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Sample records for absolute quantification method

  1. Absolute and relative quantification of RNA modifications via biosynthetic isotopomers

    PubMed Central

    Kellner, Stefanie; Ochel, Antonia; Thüring, Kathrin; Spenkuch, Felix; Neumann, Jennifer; Sharma, Sunny; Entian, Karl-Dieter; Schneider, Dirk; Helm, Mark

    2014-01-01

    In the resurging field of RNA modifications, quantification is a bottleneck blocking many exciting avenues. With currently over 150 known nucleoside alterations, detection and quantification methods must encompass multiple modifications for a comprehensive profile. LC–MS/MS approaches offer a perspective for comprehensive parallel quantification of all the various modifications found in total RNA of a given organism. By feeding 13C-glucose as sole carbon source, we have generated a stable isotope-labeled internal standard (SIL-IS) for bacterial RNA, which facilitates relative comparison of all modifications. While conventional SIL-IS approaches require the chemical synthesis of single modifications in weighable quantities, this SIL-IS consists of a nucleoside mixture covering all detectable RNA modifications of Escherichia coli, yet in small and initially unknown quantities. For absolute in addition to relative quantification, those quantities were determined by a combination of external calibration and sample spiking of the biosynthetic SIL-IS. For each nucleoside, we thus obtained a very robust relative response factor, which permits direct conversion of the MS signal to absolute amounts of substance. The application of the validated SIL-IS allowed highly precise quantification with standard deviations <2% during a 12-week period, and a linear dynamic range that was extended by two orders of magnitude. PMID:25129236

  2. Development of an HPLC Method for Absolute Quantification and QAMS of Flavonoids Components in Psoralea corylifolia L.

    PubMed Central

    Yan, Cuiping; Wu, Yu; Weng, Zebin; Gao, Qianqian; Yang, Guangming; Chen, Zhipeng; Cai, Baochang; Li, Weidong

    2015-01-01

    The seeds of Psoralea corylifolia L. (Fabaceae) are a commonly used medicinal herb in eastern Asia with many beneficial effects in clinical therapies. In this study, a simple, sensitive, precise, and specific reverse phase high-performance liquid chromatography (HPLC) method was established for quantification of 9 flavonoids in P. corylifolia, including isobavachin, neobavaisoflavone, bavachin, corylin, bavachalcone, bavachinin, isobavachalcone, corylifol A, and 4′-O-methylbavachalcone. Based on this method, a quantitative analysis of multicomponents by single marker (QAMS) was carried out, and the relative correction factors (RCFs) were calculated for determining the contents of other flavonoids. The accuracy of QAMS method was verified by comparing with the results of external standard method, as well as the feasibility and adaptability of the method applied on quality control of P. corylifolia. The 9 compounds were baseline separated in 60 min with a good linearity of regression coefficient (R2) over 0.9991. The accuracies of QAMS were between 92.89% and 109.5%. The RSD values of f in different injection volume were between 2.3% and 3.6%. The results obtained from QAMS suggested that it was a convenient and accurate method to determine multicomponents especially when some authentic standard substances were unavailable. It can be used to control the quality of P. corylifolia. PMID:26587307

  3. 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...

  4. Absolute Quantification of Individual Biomass Concentrations in a Methanogenic Coculture

    PubMed Central

    2014-01-01

    Identification of individual biomass concentrations is a crucial step towards an improved understanding of anaerobic digestion processes and mixed microbial conversions in general. The knowledge of individual biomass concentrations allows for the calculation of biomass specific conversion rates which form the basis of anaerobic digestion models. Only few attempts addressed the absolute quantification of individual biomass concentrations in methanogenic microbial ecosystems which has so far impaired the calculation of biomass specific conversion rates and thus model validation. This study proposes a quantitative PCR (qPCR) approach for the direct determination of individual biomass concentrations in methanogenic microbial associations by correlating the native qPCR signal (cycle threshold, Ct) to individual biomass concentrations (mg dry matter/L). Unlike existing methods, the proposed approach circumvents error-prone conversion factors that are typically used to convert gene copy numbers or cell concentrations into actual biomass concentrations. The newly developed method was assessed and deemed suitable for the determination of individual biomass concentrations in a defined coculture of Desulfovibrio sp. G11 and Methanospirillum hungatei JF1. The obtained calibration curves showed high accuracy, indicating that the new approach is well suited for any engineering applications where the knowledge of individual biomass concentrations is required. PMID:24949269

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

    PubMed

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

    2015-01-01

    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. PMID:26325288

  6. 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.

  7. Absolute Quantification of Selected Proteins in the Human Osteoarthritic Secretome

    PubMed Central

    Peffers, Mandy J.; Beynon, Robert J.; Clegg, Peter D.

    2013-01-01

    Osteoarthritis (OA) is characterized by a loss of extracellular matrix which is driven by catabolic cytokines. Proteomic analysis of the OA cartilage secretome enables the global study of secreted proteins. These are an important class of molecules with roles in numerous pathological mechanisms. Although cartilage studies have identified profiles of secreted proteins, quantitative proteomics techniques have been implemented that would enable further biological questions to be addressed. To overcome this limitation, we used the secretome from human OA cartilage explants stimulated with IL-1β and compared proteins released into the media using a label-free LC-MS/MS-based strategy. We employed QconCAT technology to quantify specific proteins using selected reaction monitoring. A total of 252 proteins were identified, nine were differentially expressed by IL-1 β stimulation. Selected protein candidates were quantified in absolute amounts using QconCAT. These findings confirmed a significant reduction in TIMP-1 in the secretome following IL-1β stimulation. Label-free and QconCAT analysis produced equivocal results indicating no effect of cytokine stimulation on aggrecan, cartilage oligomeric matrix protein, fibromodulin, matrix metalloproteinases 1 and 3 or plasminogen release. This study enabled comparative protein profiling and absolute quantification of proteins involved in molecular pathways pertinent to understanding the pathogenesis of OA. PMID:24132152

  8. Qualification of a surrogate matrix-based absolute quantification method for amyloid-β₄₂ in human cerebrospinal fluid using 2D UPLC-tandem mass spectrometry.

    PubMed

    Korecka, Magdalena; Waligorska, Teresa; Figurski, Michal; Toledo, Jon B; Arnold, Steven E; Grossman, Murray; Trojanowski, John Q; Shaw, Leslie M

    2014-01-01

    The primary aims of this work were to: 1) establish a calibrator surrogate matrix for quantification of amyloid-β (Aβ)42 in human cerebrospinal fluid (CSF) and preparation of quality control samples for LC-MS-MS methodology, 2) validate analytical performance of the assay, and 3) evaluate its diagnostic utility and compare it with the AlzBio3 immunoassay. The analytical methodology was based on a 2D-UPLC-MS-MS platform. Sample pretreatment used 5 M guanidine hydrochloride and extraction on μElution SPE columns as previously described. A column cleaning procedure involved gradual removal of aqueous solvents by acetonitrile assured consistent long-term chromatography performance. Receiver-operator characteristic (ROC) curve and correlation analyses evaluated the diagnostic utility of UPLC-MS-MS compared to AlzBio3 immunoassay for detection of Alzheimer's disease (AD). The surrogate matrix, artificial CSF containing 4 mg/mL of BSA, provides linear and reproducible calibration comparable to human pooled CSF as calibration matrix. Appropriate cleaning of the trapping and analytical columns provided every-day, trouble-free runs. Analyses of CSF Aβ42 showed that UPLC-MS-MS distinguished neuropathologically-diagnosed AD subjects from healthy controls with at least equivalent diagnostic utility to AlzBio3. Comparison of ROC curves for these two assays showed no statistically significant difference (p = 0.2229). Linear regression analysis of Aβ42 concentrations measured by this mass spectrometry-based method compared to the AlzBio3 immunoassay showed significantly higher but highly correlated results. In conclusion, the newly established surrogate matrix for 2D-UPLC-MS-MS measurement of Aβ42 provides selective, reproducible, and accurate results. The documented analytical performance and diagnostic performance for AD versus controls supports consideration as a candidate reference method. PMID:24625802

  9. 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. PMID:27098933

  10. Relative and absolute quantification of postsynaptic density proteome isolated from rat forebrain and cerebellum.

    PubMed

    Cheng, Dongmei; Hoogenraad, Casper C; Rush, John; Ramm, Elizabeth; Schlager, Max A; Duong, Duc M; Xu, Ping; Wijayawardana, Sameera R; Hanfelt, John; Nakagawa, Terunaga; Sheng, Morgan; Peng, Junmin

    2006-06-01

    The postsynaptic density (PSD) of central excitatory synapses is essential for postsynaptic signaling, and its components are heterogeneous among different neuronal subtypes and brain structures. Here we report large scale relative and absolute quantification of proteins in PSDs purified from adult rat forebrain and cerebellum. PSD protein profiles were determined using the cleavable ICAT strategy and LC-MS/MS. A total of 296 proteins were identified and quantified with 43 proteins exhibiting statistically significant abundance change between forebrain and cerebellum, indicating marked molecular heterogeneity of PSDs between different brain regions. Moreover we utilized absolute quantification strategy, in which synthetic isotope-labeled peptides were used as internal standards, to measure the molar abundance of 32 key PSD proteins in forebrain and cerebellum. These data confirm the abundance of calcium/calmodulin-dependent protein kinase II and PSD-95 and reveal unexpected stoichiometric ratios between glutamate receptors, scaffold proteins, and signaling molecules in the PSD. Our data also demonstrate that the absolute quantification method is well suited for targeted quantitative proteomic analysis. Overall this study delineates a crucial molecular difference between forebrain and cerebellar PSDs and provides a quantitative framework for measuring the molecular stoichiometry of the PSD. PMID:16507876

