmid DNA conformation has significant impact on the accuracy of absolute quantification by qPCR. DNA standard curves shifted significantly among plasmid standards with different DNA conformations. Moreover, the choice of DNA measurement method and plasmid DNA conformation may also contribute to the measurement error of DNA standard curves. Due to the multiple effects of plasmid DNA conformation on the accuracy of qPCR, efforts should be made to assure the highest consistency of plasmid standards for qPCR. Thus, we suggest that the conformation, preparation, quantification, purification, handling, and storage of standard plasmid DNA should be described and defined in the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) to assure the reproducibility and accuracy of qPCR absolute quantification. PMID:22194997

  17. A stochastic collocation method for uncertainty quantification and propagation in cardiovascular simulations.

    PubMed

    Sankaran, Sethuraman; Marsden, Alison L

    2011-03-01

    Simulations of blood flow in both healthy and diseased vascular models can be used to compute a range of hemodynamic parameters including velocities, time varying wall shear stress, pressure drops, and energy losses. The confidence in the data output from cardiovascular simulations depends directly on our level of certainty in simulation input parameters. In this work, we develop a general set of tools to evaluate the sensitivity of output parameters to input uncertainties in cardiovascular simulations. Uncertainties can arise from boundary conditions, geometrical parameters, or clinical data. These uncertainties result in a range of possible outputs which are quantified using probability density functions (PDFs). The objective is to systemically model the input uncertainties and quantify the confidence in the output of hemodynamic simulations. Input uncertainties are quantified and mapped to the stochastic space using the stochastic collocation technique. We develop an adaptive collocation algorithm for Gauss-Lobatto-Chebyshev grid points that significantly reduces computational cost. This analysis is performed on two idealized problems--an abdominal aortic aneurysm and a carotid artery bifurcation, and one patient specific problem--a Fontan procedure for congenital heart defects. In each case, relevant hemodynamic features are extracted and their uncertainty is quantified. Uncertainty quantification of the hemodynamic simulations is done using (a) stochastic space representations, (b) PDFs, and (c) the confidence intervals for a specified level of confidence in each problem.

  18. New methods and results for quantification of lightning-aircraft electrodynamics

    NASA Technical Reports Server (NTRS)

    Pitts, Felix L.; Lee, Larry D.; Perala, Rodney A.; Rudolph, Terence H.

    1987-01-01

    The NASA F-106 collected data on the rates of change of electromagnetic parameters on the aircraft surface during over 700 direct lightning strikes while penetrating thunderstorms at altitudes from 15,000 t0 40,000 ft (4,570 to 12,190 m). These in situ measurements provided the basis for the first statistical quantification of the lightning electromagnetic threat to aircraft appropriate for determining indirect lightning effects on aircraft. These data are used to update previous lightning criteria and standards developed over the years from ground-based measurements. The proposed standards will be the first which reflect actual aircraft responses measured at flight altitudes. Nonparametric maximum likelihood estimates of the distribution of the peak electromagnetic rates of change for consideration in the new standards are obtained based on peak recorder data for multiple-strike flights. The linear and nonlinear modeling techniques developed provide means to interpret and understand the direct-strike electromagnetic data acquired on the F-106. The reasonable results obtained with the models, compared with measured responses, provide increased confidence that the models may be credibly applied to other aircraft.

  19. Quantification of intraoral pressures during nutritive sucking: methods with normal infants.

    PubMed

    Lang, William Christopher; Buist, Neil R M; Geary, Annmarie; Buckley, Scott; Adams, Elizabeth; Jones, Albyn C; Gorsek, Stephen; Winter, Susan C; Tran, Hanh; Rogers, Brian R

    2011-09-01

    We report quantitative measurements of ten parameters of nutritive sucking behavior in 91 normal full-term infants obtained using a novel device (an Orometer) and a data collection/analytical system (Suck Editor). The sucking parameters assessed include the number of sucks, mean pressure amplitude of sucks, mean frequency of sucks per second, mean suck interval in seconds, sucking amplitude variability, suck interval variability, number of suck bursts, mean number of sucks per suck burst, mean suck burst duration, and mean interburst gap duration. For analyses, test sessions were divided into 4 × 2-min segments. In single-study tests, 36 of 60 possible comparisons of ten parameters over six pairs of 2-min time intervals showed a p value of 0.05 or less. In 15 paired tests in the same infants at different ages, 33 of 50 possible comparisons of ten parameters over five time intervals showed p values of 0.05 or less. Quantification of nutritive sucking is feasible, showing statistically valid results for ten parameters that change during a feed and with age. These findings suggest that further research, based on our approach, may show clinical value in feeding assessment, diagnosis, and clinical management.

  20. Validation of an LC-MS/MS method for the quantification of mycophenolic acid in human kidney transplant biopsies.

    PubMed

    Md Dom, Zaipul I; Noll, Benjamin D; Coller, Janet K; Somogyi, Andrew A; Russ, Graeme R; Hesselink, Dennis A; van Gelder, Teun; Sallustio, Benedetta C

    2014-01-15

    Mycophenolic acid (MPA) has a low therapeutic index and large inter-individual pharmacokinetic variability necessitating therapeutic drug monitoring to individualise dosing after transplantation. There is an ongoing discrepancy as to whether plasma MPA concentrations sufficiently predict kidney rejection or toxicity and whether immunosuppressant concentrations within the graft tissue may better predict transplant outcomes. The aim of the study was to develop an LC-MS/MS method for the quantification of MPA concentrations in human kidney biopsies taken as part of routine clinical procedures. A total of 4 surplus human kidney biopsies obtained from 4 different kidney transplant recipients were available to use for this study. MPA was also quantified in 2 kidney samples from rats administered MPA to assess tissue extraction reproducibility. Human kidney biopsies and rat kidneys were homogenised mechanically and underwent liquid-liquid extraction before analysis by LC-MS/MS. MPA-free human kidney tissue was used in calibrators and quality control samples. Analyte detection was achieved from multiple reaction monitoring of the ammonium adducts of both MPA (m/z 321.1→207.3) and N-phthaloyl-l-phenylalanine (PPA, internal standard, m/z 296.2→250.2) using positive electrospray ionisation. The method was linear (calibration curves R(2)>0.99, n=10), precise, and accurate with coefficients of variation and bias less than 15%. Extraction efficiencies for MPA and PPA were approximately 97% and 86%, respectively, and matrix effects were minimal. In 4 kidney transplant recipients, tissue MPA concentrations ranged from 1.3 to 7.7ng/mg of tissue, however, the correlation between blood (C0) and tissue MPA concentrations could not be established. The method was successfully applied to the quantification of MPA in human kidney biopsies without the need to alter current clinical protocols.