  11. Mass spectrometry-based absolute quantification reveals rhythmic variation of mouse circadian clock proteins.

    PubMed

    Narumi, Ryohei; Shimizu, Yoshihiro; Ukai-Tadenuma, Maki; Ode, Koji L; Kanda, Genki N; Shinohara, Yuta; Sato, Aya; Matsumoto, Katsuhiko; Ueda, Hiroki R

    2016-06-14

    Absolute values of protein expression levels in cells are crucial information for understanding cellular biological systems. Precise quantification of proteins can be achieved by liquid chromatography (LC)-mass spectrometry (MS) analysis of enzymatic digests of proteins in the presence of isotope-labeled internal standards. Thus, development of a simple and easy way for the preparation of internal standards is advantageous for the analyses of multiple target proteins, which will allow systems-level studies. Here we describe a method, termed MS-based Quantification By isotope-labeled Cell-free products (MS-QBiC), which provides the simple and high-throughput preparation of internal standards by using a reconstituted cell-free protein synthesis system, and thereby facilitates both multiplexed and sensitive quantification of absolute amounts of target proteins. This method was applied to a systems-level dynamic analysis of mammalian circadian clock proteins, which consist of transcription factors and protein kinases that govern central and peripheral circadian clocks in mammals. Sixteen proteins from 20 selected circadian clock proteins were successfully quantified from mouse liver over a 24-h time series, and 14 proteins had circadian variations. Quantified values were applied to detect internal body time using a previously developed molecular timetable method. The analyses showed that single time-point data from wild-type mice can predict the endogenous state of the circadian clock, whereas data from clock mutant mice are not applicable because of the disappearance of circadian variation. PMID:27247408

  12. Evaluation of serum phosphopeptides as potential cancer biomarkers by mass spectrometric absolute quantification.

    PubMed

    Zhai, Guijin; Wu, Xiaoyan; Luo, Qun; Wu, Kui; Zhao, Yao; Liu, Jianan; Xiong, Shaoxiang; Feng, Yu-Qi; Yang, Liping; Wang, Fuyi

    2014-07-01

    Mass spectrometric quantification of phosphopeptides is a challenge due to the ion suppression effect of highly abundant non-phosphorylated peptides in complex samples such as serum. Several strategies for relative quantification of serum phosphopeptides based on MS have been developed, but the power of relative quantities was limited when making direct comparisons between two groups of samples or acting as a clinical examination index. Herein, we describe an MS absolute quantification strategy combined with Titania Coated Magnetic Hollow Mesoporous Silica Microspheres (TiO2/MHMSM) enrichment and stable isotopic acetyl labeling for phosphopeptides in human serum. Four endogenous serum phosphopeptides generated by degradation of fibrinogen were identified by LC-ESI-MS/MS following TiO2/MHMSM enrichment. The ESI-MS signal intensity ratios of the four phosphopeptide standards labeled with N-acetoxy-H3-succinimide (H3-NAS) and N-acetoxy-D3-succinimide (D3-NAS), following TiO2/MHMSM capture are linearly correlated with the molar ratios of the "light" to "heavy" phosphopeptides over the range of 0.1-4 with an r(2) of up to 0.998 and a slope of close to 1. The recovery of the four phosphopeptides spiked at low, medium and high levels in human sera were 98.4-111.9% with RSDs ranging 2.0-10.1%. The absolute quantification of the phosphopeptides in serum samples of 20 healthy persons and 20 gastric cancer patients by the developed method demonstrated that 3 out of the 4 phosphopeptides showed remarkable variation in serum level between healthy and cancer groups, and the phosphopeptide DpSGEGDFLAEGGGVR is significantly down-regulated in the serum of patients, being a potential biomarker for gastric cancer diagnosis. PMID:24840465

  13. 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

  14. 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. PMID:27074005

  15. Absolute quantification of UGT1A1 in various tissues and cell lines using isotope label-free UPLC-MS/MS method determines its turnover number and correlates with its glucuronidation activities.

    PubMed

    Xu, Beibei; Gao, Song; Wu, Baojian; Yin, Taijun; Hu, Ming

    2014-01-01

    Uridine 5'-diphosphate-glucuronosyltransferase (UGT)1A1 is a major phase II metabolism enzyme responsible for glucuronidation of drugs and endogenous compounds. The purpose of this study was to determine the expression level of UGT1A1 in human liver microsomes and human cell lines by using an isotope label-free LC-MS/MS method. A Waters Ultra performance liquid chromatography (UPLC) system coupled with an API 5500Qtrap mass spectrometer was used for the analysis. Two signature peptides (Pep-1, and Pep-2) were employed to quantify UGT1A1 by multiple reaction monitoring (MRM) approach. Standard addition method was used to validate the assay to account for the matrix effect. 17β-Estradiol was used as the marker substrate to determine UGT1A1 activities. The validated method has a linear range of 200-0.0195nM for both signature peptides. The precision, accuracy, and matrix effect were in acceptable ranges. UGT1A1 expression levels were then determined using 8 individual human liver microsomes, a pooled human liver microsomes, three UGT1A1 genotyped human liver microsomes, and four cell lines (Caco-2, MCF-7, Hela, and HepG2). The correlations study showed that the UGT1A1 protein levels were strongly correlated with its glucuronidation activities in human liver microsomes (R(2)=0.85) and in microsomes prepared from cell lines (R(2)=0.95). Isotope-labeled peptides were not necessary for LC-MS/MS quantitation of proteins. The isotope label-free absolute quantification method used here had good accuracy, sensitivity, linear range, and reproducibility, and were used successfully for the accurate determination of UGT1A1 from tissues and cell lines. PMID:24055854

  16. Non-invasive quantification of brain glycogen absolute concentration

    PubMed Central

    van Heeswijk, Ruud B.; Xin, Lijing; Laus, Sabrina; Frenkel, Hanne; Lei, Hongxia; Gruetter, Rolf

    2009-01-01

    The only currently available method to measure brain glycogen in vivo is 13C NMR spectroscopy. Incorporation of 13C-labeled glucose (Glc) is necessary to allow glycogen measurement, but might be affected by turnover changes. Our aim was to measure glycogen absolute concentration in the rat brain by eliminating label turnover as variable. The approach is based on establishing an increased, constant 13C isotopic enrichment (IE). 13C-Glc infusion is then performed at the IE of brain glycogen. As glycogen IE cannot be assessed in vivo, we validated that it can be inferred from that of N-acetyl-aspartate IE in vivo: After [1-13C]-Glc ingestion, glycogen IE was 2.2 ± 0.1 fold that of N-acetyl-aspartate (n = 11, R2 = 0.77). After subsequent Glc infusion, glycogen IE equaled brain Glc IE (n = 6, paired t-test, p = 0.37), implying isotopic steady-state achievement and complete turnover of the glycogen molecule. Glycogen concentration measured in vivo by 13C NMR (mean ± SD: 5.8 ± 0.7 μmol/g) was in excellent agreement with that in vitro (6.4 ± 0.6 μmol/g, n = 5). When insulin was administered, the stability of glycogen concentration was analogous to previous biochemical measurements implying that glycogen turnover is activated by insulin. We conclude that the entire glycogen molecule is turned over and that insulin activates glycogen turnover. PMID:19013831

  17. 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. PMID:20163147

  18. 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

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

    PubMed

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

    2016-08-01

    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

  20. 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. PMID:22967562

  1. A strategy for absolute proteome quantification with mass spectrometry by hierarchical use of peptide-concatenated standards.

    PubMed

    Kito, Keiji; Okada, Mitsuhiro; Ishibashi, Yuko; Okada, Satoshi; Ito, Takashi

    2016-05-01

    The accurate and precise absolute abundance of proteins can be determined using mass spectrometry by spiking the sample with stable isotope-labeled standards. In this study, we developed a strategy of hierarchical use of peptide-concatenated standards (PCSs) to quantify more proteins over a wider dynamic range. Multiple primary PCSs were used for quantification of many target proteins. Unique "ID-tag peptides" were introduced into individual primary PCSs, allowing us to monitor the exact amounts of individual PCSs using a "secondary PCS" in which all "ID-tag peptides" were concatenated. Furthermore, we varied the copy number of the "ID-tag peptide" in each PCS according to a range of expression levels of target proteins. This strategy accomplished absolute quantification over a wider range than that of the measured ratios. The quantified abundance of budding yeast proteins showed a high reproducibility for replicate analyses and similar copy numbers per cell for ribosomal proteins, demonstrating the accuracy and precision of this strategy. A comparison with the absolute abundance of transcripts clearly indicated different post-transcriptional regulation of expression for specific functional groups. Thus, the approach presented here is a faithful method for the absolute quantification of proteomes and provides insights into biological mechanisms, including the regulation of expressed protein abundance. PMID:27030420

  2. 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.

  3. 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

  4. 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

  5. Mass spectrometry based proteomics for absolute quantification of proteins from tumor cells

    PubMed Central

    Wang, Hong; Hanash, Sam

    2015-01-01

    In-depth quantitative profiling of the proteome and sub-proteomes of tumor cells has relevance to tumor classification, the development of novel therapeutics, and of prognostic and predictive markers and to disease monitoring. In particular the tumor cell surface represents a highly relevant compartment for the development of targeted therapeutics and immunotherapy. We have developed a proteomic platform to profile tumor cells that encompasses enrichment of surface membrane proteins, intact protein fractionation and label-free mass spectrometry based absolute quantification. Here we describe the methodology for capture, identification and quantification of cell surface proteins using biotinylation for labeling of the cell surface, avidin for capture of biotinylated proteins and ion mobility mass spectrometry for protein identification and quantification. PMID:25794949

  6. 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.

  7. 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.

  8. Absolute Quantification of the Host-To-Parasite DNA Ratio in Theileria parva-Infected Lymphocyte Cell Lines

    PubMed Central

    Gotia, Hanzel T.; Munro, James B.; Knowles, Donald P.; Daubenberger, Claudia A.; Bishop, Richard P.; Silva, Joana C.

    2016-01-01

    Theileria parva is a tick-transmitted intracellular apicomplexan pathogen of cattle in sub-Saharan Africa that causes East Coast fever (ECF). ECF is an acute fatal disease that kills over one million cattle annually, imposing a tremendous burden on African small-holder cattle farmers. The pathology and level of T. parva infections in its wildlife host, African buffalo (Syncerus caffer), and in cattle are distinct. We have developed an absolute quantification method based on quantitative PCR (qPCR) in which recombinant plasmids containing single copy genes specific to the parasite (apical membrane antigen 1 gene, ama1) or the host (hypoxanthine phosphoribosyltransferase 1, hprt1) are used as the quantification reference standards. Our study shows that T. parva and bovine cells are present in similar numbers in T. parva-infected lymphocyte cell lines and that consequently, due to its much smaller genome size, T. parva DNA comprises between 0.9% and 3% of the total DNA samples extracted from these lines. This absolute quantification assay of parasite and host genome copy number in a sample provides a simple and reliable method of assessing T. parva load in infected bovine lymphocytes, and is accurate over a wide range of host-to-parasite DNA ratios. Knowledge of the proportion of target DNA in a sample, as enabled by this method, is essential for efficient high-throughput genome sequencing applications for a variety of intracellular pathogens. This assay will also be very useful in future studies of interactions of distinct host-T. parva stocks and to fully characterize the dynamics of ECF infection in the field. PMID:26930209

  9. Absolute Quantification of the Host-To-Parasite DNA Ratio in Theileria parva-Infected Lymphocyte Cell Lines.

    PubMed

    Gotia, Hanzel T; Munro, James B; Knowles, Donald P; Daubenberger, Claudia A; Bishop, Richard P; Silva, Joana C

    2016-01-01

    Theileria parva is a tick-transmitted intracellular apicomplexan pathogen of cattle in sub-Saharan Africa that causes East Coast fever (ECF). ECF is an acute fatal disease that kills over one million cattle annually, imposing a tremendous burden on African small-holder cattle farmers. The pathology and level of T. parva infections in its wildlife host, African buffalo (Syncerus caffer), and in cattle are distinct. We have developed an absolute quantification method based on quantitative PCR (qPCR) in which recombinant plasmids containing single copy genes specific to the parasite (apical membrane antigen 1 gene, ama1) or the host (hypoxanthine phosphoribosyltransferase 1, hprt1) are used as the quantification reference standards. Our study shows that T. parva and bovine cells are present in similar numbers in T. parva-infected lymphocyte cell lines and that consequently, due to its much smaller genome size, T. parva DNA comprises between 0.9% and 3% of the total DNA samples extracted from these lines. This absolute quantification assay of parasite and host genome copy number in a sample provides a simple and reliable method of assessing T. parva load in infected bovine lymphocytes, and is accurate over a wide range of host-to-parasite DNA ratios. Knowledge of the proportion of target DNA in a sample, as enabled by this method, is essential for efficient high-throughput genome sequencing applications for a variety of intracellular pathogens. This assay will also be very useful in future studies of interactions of distinct host-T. parva stocks and to fully characterize the dynamics of ECF infection in the field. PMID:26930209

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

    PubMed

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

    2015-01-01

    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. PMID:26333806

  11. 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.

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

    PubMed Central

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

    2015-01-01

    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. PMID:26333806

  13. Novel isotopic N, N-dimethyl leucine (iDiLeu) reagents enable absolute quantification of peptides and proteins using a standard curve approach

    PubMed Central

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

    2014-01-01

    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 due to 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 due to their synthetic simplicity, and have increased throughput compared to 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) while the second enables standard curve creation and analyte quantification in one run (<8% error). PMID:25377360

  14. 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).

  15. Within-day repeatability for absolute quantification of Lawsonia intracellularis bacteria in feces from growing pigs.

    PubMed

    Pedersen, Ken Steen; Pedersen, Klaus H; Hjulsager, Charlotte; Larsen, Lars Erik; Ståhl, Marie; Stege, Helle; Angen, Øystein; Nielsen, Jens Peter

    2012-09-01

    Absolute quantification of Lawsonia intracellularis by real-time polymerase chain reaction (PCR) is now possible on a routine basis. Poor repeatability of quantification can result in disease status misclassification of individual pigs when a single fecal sample is obtained. The objective of the current study was to investigate overall variation within a day for fecal numbers of L. intracellularis bacteria determined by real-time PCR in growing pigs. From each of 30 pigs with an infection of L. intracellularis, 5 fecal samples were collected within 1 day. A total of 150 fecal samples were obtained and subjected to quantitative PCR (qPCR) testing. Mean fecal dry matter content was 14.3% (standard deviation = 4.5%). Two pigs (6.7%) alternated between being L. intracellularis qPCR positive and negative. For 28 pigs, the excreting levels of L. intracellularis were within the dynamic range of the qPCR assay at all sampling points. For these 28 pigs, the mean excretion level of L. intracellularis was 6.1 log(10) bacteria/g feces (standard deviation = 1.2 log(10) bacteria/g feces). The maximum observed difference between 2 fecal samples from the same pig was 1.1 log(10) bacteria/g feces. The average standard deviation for individual pigs was 0.27 log(10) bacteria/g feces. The average coefficient of variation within pigs was 0.04, ranging from 0.006 to 0.08. The results imply that absolute quantification of L. intracellularis by qPCR has acceptable repeatability within 1 day. However, a population-specific proportion of pigs alternating between positive and negative test results must be expected. PMID:22786973

  16. Direct and Absolute Quantification of over 1800 Yeast Proteins via Selected Reaction Monitoring.

    PubMed

    Lawless, Craig; Holman, Stephen W; Brownridge, Philip; Lanthaler, Karin; Harman, Victoria M; Watkins, Rachel; Hammond, Dean E; Miller, Rebecca L; Sims, Paul F G; Grant, Christopher M; Eyers, Claire E; Beynon, Robert J; Hubbard, Simon J

    2016-04-01

    Defining intracellular protein concentration is critical in molecular systems biology. Although strategies for determining relative protein changes are available, defining robust absolute values in copies per cell has proven significantly more challenging. Here we present a reference data set quantifying over 1800Saccharomyces cerevisiaeproteins by direct means using protein-specific stable-isotope labeled internal standards and selected reaction monitoring (SRM) mass spectrometry, far exceeding any previous study. This was achieved by careful design of over 100 QconCAT recombinant proteins as standards, defining 1167 proteins in terms of copies per cell and upper limits on a further 668, with robust CVs routinely less than 20%. The selected reaction monitoring-derived proteome is compared with existing quantitative data sets, highlighting the disparities between methodologies. Coupled with a quantification of the transcriptome by RNA-seq taken from the same cells, these data support revised estimates of several fundamental molecular parameters: a total protein count of ∼100 million molecules-per-cell, a median of ∼1000 proteins-per-transcript, and a linear model of protein translation explaining 70% of the variance in translation rate. This work contributes a "gold-standard" reference yeast proteome (including 532 values based on high quality, dual peptide quantification) that can be widely used in systems models and for other comparative studies. PMID:26750110

  17. Direct and Absolute Quantification of over 1800 Yeast Proteins via Selected Reaction Monitoring*

    PubMed Central

    Lawless, Craig; Holman, Stephen W.; Brownridge, Philip; Lanthaler, Karin; Harman, Victoria M.; Watkins, Rachel; Hammond, Dean E.; Miller, Rebecca L.; Sims, Paul F. G.; Grant, Christopher M.; Eyers, Claire E.; Beynon, Robert J.

    2016-01-01

    Defining intracellular protein concentration is critical in molecular systems biology. Although strategies for determining relative protein changes are available, defining robust absolute values in copies per cell has proven significantly more challenging. Here we present a reference data set quantifying over 1800 Saccharomyces cerevisiae proteins by direct means using protein-specific stable-isotope labeled internal standards and selected reaction monitoring (SRM) mass spectrometry, far exceeding any previous study. This was achieved by careful design of over 100 QconCAT recombinant proteins as standards, defining 1167 proteins in terms of copies per cell and upper limits on a further 668, with robust CVs routinely less than 20%. The selected reaction monitoring-derived proteome is compared with existing quantitative data sets, highlighting the disparities between methodologies. Coupled with a quantification of the transcriptome by RNA-seq taken from the same cells, these data support revised estimates of several fundamental molecular parameters: a total protein count of ∼100 million molecules-per-cell, a median of ∼1000 proteins-per-transcript, and a linear model of protein translation explaining 70% of the variance in translation rate. This work contributes a “gold-standard” reference yeast proteome (including 532 values based on high quality, dual peptide quantification) that can be widely used in systems models and for other comparative studies. PMID:26750110

  18. Testing the quasi-absolute method in photon activation analysis

    SciTech Connect

    Sun, Z. J.; Wells, D.; Starovoitova, V.; Segebade, C.

    2013-04-19

    In photon activation analysis (PAA), relative methods are widely used because of their accuracy and precision. Absolute methods, which are conducted without any assistance from calibration materials, are seldom applied for the difficulty in obtaining photon flux in measurements. This research is an attempt to perform a new absolute approach in PAA - quasi-absolute method - by retrieving photon flux in the sample through Monte Carlo simulation. With simulated photon flux and database of experimental cross sections, it is possible to calculate the concentration of target elements in the sample directly. The QA/QC procedures to solidify the research are discussed in detail. Our results show that the accuracy of the method for certain elements is close to a useful level in practice. Furthermore, the future results from the quasi-absolute method can also serve as a validation technique for experimental data on cross sections. The quasi-absolute method looks promising.