  1. A rapid, reproducible, on-the-fly orthogonal array optimization method for targeted protein quantification by LC/MS and its application for accurate and sensitive quantification of carbonyl reductases in human liver.

    PubMed

    Cao, Jin; Gonzalez-Covarrubias, Vanessa; Covarrubias, Vanessa M; Straubinger, Robert M; Wang, Hao; Duan, Xiaotao; Yu, Haoying; Qu, Jun; Blanco, Javier G

    2010-04-01

    Liquid chromatography (LC)/mass spectrometry (MS) in selected-reactions-monitoring (SRM) mode provides a powerful tool for targeted protein quantification. However, efficient, high-throughput strategies for proper selection of signature peptides (SP) for protein quantification and accurate optimization of their SRM conditions remain elusive. Here we describe an on-the-fly, orthogonal array optimization (OAO) approach that enables rapid, comprehensive, and reproducible SRM optimization of a large number of candidate peptides in a single nanoflow-LC/MS run. With the optimized conditions, many peptide candidates can be evaluated in biological matrixes for selection of the final SP. The OAO strategy employs a systematic experimental design that strategically varies product ions, declustering energy, and collision energy in a cycle of 25 consecutive SRM trials, which accurately reveals the effects of these factors on the signal-to-noise ratio of a candidate peptide and optimizes each. As proof of concept, we developed a highly sensitive, accurate, and reproducible method for the quantification of carbonyl reductases CBR1 and CBR3 in human liver. Candidate peptides were identified by nano-LC/LTQ/Orbitrap, filtered using a stringent set of criteria, and subjected to OAO. After evaluating both sensitivity and stability of the candidates, two SP were selected for quantification of each protein. As a result of the accurate OAO of assay conditions, sensitivities of 80 and 110 amol were achieved for CBR1 and CBR3, respectively. The method was validated and used to quantify the CBRs in 33 human liver samples. The mean level of CBR1 was 93.4 +/- 49.7 (range: 26.2-241) ppm of total protein, and of CBR3 was 7.69 +/- 4.38 (range: 1.26-17.9) ppm. Key observations of this study: (i) evaluation of peptide stability in the target matrix is essential for final selection of the SP; (ii) utilization of two unique SP contributes to high reliability of target protein quantification; (iii

  2. Absolute and relative blindsight.

    PubMed

    Balsdon, Tarryn; Azzopardi, Paul

    2015-03-01

    The concept of relative blindsight, referring to a difference in conscious awareness between conditions otherwise matched for performance, was introduced by Lau and Passingham (2006) as a way of identifying the neural correlates of consciousness (NCC) in fMRI experiments. By analogy, absolute blindsight refers to a difference between performance and awareness regardless of whether it is possible to match performance across conditions. Here, we address the question of whether relative and absolute blindsight in normal observers can be accounted for by response bias. In our replication of Lau and Passingham's experiment, the relative blindsight effect was abolished when performance was assessed by means of a bias-free 2AFC task or when the criterion for awareness was varied. Furthermore, there was no evidence of either relative or absolute blindsight when both performance and awareness were assessed with bias-free measures derived from confidence ratings using signal detection theory. This suggests that both relative and absolute blindsight in normal observers amount to no more than variations in response bias in the assessment of performance and awareness. Consideration of the properties of psychometric functions reveals a number of ways in which relative and absolute blindsight could arise trivially and elucidates a basis for the distinction between Type 1 and Type 2 blindsight.

  3. Lower and upper bounds for the absolute free energy by the hypothetical scanning Monte Carlo method: application to liquid argon and water.

    PubMed

    White, Ronald P; Meirovitch, Hagai

    2004-12-08

    The hypothetical scanning (HS) method is a general approach for calculating the absolute entropy S and free energy F by analyzing Boltzmann samples obtained by Monte Carlo or molecular dynamics techniques. With HS applied to a fluid, each configuration i of the sample is reconstructed by gradually placing the molecules in their positions at i using transition probabilities (TPs). At each step of the process the system is divided into two parts, the already treated molecules (the "past"), which are fixed, and the as yet unspecified (mobile) "future" molecules. Obtaining the TP exactly requires calculating partition functions over all positions of the future molecules in the presence of the frozen past, thus it is customary to invoke various approximations to best represent these quantities. In a recent publication [Proc. Natl. Acad. Sci. USA 101, 9235 (2004)] we developed a version of HS called complete HSMC, where each TP is calculated from an MC simulation involving all of the future molecules (the complete future); the method was applied very successfully to Lennard-Jones systems (liquid argon) and a box of TIP3P water molecules. In its basic implementation the method provides lower and upper bounds for F, where the latter can be evaluated only for relatively small systems. Here we introduce a new expression for an upper bound, which can be evaluated for larger systems. We also propose a new exact expression for F and verify its effectiveness. These free energy functionals lead to significantly improved accuracy (as applied to the liquid systems above) which is comparable to our thermodynamic integration results. We formalize and discuss theoretical aspects of HSMC that have not been addressed in previous studies. Additionally, several functionals are developed and shown to provide the free energy through the analysis of a single configuration.

  4. Quantification of 4'-geranyloxyferulic acid (GOFA) in honey samples of different origin by validated RP-HPLC-UV method.

    PubMed

    Genovese, Salvatore; Taddeo, Vito Alessandro; Fiorito, Serena; Epifano, Francesco

    2016-01-05

    Natural honey has been employed as a nutraceutical agent with benefits and therapeutic promises for humans for many centuries. It has been largely used as food and medicine by all generations, traditions, and civilizations, both ancient and modern. Several chemicals having beneficial effects for human health have been reported as components of natural honey and these include sugars, organic acids, aminoacids, minerals, and vitamins. Also some important phytochemicals have been described and these comprise tannins, flavonoids, terpenes, saponins, and alkaloids. In this note it is described the successful application of a RP HPLC-UV-vis method for the separation and quantification of 4'-geranyloxyferulic acid (GOFA) in four honey samples of different origin. Concentration values showed a great variation between the four samples tested, being chestnut honey the one richest in GOFA (7.87 mg/g). The findings described herein represent the first example reported in the literature of the characterization of an oxyprenylated phenylpropanoid in honey.