  19. 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. PMID:27451195

  20. A simple and accurate protocol for absolute polar metabolite quantification in cell cultures using quantitative nuclear magnetic resonance.

    PubMed

    Goldoni, Luca; Beringhelli, Tiziana; Rocchia, Walter; Realini, Natalia; Piomelli, Daniele

    2016-05-15

    Absolute analyte quantification by nuclear magnetic resonance (NMR) spectroscopy is rarely pursued in metabolomics, even though this would allow researchers to compare results obtained using different techniques. Here we report on a new protocol that permits, after pH-controlled serum protein removal, the sensitive quantification (limit of detection [LOD] = 5-25 μM) of hydrophilic nutrients and metabolites in the extracellular medium of cells in cultures. The method does not require the use of databases and uses PULCON (pulse length-based concentration determination) quantitative NMR to obtain results that are significantly more accurate and reproducible than those obtained by CPMG (Carr-Purcell-Meiboom-Gill) sequence or post-processing filtering approaches. Three practical applications of the method highlight its flexibility under different cell culture conditions. We identified and quantified (i) metabolic differences between genetically engineered human cell lines, (ii) alterations in cellular metabolism induced by differentiation of mouse myoblasts into myotubes, and (iii) metabolic changes caused by activation of neurotransmitter receptors in mouse myoblasts. Thus, the new protocol offers an easily implementable, efficient, and versatile tool for the investigation of cellular metabolism and signal transduction. PMID:26898303

  1. 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. PMID:25864956

  2. A practical method for sensor absolute calibration.

    PubMed

    Meisenholder, G W

    1966-04-01

    This paper describes a method of performing sensor calibrations using an NBS standard of spectral irradiance. The method shown, among others, was used for calibration of the Mariner IV Canopus sensor. Agreement of inflight response to preflight calibrations performed by this technique has been found to be well within 10%. PMID:20048890

  3. Counting numbers of synaptic proteins: absolute quantification and single molecule imaging techniques.

    PubMed

    Patrizio, Angela; Specht, Christian G

    2016-10-01

    The ability to count molecules is essential to elucidating cellular mechanisms, as these often depend on the absolute numbers and concentrations of molecules within specific compartments. Such is the case at chemical synapses, where the transmission of information from presynaptic to postsynaptic terminals requires complex interactions between small sets of molecules. Be it the subunit stoichiometry specifying neurotransmitter receptor properties, the copy numbers of scaffold proteins setting the limit of receptor accumulation at synapses, or protein packing densities shaping the molecular organization and plasticity of the postsynaptic density, all of these depend on exact quantities of components. A variety of proteomic, electrophysiological, and quantitative imaging techniques have yielded insights into the molecular composition of synaptic complexes. In this review, we compare the different quantitative approaches and consider the potential of single molecule imaging techniques for the quantification of synaptic components. We also discuss specific neurobiological data to contextualize the obtained numbers and to explain how they aid our understanding of synaptic structure and function. PMID:27335891

  4. Selective and absolute quantification of endogenous hypochlorous acid with quantum-dot conjugated microbeads.

    PubMed

    Yang, Yi-Cyun; Lu, Hsueh-Han; Wang, Wei-Ti; Liau, Ian

    2011-11-01

    Endogenous hypochlorous acid (HOCl) secreted by leukocytes plays a critical role in both the immune defense of mammalians and the pathogenesis of various diseases intimately related to inflammation. We report the first selective and absolute quantification of endogenous HOCl produced by leukocytes in vitro and in vivo with a novel quantum dot-based sensor. An activated human neutrophil secreted 6.5 ± 0.9 × 10(8) HOCl molecules into its phagosome, and kinetic measurement for the secretions showed that the extracellular generation of HOCl was temporally retarded, but the quantity eventually attained a level comparable with its intraphagosomal counterpart with a delay of about 1.5 h. The quantity of HOCl secreted from the hepatic leukocytes of rats with or without stimulation of lipopolysaccharide was also determined. These results indicate a possibility to extend our approach to not only clinical settings for quantitative assessment of the bactericidal capability of isolated leukocytes of patients but also fundamental biomedical research that requires critical evaluation of the inflammatory response of animals. PMID:21950322

  5. 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

    2015-07-01

    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. PMID:25947077

  6. Validation of RPS13 as a reference gene for absolute quantification of SIV RNA in tissue of rhesus macaques.

    PubMed

    Robichaux, Spencer; Lacour, Nedra; Bagby, Gregory J; Amedee, Angela M

    2016-10-01

    Persistent HIV reservoirs and the absolute quantification of viral RNA copies in tissues have become a prominent focus of multiple areas ofHIV/SIV research. Absolute quantification of viral RNA via reverse transcription, quantitative PCR (RT-qPCR) necessitates the use of an appropriate RNA reference gene whose expression is unaffected by both experimental and confounding conditions. In this study, we demonstrate the utility of ribosomal protein S13 mRNA (RPS13) as a stable, medium abundance reference gene for RT-qPCR normalization of HIV/SIV RNA copy number. We developed a RPS13 RNA standard assay utilizing an in vitro RNA transcript for normalization of absolute SIV RNA quantities in tissues reservoirs. The RT-qPCR assay showed a high degree of repeatability and reproducibility across RNA levels appropriate for absolute SIV quantification. In assessing the utility of RPS13 as a reference gene, limited variation in the absolute, inter-tissue quantities of RPS13 mRNA was observed within multiple tissue samples obtained from rhesus macaques (average CV=2.86%). We demonstrate rhesus macaque RPS13 mRNA expression is not affected by alcohol administration, SIV infection, or antiviral therapy (PMPA/FTC). Additionally, assay functionality was validated for normalization of SIV copy number using cellular RNA prepared from samples of variable RNA integrity. RPS13 is a suitable reference gene for normalization of absolute SIV RNA quantities in tissues and is most appropriate for intra-tissue or similar tissue type comparisons of SIV copy number. PMID:27510462

  7. An improved generalized Newton method for absolute value equations.

    PubMed

    Feng, Jingmei; Liu, Sanyang

    2016-01-01

    In this paper, we suggest and analyze an improved generalized Newton method for solving the NP-hard absolute value equations [Formula: see text] when the singular values of A exceed 1. We show that the global and local quadratic convergence of the proposed method. Numerical experiments show the efficiency of the method and the high accuracy of calculation. PMID:27462490

  8. Harnessing immunomagnetic separation and quantum dot-based quantification capacities for the enumeration of absolute levels of biomarker

    NASA Astrophysics Data System (ADS)

    Park, Hoyoung; Hwang, Mintai P.; Lee, Jong-Wook; Choi, Jonghoon; Hyi Lee, Kwan

    2013-07-01

    The field of biomarker quantification has experienced a growth parallel to the discovery of new materials. In this paper, we propose an innovative system for the separation and quantification of biomarkers using a simple magnetic bead (MB)-quantum dot (QD) sandwich assay. The basis of the system lies in the interaction between histidine residues on protein G and Ni ions on QDs, and the use of imidazole to selectively detach QDs bound to target biomarkers, in effect enumerating the absolute number of biomarker units. We used C-reactive protein (CRP) as a proof-of-concept and demonstrated a detection sensitivity of 82.5 fmoles in 50 μl of sample volume, a commonly used analytical volume (e.g. ELISA). Although CRP was used as a model to conduct this study, the sensitivity and simplicity of this detachable system make it a viable approach in the quantification of other target analytes.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. Invalid phase values removal method for absolute phase recovery.

    PubMed

    Lu, Jin; Mo, Rong; Sun, Huibin; Chang, Zhiyong; Zhao, Xiaxia

    2016-01-10

    A novel approach is presented for more effectively removing invalid phase values in absolute phase recovery. The approach is based on a detailed study involving the types and cases of invalid phase values. Meanwhile, some commonalities of the existing removal algorithms also are thoroughly analyzed. It is well known that rough absolute phase and fringe order maps can very easily be obtained by temporal phase unwrapping techniques. After carefully analyzing the components and fringe order distribution of the rough fringe order map, the proposed method chiefly adopts an entirely new strategy to refine a pure fringe order map. The strategy consists of three parts: (1) the square of an image gradient, (2) subregion areas of the binary image, and (3) image decomposition and composition. In combination with the pure fringe order map and a removal criterion, the invalid phase values can be identified and filtered out from the rough absolute phase map. This new strategy not only gets rid of the limitations of traditional removal methods but also has a two-fold function. The paper also offers different metrics from the experiment to evaluate the quality of the final absolute phase. In contrast with other removal methods, experimental results have verified the feasibility, effectiveness, and superiority of the proposed method. PMID:26835776

  14. Quantification of Absolute Fat Mass by Magnetic Resonance Imaging: a Validation Study against Chemical Analysis

    PubMed Central

    Hu, Houchun H.; Li, Yan; Nagy, Tim R.; Goran, Michael I.; Nayak, Krishna S.