  5. Development and validation of LC-MS methods for peptaibol quantification in fungal extracts according to their lengths.

    PubMed

    Van Bohemen, Anne-Isaline; Zalouk-Vergnoux, Aurore; Poirier, Laurence; Phuong, Nam Ngoc; Inguimbert, Nicolas; Ben Haj Salah, Khoubaib; Ruiz, Nicolas; Pouchus, Yves François

    2016-01-15

    Some terrestrial Trichoderma sp. strains are already used as biological control agents (BCAs). They all produce peptaibols, small antimicrobial peptides which are supposed to play a role in the anti-phytopathogenic activity of Trichoderma sp. Trichoderma strains producing high amounts of peptaibols could represent new potential BCAs. In this context, marine-derived Trichoderma strains from the marine fungal strain collection of the "Mer, Molécules, Santé" (MMS) laboratory were investigated for their peptaibol production. Previously, the quantification of peptaibols was performed using alamethicin, as standard (20-amino acid residues peptaibol). In this study, the development and validation of quantification LC/ESI-TI-MS methods using different standards of peptaibols (11-, 14- and 20-amino acid residues) was performed in order to quantify all of them, in a single analysis, in Trichoderma crude extracts according to their chain length. The developed and validated methods were used to study the peptaibol production kinetic of a marine-derived Trichoderma strain, i.e., Trichoderma longibrachiatum (MMS 151). The results showed the optimal culture time at the 9th day with concentrations reaching 1.4±0.2% and 2.3±0.4% of the fungal biomass respectively for 11- and 20-residue peptaibols. Then, the different peptaibol subgroups produced by 13 Trichoderma strains were quantified. According to their 18-, 19- and 20-residue peptaibol production, three strains referenced as MMS 1541, MMS 639 and MMS 151 seemed to be good candidates as potential new biological control agents with respective production of 0.4, 0.4 and 2.1%.

  6. Normalized Tritium Quantification Approach (NoTQA) a Method for Quantifying Tritium Contaminated Trash and Debris at LLNL

    SciTech Connect

    Dominick, J L; Rasmussen, C L

    2008-07-23

    Several facilities and many projects at LLNL work exclusively with tritium. These operations have the potential to generate large quantities of Low-Level Radioactive Waste (LLW) with the same or similar radiological characteristics. A standardized documented approach to characterizing these waste materials for disposal as radioactive waste will enhance the ability of the Laboratory to manage them in an efficient and timely manner while ensuring compliance with all applicable regulatory requirements. This standardized characterization approach couples documented process knowledge with analytical verification and is very conservative, overestimating the radioactivity concentration of the waste. The characterization approach documented here is the Normalized Tritium Quantification Approach (NoTQA). This document will serve as a Technical Basis Document which can be referenced in radioactive waste characterization documentation packages such as the Information Gathering Document. In general, radiological characterization of waste consists of both developing an isotopic breakdown (distribution) of radionuclides contaminating the waste and using an appropriate method to quantify the radionuclides in the waste. Characterization approaches require varying degrees of rigor depending upon the radionuclides contaminating the waste and the concentration of the radionuclide contaminants as related to regulatory thresholds. Generally, as activity levels in the waste approach a regulatory or disposal facility threshold the degree of required precision and accuracy, and therefore the level of rigor, increases. In the case of tritium, thresholds of concern for control, contamination, transportation, and waste acceptance are relatively high. Due to the benign nature of tritium and the resulting higher regulatory thresholds, this less rigorous yet conservative characterization approach is appropriate. The scope of this document is to define an appropriate and acceptable

  7. Quantification of carbon nanotubes in different environmental matrices by a microwave induced heating method.

    PubMed

    He, Yang; Al-Abed, Souhail R; Dionysiou, Dionysios D

    2017-02-15

    Carbon nanotubes (CNTs) have been incorporated into numerous consumer products, and have also been employed in various industrial areas because of their extraordinary properties. The large scale production and wide applications of CNTs make their release into the environment a major concern. Therefore, it is crucial to determine the degree of potential CNT contamination in the environment, which requires a sensitive and accurate technique for selectively detecting and quantifying CNTs in environmental matrices. In this study, a simple device based on utilizing heat generated/temperature increase from CNTs under microwave irradiation was built to quantify single-walled CNTs (SWCNTs), multi-walled CNTs (MWCNTs) and carboxylated CNTs (MWCNT-COOH) in three environmentally relevant matrices (sand, soil and sludge). Linear temperature vs CNT mass relationships were developed for the three environmental matrices spiked with known amounts of different types of CNTs that were then irradiated in a microwave at low energies (70-149W) for a short time (15-30s). MWCNTs had a greater microwave response in terms of heat generated/temperature increase than SWCNTs and MWCNT-COOH. An evaluation of microwave behavior of different carbonaceous materials showed that the microwave measurements of CNTs were not affected even with an excess of other organic, inorganic carbon or carbon based nanomaterials (fullerene, granular activated carbon and graphene oxide), mainly because microwave selectively heats materials such as CNTs that have a higher dielectric loss factor. Quantification limits using this technique for the sand, soil and sludge were determined as low as 18.61, 27.92, 814.4μg/g for MWCNTs at a microwave power of 133W and exposure time of 15s.

  8. Absolute neutrino mass scale

    NASA Astrophysics Data System (ADS)

    Capelli, Silvia; Di Bari, Pasquale

    2013-04-01

    Neutrino oscillation experiments firmly established non-vanishing neutrino masses, a result that can be regarded as a strong motivation to extend the Standard Model. In spite of being the lightest massive particles, neutrinos likely represent an important bridge to new physics at very high energies and offer new opportunities to address some of the current cosmological puzzles, such as the matter-antimatter asymmetry of the Universe and Dark Matter. In this context, the determination of the absolute neutrino mass scale is a key issue within modern High Energy Physics. The talks in this parallel session well describe the current exciting experimental activity aiming to determining the absolute neutrino mass scale and offer an overview of a few models beyond the Standard Model that have been proposed in order to explain the neutrino masses giving a prediction for the absolute neutrino mass scale and solving the cosmological puzzles.

  9. Validation of a fast and accurate chromatographic method for detailed quantification of vitamin E in green leafy vegetables.

    PubMed

    Cruz, Rebeca; Casal, Susana

    2013-11-15

    Vitamin E analysis in green vegetables is performed by an array of different methods, making it difficult to compare published data or choosing the adequate one for a particular sample. Aiming to achieve a consistent method with wide applicability, the current study reports the development and validation of a fast micro-method for quantification of vitamin E in green leafy vegetables. The methodology uses solid-liquid extraction based on the Folch method, with tocol as internal standard, and normal-phase HPLC with fluorescence detection. A large linear working range was confirmed, being highly reproducible, with inter-day precisions below 5% (RSD). Method sensitivity was established (below 0.02 μg/g fresh weight), and accuracy was assessed by recovery tests (>96%). The method was tested in different green leafy vegetables, evidencing diverse tocochromanol profiles, with variable ratios and amounts of α- and γ-tocopherol, and other minor compounds. The methodology is adequate for routine analyses, with a reduced chromatographic run (<7 min) and organic solvent consumption, and requires only standard chromatographic equipment available in most laboratories.

  10. A single reversed-phase UPLC method for quantification of levofloxacin in aqueous humour and pharmaceutical dosage forms.

    PubMed

    Gupta, Himanshu; Aqil, M; Khar, R K; Ali, Asgar; Chander, Prakash

    2010-07-01

    An attempt was made to develop a single, rapid, specific, and sensitive gradient reversed-phase ultra-performance liquid chromatographic method for quantitative analysis of levofloxacin. The single method thus developed is applied for the quantification of levofloxacin both in aqueous humour as well as pharmaceutical dosage forms (i.e., tablets and eye drops). The newly developed method is applicable for pharmacokinetic studies of eye formulations. The chromatographic separation of levofloxacin was achieved on a Waters Acquity HSS T-3 column (100 x 2.1 mm, 1.8 microm) within a short run-time of 5 min. The method was validated according to the ICH guidelines with respect to system suitability, linearity, limit of quantitation and detection, precision, accuracy, robustness, and specificity. Forced degradation studies were also performed in levofloxacin bulk drug samples to demonstrate the stability-indicating power of the developed ultra-performance liquid chromatography method. The developed method was then successfully applied for the ocular pharmacokinetic study of levofloxacin eye formulations and assay of levofloxacin pharmaceutical dosage form.

  11. High resolution LC-ESI-TOF-mass spectrometry method for fast separation, identification, and quantification of 12 isoflavones in soybeans and soybean products.

    PubMed

    Lee, Mi Jin; Chung, Ill-Min; Kim, Hunjung; Jung, Mun Yhung

    2015-06-01

    A high resolution LC-ESI-TOF-MS analytical method was established for the rapid isolation, identification, and quantification of 12 isoflavones in soybean and soybean products. Individual isoflavones were identified on the basis of the accurate mass data of their respective protonated mass ions, Na or K adduct ions, fragment ions, and isotope ion patterns. The protonated mass ions of isoflavones were extracted for their quantification in soybean products. Twelve different isoflavones in the soybean products were fully separated, identified and quantified within 12min separation time. The established LC-TOF/MS was an effective analytical method for the simultaneous characterization and quantification of isoflavones with exceptionally short analytical time, high selectivity, a high linearity (r(2)>0.992) in a wide range, low LOD and LOQ, high precision, inter-and intra-day repeatability, and no significant matrix effect. Furthermore, it requires simple sample preparation procedure (solvent extraction, dilution, and syringe filtration).

  12. The absolute path command

    SciTech Connect

    Moody, A.

    2012-05-11

    The ap command traveres all symlinks in a given file, directory, or executable name to identify the final absolute path. It can print just the final path, each intermediate link along with the symlink chan, and the permissions and ownership of each directory component in the final path. It has functionality similar to "which", except that it shows the final path instead of the first path. It is also similar to "pwd", but it can provide the absolute path to a relative directory from the current working directory.

  13. A method for determining weights for excess relative risk and excess absolute risk when applied in the calculation of lifetime risk of cancer from radiation exposure.

    PubMed

    Walsh, Linda; Schneider, Uwe

    2013-03-01

    Radiation-related risks of cancer can be transported from one population to another population at risk, for the purpose of calculating lifetime risks from radiation exposure. Transfer via excess relative risks (ERR) or excess absolute risks (EAR) or a mixture of both (i.e., from the life span study (LSS) of Japanese atomic bomb survivors) has been done in the past based on qualitative weighting. Consequently, the values of the weights applied and the method of application of the weights (i.e., as additive or geometric weighted means) have varied both between reports produced at different times by the same regulatory body and also between reports produced at similar times by different regulatory bodies. Since the gender and age patterns are often markedly different between EAR and ERR models, it is useful to have an evidence-based method for determining the relative goodness of fit of such models to the data. This paper identifies a method, using Akaike model weights, which could aid expert judgment and be applied to help to achieve consistency of approach and quantitative evidence-based results in future health risk assessments. The results of applying this method to recent LSS cancer incidence models are that the relative EAR weighting by cancer solid cancer site, on a scale of 0-1, is zero for breast and colon, 0.02 for all solid, 0.03 for lung, 0.08 for liver, 0.15 for thyroid, 0.18 for bladder and 0.93 for stomach. The EAR weighting for female breast cancer increases from 0 to 0.3, if a generally observed change in the trend between female age-specific breast cancer incidence rates and attained age, associated with menopause, is accounted for in the EAR model. Application of this method to preferred models from a study of multi-model inference from many models fitted to the LSS leukemia mortality data, results in an EAR weighting of 0. From these results it can be seen that lifetime risk transfer is most highly weighted by EAR only for stomach cancer. However

  14. Quantification of santonin in eight species of Artemisia from Kazakhstan by means of HPLC-UV: Method development and validation

    PubMed Central

    Bekezhanova, Tolkyn; Sadykova; Shukirbekova, Alma

    2017-01-01

    Santonin, a powerful anthelmintic drug that was formely used to treat worms, is Artemisia cina's main constituent. However, due to its toxicity to humans, it is no longer in use. Kazakhstan is looking to introduce this plant as an anthelmintic drug for veterinary purposes, despite the known toxic properties of the santonin. The objective of this study was to develop a fast and specific method for the identification of santonin and its precise quantitation using HPLC-UV in order to avoid unnecessary intoxication, which is paramount for the development of veterinary medicines. The results obtained showed that santonin appears at around 5.7 minutes in this very reliable HPLC method. The validation of the method was performed by the investigation of parameters such as precision, accuracy, reproducibility and recovery. The method was used to identify and quantify santonin in leaves of A. scoparia, A. foetida, A. gmelinni, A. schrenkiana, A. frigida, A. sublesingiana, A terra-albae, and A. absinthium from Kazakhstan as well as in three different extracts of leaves of A. cina. This study has provided a faster and simpler method for the identification and quantification of this compound in other species of Artemisia of economic importance. PMID:28301522

  15. Quantification of santonin in eight species of Artemisia from Kazakhstan by means of HPLC-UV: Method development and validation.