    2011-01-01

    Objective To develop a magnetic resonance imaging (MRI)-based approach for quantifying absolute fat mass in organs, muscles, and adipose tissues, and to validate its accuracy against reference chemical analysis (CA). Methods Chemical-shift imaging can accurately decompose water and fat signals from the acquired MRI data. A proton density fat fraction (PDFF) can be computed from the separated images, and reflects the relative fat content on a voxel-by-voxel basis. The PDFF is mathematically closely related to the fat mass fraction and can be converted to absolute fat mass in grams by multiplying by the voxel volume and the mass density of fat. In this validation study, 97 freshly excised and unique samples from four pigs, comprising of organs, muscles, and adipose and lean tissues were imaged by MRI and then analyzed independently by CA. Linear regression was used to assess correlation, agreement, and measurement differences between MRI and CA. Results Considering all 97 samples, a strong correlation and agreement was obtained between MRI and CA-derived fat mass (slope = 1.01, intercept = 1.99g, r2 = 0.98, p < 0.01). The mean difference d between MRI and CA was 2.17±3.40g. MRI did not exhibit any tendency to under or overestimate CA (p > 0.05). When considering samples from each pig separately, the results were (slope = 1.05, intercept = 1.11g, r2 = 0.98, d = 2.66±4.36g), (slope = 0.99, intercept = 2.33g, r2 = 0.99, d = 1.88±2.68g), (slope = 1.07, intercept = 1.52g, r2 = 0.96, d = 2.73±2.50g), and (slope=0.92, intercept=2.84g, r2 = 0.97, d = 1.18±3.90g), respectively. Conclusion Chemical-shift MRI and PDFF provides an accurate means of determining absolute fat mass in organs, muscles, and adipose and lean tissues. PMID:23204926

  15. 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.

  16. Simultaneous absolute quantification of 11 cytochrome P450 isoforms in human liver microsomes by liquid chromatography tandem mass spectrometry with in silico target peptide selection.

    PubMed

    Kawakami, Hirotaka; Ohtsuki, Sumio; Kamiie, Junichi; Suzuki, Takashi; Abe, Takaaki; Terasaki, Tetsuya

    2011-01-01

    Cytochrome P450 (CYP) proteins are involved in the biological oxidation and reduction of xenobiotics, affecting the pharmacological efficiency of drugs. This study aimed to establish a method to simultaneously quantify 11 CYP isoforms by multiplexed-multiple reaction monitoring analysis with liquid chromatography tandem mass spectrometry and in silico peptide selection to clarify CYP isoform expression profiles in human liver tissue. CYP1A2, 2A6, and 2D6 target peptides were identified by shot-gun proteomic analysis, and those of other isoforms were selected by in silico peptide selection criteria. The established quantification method detected target peptides at 10  fmol, and the dynamic range of calibration curves was at least 500-fold. The quantification value of CYP1A2 in Supersomes was not significantly different between the established method and quantitative immunoblot analysis. The absolute protein expression levels of 11 CYP isoforms were determined from one pooled and 10 individual human liver microsomes. In the individual microsomes, CYP2C9 showed the highest protein expression level, and CYP1A2, 2A6, 2C19, and 3A4 protein expression exhibited more than a 20-fold difference among individuals. This highly sensitive and selective quantification method is a useful tool for the analysis of highly homologous CYP isoforms and the contribution made by each CYP isoform to drug metabolism. PMID:20564338

  17. Peptide Biosynthesis with Stable Isotope Labeling from a Cell-free Expression System for Targeted Proteomics with Absolute Quantification.

    PubMed

    Xian, Feng; Zi, Jin; Wang, Quanhui; Lou, Xiaomin; Sun, Haidan; Lin, Liang; Hou, Guixue; Rao, Weiqiao; Yin, Changcheng; Wu, Lin; Li, Shuwei; Liu, Siqi

    2016-08-01

    Because of its specificity and sensitivity, targeted proteomics using mass spectrometry for multiple reaction monitoring is a powerful tool to detect and quantify pre-selected peptides from a complex background and facilitates the absolute quantification of peptides using isotope-labeled forms as internal standards. How to generate isotope-labeled peptides remains an urgent challenge for accurately quantitative targeted proteomics on a large scale. Herein, we propose that isotope-labeled peptides fused with a quantitative tag could be synthesized through an expression system in vitro, and the homemade peptides could be enriched by magnetic beads with tag-affinity and globally quantified based on the corresponding multiple reaction monitoring signals provided by the fused tag. An Escherichia coli cell-free protein expression system, protein synthesis using recombinant elements, was adopted for the synthesis of isotope-labeled peptides fused with Strep-tag. Through a series of optimizations, we enabled efficient expression of the labeled peptides such that, after Strep-Tactin affinity enrichment, the peptide yield was acceptable in scale for quantification, and the peptides could be completely digested by trypsin to release the Strep-tag for quantification. Moreover, these recombinant peptides could be employed in the same way as synthetic peptides for multiple reaction monitoring applications and are likely more economical and useful in a laboratory for the scale of targeted proteomics. As an application, we synthesized four isotope-labeled glutathione S-transferase (GST) peptides and added them to mouse sera pre-treated with GST affinity resin as internal standards. A quantitative assay of the synthesized GST peptides confirmed the absolute GST quantification in mouse sera to be measurable and reproducible. PMID:27234506

  18. 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.

  19. The advantage of absolute quantification in comparative hormone research as indicated by a newly established real-time RT-PCR: GH, IGF-I, and IGF-II gene expression in the tilapia, Oreochromis niloticus.

    PubMed

    Eppler, Elisabeth; Caelers, Antje; Berishvili, Giorgi; Reinecke, Manfred

    2005-04-01

    We have developed a real-time RT-PCR that absolutely quantifies the gene expression of hormones using the standard curve method. The method avoids cloning procedures by using primer extension to create templates containing a T7 promoter gene sequence. It is rapid since neither separate reverse transcriptions nor postamplification steps are necessary, and its low detection level (2 pg/mug total RNA) allows precise absolute quantification. Using the method, we have quantified the gene expression of GH, IGF-I, and IGF-II in the tilapia. PMID:15891047

  20. Absolute Quantification of Enterococcal 23S rRNA Gene Using Digital PCR.

    PubMed

    Wang, Dan; Yamahara, Kevan M; Cao, Yiping; Boehm, Alexandria B

    2016-04-01

    We evaluated the ability of chip-based digital PCR (dPCR) to quantify enterococci, the fecal indicator recommended by the United States Environmental Protection Agency (USEPA) for water-quality monitoring. dPCR uses Poisson statistics to estimate the number of DNA fragments in a sample with a specific sequence. Underestimation may occur when a gene is redundantly encoded in the genome and multiple copies of that gene are on one DNA fragment. When genomic DNA (gDNA) was extracted using two commercial DNA extraction kits, we confirmed that dPCR could discern individual copies of the redundant 23s rRNA gene in the enterococcal genome. dPCR quantification was accurate when compared to the nominal concentration inferred from fluorometer measurements (linear regression slope = 0.98, intercept = 0.03, R(2) = 0.99, and p value <0.0001). dPCR quantification was also consistent with quantitative PCR (qPCR) measurements as well as cell counts for BioBall reference standard and 24 environmental water samples. qPCR and dPCR quantification of enterococci in the 24 environmental samples were significantly correlated (linear regression slope =1.08, R(2) of 0.96, and p value <0.0001); the group mean of the qPCR measurements was 0.19 log units higher than that of the dPCR measurements. At environmentally relevant concentrations, dPCR quantification was more precise (i.e., had narrower 95% confidence intervals than qPCR quantification). We observed that humic acid caused a similar level of inhibition in both dPCR and qPCR, but calcium inhibited dPCR to a lesser degree than qPCR. Inhibition of dPCR was partially relieved when the number of thermal cycles was increased. Based on these results, we conclude that dPCR is a viable option for enumerating enterococci in ambient water. PMID:26903207

  1. 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.

  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

    2007-07-17

    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.

  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

    DOEpatents

    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. Toward absolute quantification of iron oxide nanoparticles as well as cell internalized fraction using multiparametric MRI

    PubMed Central

    Girard, O. M.; Ramirez, R.; McCarty, S.; Mattrey, R. F.

    2012-01-01

    Iron oxide nanoparticles (IONPs) are widely used as MR contrast agents because of their strong magnetic properties and broad range of applications. The contrast induced by IONPs typically depends on concentration, water accessibility, particle size, and heterogeneity of IONP distribution within the microenvironment. Although the latter could be a tool to assess local physiological effects at the molecular level, it renders IONP quantification from relaxation measurements challenging. This study aims to quantify IONP concentration using susceptibility measurements. In addition, further analysis of relaxation data is proposed to extract quantitative information about the IONP spatial distribution. Mesenchymal stem cells were labeled with IONPs and the IONP concentration measured by mass spectroscopy. MR relaxation parameters (T1, T2, T2*) as well as magnetic susceptibility of cylindrical samples containing serial dilutions of mixtures of free and cell-internalized IONPs were measured and correlated with IONP concentration. Unlike relaxation data, magnetic susceptibility was independent of whether IONPs were free or internalized, making it an excellent candidate for IONP quantification. Using IONP concentration derived from mass spectroscopy and measured relaxation times, free and internalized IONP fractions were accurately calculated. Magnetic susceptibility was shown to be a robust technique to measure IONP concentration in this preliminary study. Novel imaging based susceptibility mapping techniques could prove to be valuable tools to quantify IONP concentration directly by MRI, for samples of arbitrarily shape. Combined with relaxation time mapping techniques, especially T2 and T2*, this could be an efficient way to measure both IONP concentration and the internalized IONP fraction in vivo using MRI, to gain insight into tissue function and molecular imaging paradigms. PMID:22649047

  7. Absolute quantification of cerebral blood flow in neurologically normal volunteers: dynamic-susceptibility contrast MRI-perfusion compared with computed tomography (CT)-perfusion.