    PubMed

    Sakipova, Zuriyadda; Wong, Nikki Siu Hai; Bekezhanova, Tolkyn; Sadykova; Shukirbekova, Alma; Boylan, Fabio

    2017-01-01

    Santonin, a powerful anthelmintic drug that was formely used to treat worms, is Artemisia cina's main constituent. However, due to its toxicity to humans, it is no longer in use. Kazakhstan is looking to introduce this plant as an anthelmintic drug for veterinary purposes, despite the known toxic properties of the santonin. The objective of this study was to develop a fast and specific method for the identification of santonin and its precise quantitation using HPLC-UV in order to avoid unnecessary intoxication, which is paramount for the development of veterinary medicines. The results obtained showed that santonin appears at around 5.7 minutes in this very reliable HPLC method. The validation of the method was performed by the investigation of parameters such as precision, accuracy, reproducibility and recovery. The method was used to identify and quantify santonin in leaves of A. scoparia, A. foetida, A. gmelinni, A. schrenkiana, A. frigida, A. sublesingiana, A terra-albae, and A. absinthium from Kazakhstan as well as in three different extracts of leaves of A. cina. This study has provided a faster and simpler method for the identification and quantification of this compound in other species of Artemisia of economic importance.

  16. A porous graphitized carbon column HPLC method for the quantification of paracetamol, pseudoephedrine, and chlorpheniramine in a pharmaceutical formulation.

    PubMed

    Kalogria, Eleni; Koupparis, Michael; Panderi, Irene

    2010-01-01

    A simple, rapid, and stability-indicating HPLC method has been developed, fully validated, and applied to the quantification of paracetamol, pseudoephedrine hydrochloride, and chlorpheniramine maleate in a pharmaceutical formulation, using hydrochlorothiazide as an internal standard. Chromatographic separation was achieved isocratically on an RP porous graphitized carbon analytical column (125 x 2.1 mm id, particle size 5 microm) using 5.0 mM ammonium acetate-acetonitrile (35 + 65, v/v) mobile phase at a flow rate of 0.50 mL/min. UV spectrophotometric detection at 220 nm was used. The method had linear calibration curves over the range of 30-70 microg/mL for paracetamol, 1.8-4.2 microg/mL for pseudoephedrine hydrochloride, and 120-280 ng/mL for chlorpheniramine maleate. The intraday and interday RSD values were less than 3.2% for all compounds, while the relative error was less than 2.9%. Accelerated stability studies performed under various stress conditions proved the selectivity of the method. The developed method was applied successfully to QC and content uniformity tests of commercial tablets.

  17. Influence of DNA extraction methods, PCR inhibitors and quantification methods on real-time PCR assay of biotechnology-derived traits.

    PubMed

    Demeke, Tigst; Jenkins, G Ronald

    2010-03-01

    Biotechnology-derived varieties of canola, cotton, corn and soybean are being grown in the USA, Canada and other predominantly grain exporting countries. Although the amount of farmland devoted to production of biotechnology-derived crops continues to increase, lingering concerns that unintended consequences may occur provide the EU and most grain-importing countries with justification to regulate these crops. Legislation in the EU requires traceability of grains/oilseeds, food and feed products, and labelling, when a threshold level of 0.9% w/w of genetically engineered trait is demonstrated to be present in an analytical sample. The GE content is routinely determined by quantitative PCR (qPCR) and plant genomic DNA provides the template for the initial steps in this process. A plethora of DNA extraction methods exist for qPCR applications. Implementing standardized methods for detection of genetically engineered traits is necessary to facilitate grain marketing. The International Organization for Standardization draft standard 21571 identifies detergent-based methods and commercially available kits that are widely used for DNA extraction, but also indicates that adaptations may be necessary depending upon the sample matrix. This review assesses advantages and disadvantages of various commercially available DNA extraction kits, as well as modifications to published cetyltrimethylammonium bromide methods. Inhibitors are a major obstacle for efficient amplification in qPCR. The types of PCR inhibitors and techniques to minimize inhibition are discussed. Finally, accurate quantification of DNA for applications in qPCR is not trivial. Many confounders contribute to differences in analytical measurements when a particular DNA quantification method is applied and different methods do not always provide concordant results on the same DNA sample. How these differences impact measurement uncertainty in qPCR is considered.

  18. STATISTICAL VALIDATION OF SULFATE QUANTIFICATION METHODS USED FOR ANALYSIS OF ACID MINE DRAINAGE

    EPA Science Inventory

    Turbidimetric method (TM), ion chromatography (IC) and inductively coupled plasma atomic emission spectrometry (ICP-AES) with and without acid digestion have been compared and validated for the determination of sulfate in mining wastewater. Analytical methods were chosen to compa...

  19. Development and validation of automated SPE-HPLC-MS/MS methods for the quantification of asenapine, a new antipsychotic agent, and its two major metabolites in human urine.

    PubMed

    de Boer, Theo; Meulman, Erik; Meijering, Henri; Wieling, Jaap; Dogterom, Peter; Lass, Holger

    2012-12-01

    To support the evaluation of the pharmacokinetic parameters of asenapine (ASE) in urine, we developed and validated online solid-phase extraction high-performance liquid chromatography methods with tandem mass spectrometry detection (SPE-LC-MS/MS) for the quantification of ASE and two of its major metabolites, N-desmethylasenapine (DMA) and asenapine-N⁺-glucuronide (ASG). The linearity in human urine was found acceptable for quantification in a concentration range of 0.500-100 ng/mL for ASE and DMA and 10.0-3000 ng/mL for ASG, respectively.