    PubMed

    Ziegelitz, Doerthe; Starck, Göran; Mikkelsen, Irene K; Tullberg, Mats; Edsbagge, Mikael; Wikkelsö, Carsten; Forssell-Aronson, Eva; Holtås, Stig; Knutsson, Linda

    2009-07-01

    To improve the reproducibility of arterial input function (AIF) registration and absolute cerebral blood flow (CBF) quantification in dynamic-susceptibility MRI-perfusion (MRP) at 1.5T, we rescaled the AIF by use of a venous output function (VOF). We compared CBF estimates of 20 healthy, elderly volunteers, obtained by computed tomography (CT)-perfusion (CTP) and MRP on two consecutive days. MRP, calculated without the AIF correction, did not result in any significant correlation with CTP. The rescaled MRP showed fair to moderate correlation with CTP for the central gray matter (GM) and the whole brain. Our results indicate that the method used for correction of partial volume effects (PVEs) improves MRP experiments by reducing AIF-introduced variance at 1.5T. PMID:19253361

  8. Absolute mRNA quantification using the polymerase chain reaction (PCR). A novel approach by a PCR aided transcript titration assay (PATTY).

    PubMed Central

    Becker-André, M; Hahlbrock, K

    1989-01-01

    The polymerase chain reaction (PCR) is used as part of a new approach to the absolute quantification of mRNA. We describe a PCR aided transcript titration assay (PATTY) which is based on the co-amplification of an in vitro generated transcript differing by a single base exchange from the target mRNA. Identical portions of a total RNA sample are "spiked" with different amounts of this mutated standard RNA, converted to cDNA and amplified by PCR. Because the base exchange creates a novel restriction endonuclease site, the ratio of co-amplified DNA derived from target mRNA to amplified DNA derived from standard RNA can be determined after restriction endonuclease digestion and separation by gel electrophoresis. This method gives accurate results within 24 hours and is useful especially for the quantification of either low-abundance mRNA or more abundant mRNA present in very small amounts of total RNA. The low-abundance mRNA encoding 4-coumarate:CoA ligase (4CL) in cultured potato cells (Solanum tuberosum L.) was measured in a case study. About 100 molecules per assay could be accurately detected by the new method. Images PMID:2479917

  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. 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.

  11. Rapid, absolute, and simultaneous quantification of specific pathogenic strain and total bacterial cells using an ultrasensitive dual-color flow cytometer.

    PubMed

    Yang, Lingling; Wu, Lina; Zhu, Shaobin; Long, Yao; Hang, Wei; Yan, Xiaomei

    2010-02-01

    This paper describes a rapid and sensitive strategy for the absolute and simultaneous quantification of specific pathogenic strain and total bacterial cells in a mixture. A laboratory-built compact, high-sensitivity, dual channel flow cytometer (HSDCFCM) was modified to enable dual fluorescence detection. A bacterial cell mixture comprising heat-killed pathogenic Escherichia coli E. coli O157:H7 and harmless E. coli DH5alpha was used as a model system. Pathogenic E. coli O157:H7 cells were selectively labeled by red fluorescent probe via antibody-antigen interaction, and all bacterial cells were stained with membrane-permeable nucleic acid dye that fluoresces green. When each individual bacterium passes through the interrogating laser beam, E. coli O157:H7 emits both red and green fluorescence, while E. coli DH5alpha exhibits only green fluorescence. Because the fluorescence burst generated from each individual bacterial cell was easily distinguished from the background, accurate enumeration and consequently absolute quantification were achieved for both pathogenic and total bacterial cells. By using this strategy, accurate counting of bacteria at a density above 1.0 x 10(5) cells/mL can be accomplished with 1 min of data acquisition time after fluorescent staining. Excellent correlation between the concentrations measured by the HSDCFCM and the conventional plate-counting method were obtained for pure-cultured E. coli O157:H7 (R(2) = 0.9993) and E. coli DH5alpha (R(2) = 0.9998). Bacterial cell mixtures with varying proportions of E. coli O157:H7 and E. coli DH5alpha were measured with good ratio correspondence. We applied the established approach to detecting artificially contaminated drinking water samples; E. coli O157:H7 of 1.0 x 10(2) cells/mL were accurately quantified upon sample enrichment. It is believed that the proposed method will find wide applications in many fields demanding bacterial identification and quantification. PMID:20039721

  12. Method and apparatus for making absolute range measurements

    DOEpatents

    Allison, Stephen W.; Cates, Michael R.; Key, William S.; Sanders, Alvin J.; Earl, Dennis D.

    1999-01-01

    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 an object which causes it to be split (hereinafter referred to as a "beamsplitter"), and then to a target. The beam is reflected from the target onto a screen containing an aperture spaced a known distance from the beamsplitter. The aperture is sized so as to produce a Fresnel diffraction pattern. A portion of the beam travels through the aperture to a detector, spaced a known distance from the screen. The detector detects the central intensity of the beam. The distance from the object which causes the beam to be split to the target can then be calculated based upon the known wavelength, aperture radius, beam intensity, and distance from the detector to the screen. Several apparatus embodiments are disclosed for practicing the method embodiments of the present invention.

  13. Method and apparatus for making absolute range measurements

    DOEpatents

    Allison, S.W.; Cates, M.R.; Key, W.S.; Sanders, A.J.; Earl, D.D.

    1999-06-22

    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 an object which causes it to be split (hereinafter referred to as a beam splitter''), and then to a target. The beam is reflected from the target onto a screen containing an aperture spaced a known distance from the beam splitter. The aperture is sized so as to produce a Fresnel diffraction pattern. A portion of the beam travels through the aperture to a detector, spaced a known distance from the screen. The detector detects the central intensity of the beam. The distance from the object which causes the beam to be split to the target can then be calculated based upon the known wavelength, aperture radius, beam intensity, and distance from the detector to the screen. Several apparatus embodiments are disclosed for practicing the method embodiments of the present invention. 9 figs.

  14. 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

  15. RNAontheBENCH: computational and empirical resources for benchmarking RNAseq quantification and differential expression methods.

    PubMed

    Germain, Pierre-Luc; Vitriolo, Alessandro; Adamo, Antonio; Laise, Pasquale; Das, Vivek; Testa, Giuseppe

    2016-06-20

    RNA sequencing (RNAseq) has become the method of choice for transcriptome analysis, yet no consensus exists as to the most appropriate pipeline for its analysis, with current benchmarks suffering important limitations. Here, we address these challenges through a rich benchmarking resource harnessing (i) two RNAseq datasets including ERCC ExFold spike-ins; (ii) Nanostring measurements of a panel of 150 genes on the same samples; (iii) a set of internal, genetically-determined controls; (iv) a reanalysis of the SEQC dataset; and (v) a focus on relative quantification (i.e. across-samples). We use this resource to compare different approaches to each step of RNAseq analysis, from alignment to differential expression testing. We show that methods providing the best absolute quantification do not necessarily provide good relative quantification across samples, that count-based methods are superior for gene-level relative quantification, and that the new generation of pseudo-alignment-based software performs as well as established methods, at a fraction of the computing time. We also assess the impact of library type and size on quantification and differential expression analysis. Finally, we have created a R package and a web platform to enable the simple and streamlined application of this resource to the benchmarking of future methods. PMID:27190234

  16. RNAontheBENCH: computational and empirical resources for benchmarking RNAseq quantification and differential expression methods

    PubMed Central

    Germain, Pierre-Luc; Vitriolo, Alessandro; Adamo, Antonio; Laise, Pasquale; Das, Vivek; Testa, Giuseppe

    2016-01-01

    RNA sequencing (RNAseq) has become the method of choice for transcriptome analysis, yet no consensus exists as to the most appropriate pipeline for its analysis, with current benchmarks suffering important limitations. Here, we address these challenges through a rich benchmarking resource harnessing (i) two RNAseq datasets including ERCC ExFold spike-ins; (ii) Nanostring measurements of a panel of 150 genes on the same samples; (iii) a set of internal, genetically-determined controls; (iv) a reanalysis of the SEQC dataset; and (v) a focus on relative quantification (i.e. across-samples). We use this resource to compare different approaches to each step of RNAseq analysis, from alignment to differential expression testing. We show that methods providing the best absolute quantification do not necessarily provide good relative quantification across samples, that count-based methods are superior for gene-level relative quantification, and that the new generation of pseudo-alignment-based software performs as well as established methods, at a fraction of the computing time. We also assess the impact of library type and size on quantification and differential expression analysis. Finally, we have created a R package and a web platform to enable the simple and streamlined application of this resource to the benchmarking of future methods. PMID:27190234

  17. 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

  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

    2015-10-01

    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. PMID:26325666

  19. Absolute Quantification of Matrix Metabolites Reveals the Dynamics of Mitochondrial Metabolism.

    PubMed

    Chen, Walter W; Freinkman, Elizaveta; Wang, Tim; Birsoy, Kıvanç; Sabatini, David M

    2016-08-25

    Mitochondria house metabolic pathways that impact most aspects of cellular physiology. While metabolite profiling by mass spectrometry is widely applied at the whole-cell level, it is not routinely possible to measure the concentrations of small molecules in mammalian organelles. We describe a method for the rapid and specific isolation of mitochondria and use it in tandem with a database of predicted mitochondrial metabolites ("MITObolome") to measure the matrix concentrations of more than 100 metabolites across various states of respiratory chain (RC) function. Disruption of the RC reveals extensive compartmentalization of mitochondrial metabolism and signatures unique to the inhibition of each RC complex. Pyruvate enables the proliferation of RC-deficient cells but has surprisingly limited effects on matrix contents. Interestingly, despite failing to restore matrix NADH/NAD balance, pyruvate does increase aspartate, likely through the exchange of matrix glutamate for cytosolic aspartate. We demonstrate the value of mitochondrial metabolite profiling and describe a strategy applicable to other organelles. PMID:27565352

  20. Absolute quantification of the pretreatment PML-RARA transcript defines the relapse risk in acute promyelocytic leukemia.

    PubMed

    Albano, Francesco; Zagaria, Antonella; Anelli, Luisa; Coccaro, Nicoletta; Tota, Giuseppina; Brunetti, Claudia; Minervini, Crescenzio Francesco; Impera, Luciana; Minervini, Angela; Cellamare, Angelo; Orsini, Paola; Cumbo, Cosimo; Casieri, Paola; Specchia, Giorgina

    2015-05-30

    In this study we performed absolute quantification of the PML-RARA transcript by droplet digital polymerase chain reaction (ddPCR) in 76 newly diagnosed acute promyelocytic leukemia (APL) cases to verify the prognostic impact of the PML-RARA initial molecular burden. ddPCR analysis revealed that the amount of PML-RARA transcript at diagnosis in the group of patients who relapsed was higher than in that with continuous complete remission (CCR) (272 vs 89.2 PML-RARA copies/ng, p = 0.0004, respectively). Receiver operating characteristic analysis detected the optimal PML-RARA concentration threshold as 209.6 PML-RARA/ng (AUC 0.78; p < 0.0001) for discriminating between outcomes (CCR versus relapse). Among the 67 APL cases who achieved complete remission after the induction treatment, those with >209.6 PML-RARA/ng had a worse relapse-free survival (p = 0.0006). At 5-year follow-up, patients with >209.6 PML-RARA/ng had a cumulative incidence of relapse of 50.3% whereas 7.5% of the patients with suffered a relapse (p < 0.0001). Multivariate analysis identified the amount of PML-RARA before induction treatment as the sole independent prognostic factor for APL relapse.Our results show that the pretreatment PML-RARA molecular burden could therefore be used to improve risk stratification in order to develop more individualized treatment regimens for high-risk APL cases. PMID:25944686

  1. 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. PMID:26988485

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

    PubMed

    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 (V̇O(2)) in exercising skeletal muscle. Healthy subjects (n=9) performed a handgrip exercise to increase BF and V̇O(2) in forearm flexor muscles, while a hybrid optical probe on the skin surface directly monitored oxy-, deoxy-, and total hemoglobin concentrations ([HbO(2)], [Hb], and THC), tissue oxygen saturation (S(t)O(2)), relative BF (rBF), and relative oxygen consumption rate (rV̇O(2)). The rBF and rV̇O(2) signals were calibrated with absolute baseline BF and V̇O(2) 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

  3. 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

  4. 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.

  5. Absolute Quantification of Norovirus Capsid Protein in Food, Water, and Soil Using Synthetic Peptides with Electrospray and MALDI Mass Spectrometry

    PubMed Central

    Hartmann, Erica M.; Colquhoun, David R.; Schwab, Kellogg J.; Halden, Rolf U.

    2015-01-01

    Norovirus infections are one of the most prominent public health problems of microbial origin in the U.S. and other industrialized countries. Surveillance is necessary to prevent secondary infection, confirm successful cleanup after outbreaks, and track the causative agent. Quantitative mass spectrometry, based on absolute quantitation with stable-isotope labeled peptides, is a promising tool for norovirus monitoring because of its speed, sensitivity, and robustness in the face of environmental inhibitors. In the current study, we present two new methods for the detection of the norovirus genogroup I capsid protein using electrospray and matrixassisted laser desorption/ionization (MALDI) mass spectrometry. The peptide TLDPIEVPLEDVR was used to quantify norovirus-like particles down to 500 attomoles with electrospray and 100 attomoles with MALDI. With MALDI, we also demonstrate a detection limit of 1 femtomole and a quantitative dynamic range of 5 orders of magnitude in the presence of an environmental matrix effect. Due to the rapid processing time and applicability to a wide range of environmental sample types (bacterial lysate, produce, milk, soil, and groundwater), mass spectrometry-based absolute quantitation has a strong potential for use in public health and environmental sciences. PMID:25603302

  6. Absolute quantification of norovirus capsid protein in food, water, and soil using synthetic peptides with electrospray and MALDI mass spectrometry.

    PubMed

    Hartmann, Erica M; Colquhoun, David R; Schwab, Kellogg J; Halden, Rolf U

    2015-04-01

    Norovirus infections are one of the most prominent public health problems of microbial origin in the U.S. and other industrialized countries. Surveillance is necessary to prevent secondary infection, confirm successful cleanup after outbreaks, and track the causative agent. Quantitative mass spectrometry, based on absolute quantitation with stable-isotope labeled peptides, is a promising tool for norovirus monitoring because of its speed, sensitivity, and robustness in the face of environmental inhibitors. In the current study, we present two new methods for the detection of the norovirus genogroup I capsid protein using electrospray and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The peptide TLDPIEVPLEDVR was used to quantify norovirus-like particles down to 500 attomoles with electrospray and 100 attomoles with MALDI. With MALDI, we also demonstrate a detection limit of 1 femtomole and a quantitative dynamic range of 5 orders of magnitude in the presence of an environmental matrix effect. Due to the rapid processing time and applicability to a wide range of environmental sample types (bacterial lysate, produce, milk, soil, and groundwater), mass spectrometry-based absolute quantitation has a strong potential for use in public health and environmental sciences. PMID:25603302

  7. 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. PMID:27050419

  8. Absolute quantification of superoxide dismutase in cytosol and mitochondria of mice hepatic cells exposed to mercury by a novel metallomic approach.

    PubMed

    García-Sevillano, M A; García-Barrera, T; Navarro, F; Gómez-Ariza, J L

    2014-09-01

    In the last years, the development of new methods for analyzing accurate and precise individual metalloproteins is of increasing importance, since numerous metalloproteins are excellent biomarkers of oxidative stress and diseases. In that way, methods based on the use of post column isotopic dilution analysis (IDA) or enriched protein standards are required to obtain a sufficient degree of accuracy, precision and high limits of detection. This paper reports the identification and absolute quantification of Cu,Zn-superoxide dismutase (Cu,Zn-SOD) in cytosol and mitochondria from mice hepatic cells using a innovative column switching analytical approach. The method consisted of orthogonal chromatographic systems coupled to inductively coupling plasma-mass spectrometry equipped with a octopole reaction systems (ICP-ORS-MS) and UV detectors: size exclusion fractionation (SEC) of the cytosolic and mitochondrial extracts followed by online anion exchange chromatographic (AEC) separation of Cu/Zn containing species. After purification, Cu,Zn-SOD was identified after tryptic digestion by molecular mass spectrometry (MS). The MS/MS spectrum of a doubly charged peptide was used to obtain the sequence of the protein using the MASCOT searching engine. This optimized methodology reduces the time of analysis and avoids the use of sample preconcentration and clean-up procedures, such as cut-off centrifuged filters, solid phase extraction (SPE), precipitation procedures, off-line fractions insolates, etc. In this sense, the method is robust, reliable and fast with typical chromatographic run time less than 20 min. Precision in terms of relative standard deviation (n = 5) is of 3-5% and detection limits is 0.21 ngCug(-1). The application of the methodology to hepatic cells from mice exposed to inorganic mercury reveals decreased levels of Cu,Zn-SOD in cytosolic and mitochondrial extracts, as a consequence of the oxidative stress caused by this toxic metal. Additionally, the

  9. 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.

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

    PubMed Central

    Rich, Kyle T.; Mast, T. Douglas

    2015-01-01

    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. PMID:26428812

  11. 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. PMID:26428812

  12. 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. PMID:26205729

  13. Pyridoxamine-5-phosphate enzyme-linked immune mass spectrometric assay substrate for linear absolute quantification of alkaline phosphatase to the yoctomole range applied to prostate specific antigen.

    PubMed

    Florentinus-Mefailoski, Angelique; Marshall, John G

    2014-11-01

    There is a need to measure proteins that are present in concentrations below the detection limits of existing colorimetric approaches with enzyme-linked immunoabsorbent assays (ELISA). The powerful enzyme alkaline phosphatase conjugated to the highly specific bacterial protein streptavidin binds to biotinylated macromolecules like proteins, antibodies, or other ligands and receptors with a high affinity. The binding of the biotinylated detection antibody, with resulting amplification of the signal by the catalytic production of reporter molecules, is key to the sensitivity of ELISA. The specificity and amplification of the signal by the enzyme alkaline phosphatase in ELISA together with the sensitivity of liquid chromatography electrospray ionization and mass spectrometry (LC-ESI-MS) to detect femtomole to picomole amounts of reporter molecules results in an ultrasensitive enzyme-linked immune mass spectrometric assay (ELIMSA). The novel ELIMSA substrate pyridoxamine-5-phosphate (PA5P) is cleaved by the enzyme alkaline phosphatase to yield the basic and hydrophilic product pyridoxamine (PA) that elutes rapidly with symmetrical peaks and a flat baseline. Pyridoxamine (PA) and (13)C PA were both observed to show a linear relationship between log ion intensity and quantity from picomole to femtomole amounts by liquid chromatography-electrospray ionization and mass spectrometry. Four independent methods, (i) internal (13)C isotope PA dilution curves, (ii) internal (13)C isotope one-point calibration, (iii) external PA standard curve, and (iv) external (13)C PA standard curve, all agreed within 1 digit in the same order of magnitude on the linear quantification of PA. Hence, a mass spectrometer can be used to robustly detect 526 ymol of the alkaline phosphatase streptavidin probe and accurately quantify zeptomole amounts of PSA against log linear absolute standard by micro electrospray on a simple ion trap. PMID:25259405

  14. 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. PMID:24482001

  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 t...

  17. 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...