  20. Absolute entropy and free energy of fluids using the hypothetical scanning method. II. Transition probabilities from canonical Monte Carlo simulations of partial systems

    NASA Astrophysics Data System (ADS)

    White, Ronald P.; Meirovitch, Hagai

    2003-12-01

    A variant of the hypothetical scanning (HS) method for calculating the absolute entropy and free energy of fluids is developed, as applied to systems of Lennard-Jones atoms (liquid argon). As in the preceding paper (Paper I), a probability Pi approximating the Boltzmann probability of system configuration i, is calculated with a reconstruction procedure based on adding the atoms gradually to an initially empty volume, where they are placed in their positions at i; in this process the volume is divided into cubic cells, which are visited layer-by-layer, line-by-line. At each step a transition probability (TP) is calculated and the product of all the TPs leads to Pi. At step k, k-1 cells have already been treated, where among them Nk are occupied by an atom. A canonical metropolis Monte Carlo (MC) simulation is carried out over a portion of the still unvisited (future) volume thus providing an approximate representation of the N-Nk as yet untreated (future) atoms. The TP of target cell k is determined from the number of visits of future atoms to this cell during the simulation. This MC version of HS, called HSMC, is based on a relatively small number of efficiency parameters; their number does not grow and their values are not changed as the number of the treated future atoms is increased (i.e., as the approximation improves); therefore, implementing HSMC for a relatively large number of future atoms (up to 40 in this study) is straightforward. Indeed, excellent results have been obtained for the free energy and the entropy.

  1. Comparison between Manual and Automated Methods for Ki-67 Immunoexpression Quantification in Ameloblastomas

    PubMed Central

    González-González, Rogelio; López-Verdín, Sandra; Robles-Bonilla, Carlos; Pereira-Prado, Vanesa

    2016-01-01

    Ameloblastoma is a common and unpredictable odontogenic tumor with high relapse rates. Several studies assessing the proliferative capacity of these neoplasms have been published, mainly using the protein Ki-67. Cell counts must be completed to determine the cell proliferation rate. Multiple methods have been developed for this purpose. The most widely used method is the labeling index, which has undergone changes over time to better facilitate cell counting. Here, we compared manual cell counting methods with automated cell counting (ImmunoRatio) to determine the relative effectiveness of these methods. The results suggest that ImmunoRatio, a free software tool, may be highly advantageous and provide results similar to manual cell counting methods when used with the appropriate calibration. However, ImmunoRatio has flaws that may affect the labeling index results. Therefore, this automated cell counting method must be supplemented with manual cell counting methods. PMID:27843757

  2. Near-infrared microscopic methods for the detection and quantification of processed by-products of animal origin

    NASA Astrophysics Data System (ADS)

    Abbas, O.; Fernández Pierna, J. A.; Dardenne, P.; Baeten, V.

    2010-04-01

    Since the BSE crisis, researches concern mainly the detection, identification, and quantification of meat and bone meal with an important focus on the development of new analytical methods. Microscopic based spectroscopy methods (NIR microscopy - NIRM or/and NIR hyperspectral imaging) have been proposed as complementary methods to the official method; the optical microscopy. NIR spectroscopy offers the advantage of being rapid, accurate and independent of human analyst skills. The combination of an NIR detector and a microscope or a camera allows the collection of high quality spectra for small feed particles having a size larger than 50 μm. Several studies undertaken have demonstrated the clear potential of NIR microscopic methods for the detection of animal particles in both raw and sediment fractions. Samples are sieved and only the gross fraction (superior than 250 μm) is investigated. Proposed methodologies have been developed to assure, with an acceptable level of confidence (95%), the detection of at least one animal particle when a feed sample is adulterated at a level of 0.1%. NIRM and NIR hyperspectral imaging are running under accreditation ISO 17025 since 2005 at CRA-W. A quantitative NIRM approach has been developed in order to fulfill the new requirements of the European commission policies. The capacities of NIRM method have been improved; only the raw fraction is analyzed, both the gross and the fine fractions of the samples are considered, and the acquisition parameters are optimized (the aperture, the gap, and the composition of the animal feed). A mapping method for a faster collection of spectra is also developed. The aim of this work is to show the new advances in the analytical methods developed in the frame of the feed ban applied in Europe.

  3. Reliable quantification of bisphenol A and its chlorinated derivatives in human urine using UPLC-MS/MS method.

    PubMed

    Venisse, N; Grignon, C; Brunet, B; Thévenot, S; Bacle, A; Migeot, V; Dupuis, A

    2014-07-01

    Bisphenol A (BPA), a widespread man-made chemical classified as an endocrine disruptor, is increasingly considered as a major cause of concern for human health. Chlorine present in drinking water may react with BPA to form chlorinated derivatives (ClxBPA), which have demonstrated a heightened level of estrogenic activity. If many epidemiological studies report that more than 90% of people have detectable BPA levels in their urine, then no such study has been undertaken regarding ClxBPA. The purpose of this work is to propose a highly sensitive and accurate analytical method adapted to large-scale biomonitoring studies aimed at assessing exposure to BPA and ClxBPA through the use of human urine. To achieve this, we have comprehensively validated a method using salting-out assisted liquid/liquid extraction (SALLE) coupled to UPLC-MS/MS and isotope dilution quantification, to measure unconjugated BPA and ClxBPA in human urine according to the accepted guidelines. Deutered BPA as well as deutered 2,2'-DCBPA was used as internal standards. The matrix calibration curve ranged from 0.05 to 1.60 ng mL(-1) and from 0.5 to 16.0 ng mL(-1) for ClxBPA and BPA respectively, and provided good linearity (r²>0.99). This method was precise (the intra- and inter-day coefficients of variation were <20% at three different concentrations: 0.05 ng mL(-1), 0.2 ng mL(-1), 0.8 ng mL(-1) and 0.5 ng mL(-1), 2 ng mL(-1), 8 ng mL(-1) for ClxBPA and BPA, respectively) and accurate (bias ranged from -13% to +12%). The limit of quantification, validated at 0.05 ng mL(-1) and 0.5 ng mL(-1) for ClxBPA and BPA respectively when using 300 µL of urine, was found to be suitable for the concentration existing in real samples. The matrix effect and the BPA cross-contamination were also investigated in this study. The analytical method developed in this study is in accordance with the requirements applicable to biomonitoring of BPA and ClxBPA in human urine.

  4. An improved UHPLC-UV method for separation and quantification of carotenoids in vegetable crops.

    PubMed

    Maurer, Megan M; Mein, Jonathan R; Chaudhuri, Swapan K; Constant, Howard L

    2014-12-15

    Carotenoid identification and quantitation is critical for the development of improved nutrition plant varieties. Industrial analysis of carotenoids is typically carried out on multiple crops with potentially thousands of samples per crop, placing critical needs on speed and broad utility of the analytical methods. Current chromatographic methods for carotenoid analysis have had limited industrial application due to their low throughput, requiring up to 60 min for complete separation of all compounds. We have developed an improved UHPLC-UV method that resolves all major carotenoids found in broccoli (Brassica oleracea L. var. italica), carrot (Daucus carota), corn (Zea mays), and tomato (Solanum lycopersicum). The chromatographic method is completed in 13.5 min allowing for the resolution of the 11 carotenoids of interest, including the structural isomers lutein/zeaxanthin and α-/β-carotene. Additional minor carotenoids have also been separated and identified with this method, demonstrating the utility of this method across major commercial food crops.

  5. Performance review of a fast HPLC-UV method for the quantification of chlorogenic acids in green coffee bean extracts.

    PubMed

    Craig, Ana Paula; Fields, Christine; Liang, Ningjian; Kitts, David; Erickson, Aron

    2016-07-01

    The aim of this study was to test the performance of a HPLC method, designated for rapid quantification of chlorogenic acids (CGA) in green coffee extract (GCE). The precision statistics associated with the method were assessed using three independent laboratories with five samples analyzed in triplicate. Seven main CGA isomers (3-CQA, 5-CQA, 4-CQA, 5-FQA, 3,4-diCQA, 3,5-diCQA and 4,5-diCQA) were quantified. The concentration of total CGA in the samples varied from 32.24% to 52.65% w/w. The repeatability and reproducibility standard deviations for the determination of individual isomers varied, respectively, from 0.01 to 0.28 and 0.05-1.59. The repeatability and reproducibility standard deviations of the calculated total CGA, corresponding to the sum of the seven main CGA isomers, varied respectively, from 0.17 to 0.58 and 0.55-2.01. The fast HPLC method evaluated in this study was considered precise and appropriate for the determination of CGA in GCE.

  6. A reversed-phase HPLC-UV method developed and validated for simultaneous quantification of six alkaloids from Nicotiana spp.

    PubMed

    Moghbel, Nahid; Ryu, BoMi; Steadman, Kathryn J

    2015-08-01

    A reversed-phase HPLC-UV method was developed, optimized, and validated for the separation and quantitation of six target alkaloids from leaves of Nicotiana species (nicotine, nornicotine, anatabine, anabasine, myosmine, and cotinine). A bidentate reversed-phase C18 column was used as stationary phase and an alkaline ammonium formate buffer and acetonitrile as mobile phase. The alkaloids were well separated in a short run time of 13min with mobile phase pH 10.5 and a small gradient of 9-13% acetonitrile, and detected using UV at 260nm. Peak parameters were acceptable for all six closely related alkaloids. The proposed method has enough linearity with correlation coefficient >0.999 within the investigated range for all tested alkaloids. Satisfactory precision was achieved for both intra- and inter-day assay, with RSD less than 2% for all alkaloid standards. Reproducibility was also within the acceptable range of RSD <2%. Limit of detection was 1.6μg/mL for nicotine and below 1μg/mL for all other alkaloids. The limit of quantification was 2.8 and 4.8μg/mL for nornicotine and nicotine respectively, and below 2μg/mL for all other alkaloids. The method was successfully applied for simultaneous analysis of alkaloids in leaves of Nicotiana benthamiana.

  7. GC/CI-MS/MS method for the identification and quantification of volatile N-nitrosamines in meat products.

    PubMed

    Sannino, Anna; Bolzoni, Luciana

    2013-12-15

    A sensitive and selective method was developed and validated for the determination of nine N-nitrosamines in meat products. The N-nitrosamines were extracted with NaOH/methanol, partitioned into dichloromethane on a ChemElut column and cleaned-up by solid-phase extraction. All samples were spiked with (2)H isotope-labelled N-nitrosamine internal standard prior to extraction. After purification on a Florisil mini-column, the extracts were analysed by gas chromatography-chemical ionisation tandem mass spectrometry (GC-CI/MS/MS) using ammonia as reagent gas. The presence of N-nitrosamines in samples was quantified by isotope dilution mass spectrometry. The method was validated for linearity and range, accuracy, precision and sensitivity. Recoveries were calculated at three levels of concentration (0.5, 1 and 10 μg/kg) spiked in raw pork meat. The values were found between 95% and 110% with relative standard deviation (RSD) values between 5% and 11%. The excellent selectivity and sensitivity allows quantification and identification of low levels of N-nitrosamines in meat products (limits of quantitation (LOQs) 0.3-0.4 μg/kg). Finally, the method was successfully used to analyse a sample of canned meat and nine different cured meat products produced in Italy. N-Nitroso-dimethylamine was detected in all examined products in the range 0.3-1.1 μg/kg.

  8. Novel and sensitive UPLC-MS/MS method for quantification of sofosbuvir in human plasma: application to a bioequivalence study.

    PubMed

    Rezk, Mamdouh R; Basalious, Emad B; Amin, Mohammed E

    2016-09-01

    A novel and sensitive LC-MS/MS method was developed and validated for determination of sofosbuvir (SF) using eplerenone as an internal standard. The Xevo TQD LC-MS/MS was operated under the multiple-reaction monitoring mode using electrospray ionization. Extraction with tert-butyl methyl ether was used in sample preparation. The prepared samples were chromatographed on Acquity UPLC BEH C18 (50 × 2.1 mm, 1.7 μm) column by pumping 0.1% formic acid and acetonitrile in an isocratic mode at a flow rate of 0.35 mL/min. Method validation was performed as per the US Food and Drug Administration guidelines and the standard curves were found to be linear in the range of 0.25-3500 ng/mL for SF. The intra- and inter-day precision and accuracy results were within the acceptable limits. A very short run time of 1 min made it possible to analyze more than 500 human plasma samples per day. A very low quantification limit of SF allowed the applicability of the developed method for determination of SF in a bioequivalence study in human volunteers. Copyright © 2016 John Wiley & Sons, Ltd.

  9. A Nonparametric Belief Propagation Method for Uncertainty Quantification with Applications to Flow in Random Porous Media

    DTIC Science & Technology

    2012-12-10

    veloped to address UQ problems. The most widely used method is the Monte Carlo (MC) method. MC’s wide acceptance is due to the fact that it can uncover...of the random space is computationally infeasible. Gaussian approximation is a widely used technique to build the continuous model, however, the...including linear and nonlinear dimension reduction algorithms. For linear dimension reduction, the most famous and the most widely used method is the

  10. Quantification of Artifact Reduction With Real-Time Cine Four-Dimensional Computed Tomography Acquisition Methods

    SciTech Connect

    Langner, Ulrich W.; Keall, Paul J.

    2010-03-15

    Purpose: To quantify the magnitude and frequency of artifacts in simulated four-dimensional computed tomography (4D CT) images using three real-time acquisition methods- direction-dependent displacement acquisition, simultaneous displacement and phase acquisition, and simultaneous displacement and velocity acquisition- and to compare these methods with commonly used retrospective phase sorting. Methods and Materials: Image acquisition for the four 4D CT methods was simulated with different displacement and velocity tolerances for spheres with radii of 0.5 cm, 1.5 cm, and 2.5 cm, using 58 patient-measured tumors and respiratory motion traces. The magnitude and frequency of artifacts, CT doses, and acquisition times were computed for each method. Results: The mean artifact magnitude was 50% smaller for the three real-time methods than for retrospective phase sorting. The dose was {approx}50% lower, but the acquisition time was 20% to 100% longer for the real-time methods than for retrospective phase sorting. Conclusions: Real-time acquisition methods can reduce the frequency and magnitude of artifacts in 4D CT images, as well as the imaging dose, but they increase the image acquisition time. The results suggest that direction-dependent displacement acquisition is the preferred real-time 4D CT acquisition method, because on average, the lowest dose is delivered to the patient and the acquisition time is the shortest for the resulting number and magnitude of artifacts.

  11. Accurate quantification of tio2 nanoparticles collected on air filters using a microwave-assisted acid digestion method

    PubMed Central

    Mudunkotuwa, Imali A.; Anthony, T. Renée; Grassian, Vicki H.; Peters, Thomas M.

    2016-01-01

    Titanium dioxide (TiO2) particles, including nanoparticles with diameters smaller than 100 nm, are used extensively in consumer products. In a 2011 current intelligence bulletin, the National Institute of Occupational Safety and Health (NIOSH) recommended methods to assess worker exposures to fine and ultrafine TiO2 particles and associated occupational exposure limits for these particles. However, there are several challenges and problems encountered with these recommended exposure assessment methods involving the accurate quantitation of titanium dioxide collected on air filters using acid digestion followed by inductively coupled plasma optical emission spectroscopy (ICP-OES). Specifically, recommended digestion methods include the use of chemicals, such as perchloric acid, which are typically unavailable in most accredited industrial hygiene laboratories due to highly corrosive and oxidizing properties. Other alternative methods that are used typically involve the use of nitric acid or combination of nitric acid and sulfuric acid, which yield very poor recoveries for titanium dioxide. Therefore, given the current state of the science, it is clear that a new method is needed for exposure assessment. In this current study, a microwave-assisted acid digestion method has been specifically designed to improve the recovery of titanium in TiO2 nanoparticles for quantitative analysis using ICP-OES. The optimum digestion conditions were determined by changing several variables including the acids used, digestion time, and temperature. Consequently, the optimized digestion temperature of 210°C with concentrated sulfuric and nitric acid (2:1 v/v) resulted in a recovery of >90% for TiO2. The method is expected to provide for a more accurate quantification of airborne TiO2 particles in the workplace environment. PMID:26181824

  12. Accurate quantification of tio2 nanoparticles collected on air filters using a microwave-assisted acid digestion method.

    PubMed

    Mudunkotuwa, Imali A; Anthony, T Renée; Grassian, Vicki H; Peters, Thomas M

    2016-01-01

    Titanium dioxide (TiO(2)) particles, including nanoparticles with diameters smaller than 100 nm, are used extensively in consumer products. In a 2011 current intelligence bulletin, the National Institute of Occupational Safety and Health (NIOSH) recommended methods to assess worker exposures to fine and ultrafine TiO(2) particles and associated occupational exposure limits for these particles. However, there are several challenges and problems encountered with these recommended exposure assessment methods involving the accurate quantitation of titanium dioxide collected on air filters using acid digestion followed by inductively coupled plasma optical emission spectroscopy (ICP-OES). Specifically, recommended digestion methods include the use of chemicals, such as perchloric acid, which are typically unavailable in most accredited industrial hygiene laboratories due to highly corrosive and oxidizing properties. Other alternative methods that are used typically involve the use of nitric acid or combination of nitric acid and sulfuric acid, which yield very poor recoveries for titanium dioxide. Therefore, given the current state of the science, it is clear that a new method is needed for exposure assessment. In this current study, a microwave-assisted acid digestion method has been specifically designed to improve the recovery of titanium in TiO(2) nanoparticles for quantitative analysis using ICP-OES. The optimum digestion conditions were determined by changing several variables including the acids used, digestion time, and temperature. Consequently, the optimized digestion temperature of 210°C with concentrated sulfuric and nitric acid (2:1 v/v) resulted in a recovery of >90% for TiO(2). The method is expected to provide for a more accurate quantification of airborne TiO(2) particles in the workplace environment.

  13. Validation of an LC-MS bioanalytical method for quantification of phytate levels in rat, dog and human plasma.

    PubMed

    Tur, Fernando; Tur, Eva; Lentheric, Irene; Mendoza, Paula; Encabo, Maximo; Isern, Bernat; Grases, Felix; Maraschiello, Ciriaco; Perelló, Joan

    2013-06-01

    Myo-inositol hexakisphosphate (phytate, IP6) is a naturally occuring compound whose determination in biological matrices is chanllenging. Several benefitial properties have been attributed to IP6 in parallel with the development of suitable analytical methodologies for its analytical determination in urine and some tissues. However, there is a lack of appropriate tools for its determination in plasma samples. In this paper, a direct, sensitive and selective bioanalytical method for the determination of IP6 based on LC-MS is presented. It is the first method published to quantify IP6 in plasma matrices directly through its molecular weight, being consequently a highly specific methodology. The method has been validated in rat, dog and human plasma, according to the acceptance criteria laid down in the FDA guidance Bioanalytical Method Validation. Accuracy and precision were not greater than 15% at medium and high concentrations and not greater than 20% at the LLOQ concentration. The mean absolute recovery obtained ranged from 78.74 to 102.44%, 62.10 to 87.21% and 61.61 to 86.99% for rat, dog and human plasma r