  18. Comparison of DNA Quantification Methods for Next Generation Sequencing

    PubMed Central

    Robin, Jérôme D.; Ludlow, Andrew T.; LaRanger, Ryan; Wright, Woodring E.; Shay, Jerry W.

    2016-01-01

    Next Generation Sequencing (NGS) is a powerful tool that depends on loading a precise amount of DNA onto a flowcell. NGS strategies have expanded our ability to investigate genomic phenomena by referencing mutations in cancer and diseases through large-scale genotyping, developing methods to map rare chromatin interactions (4C; 5C and Hi-C) and identifying chromatin features associated with regulatory elements (ChIP-seq, Bis-Seq, ChiA-PET). While many methods are available for DNA library quantification, there is no unambiguous gold standard. Most techniques use PCR to amplify DNA libraries to obtain sufficient quantities for optical density measurement. However, increased PCR cycles can distort the library’s heterogeneity and prevent the detection of rare variants. In this analysis, we compared new digital PCR technologies (droplet digital PCR; ddPCR, ddPCR-Tail) with standard methods for the titration of NGS libraries. DdPCR-Tail is comparable to qPCR and fluorometry (QuBit) and allows sensitive quantification by analysis of barcode repartition after sequencing of multiplexed samples. This study provides a direct comparison between quantification methods throughout a complete sequencing experiment and provides the impetus to use ddPCR-based quantification for improvement of NGS quality. PMID:27048884

  19. Comparison of DNA Quantification Methods for Next Generation Sequencing.

    PubMed

    Robin, Jérôme D; Ludlow, Andrew T; LaRanger, Ryan; Wright, Woodring E; Shay, Jerry W

    2016-01-01

    Next Generation Sequencing (NGS) is a powerful tool that depends on loading a precise amount of DNA onto a flowcell. NGS strategies have expanded our ability to investigate genomic phenomena by referencing mutations in cancer and diseases through large-scale genotyping, developing methods to map rare chromatin interactions (4C; 5C and Hi-C) and identifying chromatin features associated with regulatory elements (ChIP-seq, Bis-Seq, ChiA-PET). While many methods are available for DNA library quantification, there is no unambiguous gold standard. Most techniques use PCR to amplify DNA libraries to obtain sufficient quantities for optical density measurement. However, increased PCR cycles can distort the library's heterogeneity and prevent the detection of rare variants. In this analysis, we compared new digital PCR technologies (droplet digital PCR; ddPCR, ddPCR-Tail) with standard methods for the titration of NGS libraries. DdPCR-Tail is comparable to qPCR and fluorometry (QuBit) and allows sensitive quantification by analysis of barcode repartition after sequencing of multiplexed samples. This study provides a direct comparison between quantification methods throughout a complete sequencing experiment and provides the impetus to use ddPCR-based quantification for improvement of NGS quality. PMID:27048884

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

    PubMed

    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

  1. Metrological activity determination of 133Ba by sum-peak absolute method

    NASA Astrophysics Data System (ADS)

    da Silva, R. L.; de Almeida, M. C. M.; Delgado, J. U.; Poledna, R.; Santos, A.; de Veras, E. V.; Rangel, J.; Trindade, O. L.

    2016-07-01

    The National Laboratory for Metrology of Ionizing Radiation provides gamma sources of radionuclide and standardized in activity with reduced uncertainties. Relative methods require standards to determine the sample activity while the absolute methods, as sum-peak, not. The activity is obtained directly with good accuracy and low uncertainties. 133Ba is used in research laboratories and on calibration of detectors for analysis in different work areas. Classical absolute methods don't calibrate 133Ba due to its complex decay scheme. The sum-peak method using gamma spectrometry with germanium detector standardizes 133Ba samples. Uncertainties lower than 1% to activity results were obtained.

  2. Counting the platelets: a robust and sensitive quantification method for thrombus formation.

    PubMed

    Claesson, Kjersti; Lindahl, Tomas L; Faxälv, Lars

    2016-06-01

    Flow chambers are common tools used for studying thrombus formation in vitro. However, the use of such devices is not standardised and there is a large diversity among the flow chamber systems currently used, and also in the methods used for quantifying the thrombus development. It was the study objective to evaluate a new method for analysis and quantification of platelet thrombus formation that can facilitate comparison of results between research groups. Whole blood was drawn over a collagen patch in commercial Ibid or in-house constructed PDMS flow chambers. Five percent of the platelets were fluorescently labelled and z-stack time-lapse images were captured during thrombus formation. Images were processed in a Python script in which the number of platelets and their respective x-, y- and z-positions were obtained. For comparison with existing methods the platelets were also labelled and quantified using fluorescence intensity and thrombus volume estimations by confocal microscopy. The presented method was found less sensitive to microscope and image adjustments and provides more details on thrombus development dynamics than the methods for measuring fluorescence intensity and thrombus volume estimation. The platelet count method produced comparable results with commercial and PDMS flow chambers, and could also obtain information regarding the stability of each detected platelet in the thrombus. In conclusion, quantification of thrombus formation by platelet count is a sensitive and robust method that enables measurement of platelet accumulation and platelet stability in an absolute scale that could be used for comparisons between research groups. PMID:26842994

  3. 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. PMID:27617230

  4. Absolute measurement of optical flat surface shape based on the conjugate differential method.

    PubMed

    Huang, Ya; Ma, Jun; Zhu, Rihong; Yuan, Caojin; Chen, Lei; Cai, Huijuan; Sun, Weiyuan

    2015-11-16

    In this paper the conjugate differential method is proposed to measure the absolute surface shape of the flat mirror using a phase-shifting interferometer. The conjugate differential method is derived from the differential method, which extracts absolute phase differences by introducing the slight transverse shifts of the optic. It employs the measurement schemes making transverse shifts on the orthogonally bilateral symmetry positions. So the measurement procedures have been changed into four-step tests to get the phase difference map instead of three-step tests for the differential method. The precision of the slope approximation is enhanced by reducing couplings between multi-step tests, and the reliability of the measurements can be improved. Several differential wavefront reconstruction methods, such as Fourier transform, Zernike polynomial fitting and Hudgin model method, can be applied to reconstruct the absolute surface shape from the differencing phase maps in four different simulation environment. They were also used to reconstruct the absolute surface shape with the conjugate differential method in the experiment. Our method accords with the classical three-flat test better than the traditional differential method, where the deviation of RMS value between the conjugate differential method and the three-flat test is less than 0.3 nm. PMID:26698450

  5. Astigmatism error modification for absolute shape reconstruction using Fourier transform method

    NASA Astrophysics Data System (ADS)

    He, Yuhang; Li, Qiang; Gao, Bo; Liu, Ang; Xu, Kaiyuan; Wei, Xiaohong; Chai, Liqun

    2014-12-01

    A method is proposed to modify astigmatism errors in absolute shape reconstruction of optical plane using Fourier transform method. If a transmission and reflection flat are used in an absolute test, two translation measurements lead to obtain the absolute shapes by making use of the characteristic relationship between the differential and original shapes in spatial frequency domain. However, because the translation device cannot guarantee the test and reference flats rigidly parallel to each other after the translations, a tilt error exists in the obtained differential data, which caused power and astigmatism errors in the reconstructed shapes. In order to modify the astigmatism errors, a rotation measurement is added. Based on the rotation invariability of the form of Zernike polynomial in circular domain, the astigmatism terms are calculated by solving polynomial coefficient equations related to the rotation differential data, and subsequently the astigmatism terms including error are modified. Computer simulation proves the validity of the proposed method.

  6. Quantification of viral DNA during HIV-1 infection: A review of relevant clinical uses and laboratory methods.

    PubMed

    Alidjinou, E K; Bocket, L; Hober, D

    2015-02-01

    Effective antiretroviral therapy usually leads to undetectable HIV-1 RNA in the plasma. However, the virus persists in some cells of infected patients as various DNA forms, both integrated and unintegrated. This reservoir represents the greatest challenge to the complete cure of HIV-1 infection and its characteristics highly impact the course of the disease. The quantification of HIV-1 DNA in blood samples constitutes currently the most practical approach to measure this residual infection. Real-time quantitative PCR (qPCR) is the most common method used for HIV-DNA quantification and many strategies have been developed to measure the different forms of HIV-1 DNA. In the literature, several "in-house" PCR methods have been used and there is a need for standardization to have comparable results. In addition, qPCR is limited for the precise quantification of low levels by background noise. Among new assays in development, digital PCR was shown to allow an accurate quantification of HIV-1 DNA. Total HIV-1 DNA is most commonly measured in clinical routine. The absolute quantification of proviruses and unintegrated forms is more often used for research purposes. PMID:25201144

  7. Absolute quantification of Medicago truncatula sucrose synthase isoforms and N-metabolism enzymes in symbiotic root nodules and the detection of novel nodule phosphoproteins by mass spectrometry

    PubMed Central

    Wienkoop, Stefanie; Larrainzar, Estíbaliz; Glinski, Mirko; González, Esther M.; Arrese-Igor, Cesar; Weckwerth, Wolfram

    2008-01-01

    Mass spectrometry (MS) has become increasingly important for tissue specific protein quantification at the isoform level, as well as for the analysis of protein post-translational regulation mechanisms and turnover rates. Thanks to the development of high accuracy mass spectrometers, peptide sequencing without prior knowledge of the amino acid sequence—de novo sequencing—can be performed. In this work, absolute quantification of a set of key enzymes involved in carbon and nitrogen metabolism in Medicago truncatula ‘Jemalong A17’ root nodules is presented. Among them, sucrose synthase (SuSy; EC 2.4.1.13), one of the central enzymes in sucrose cleavage in root nodules, has been further characterized and the relative phosphorylation state of the three most abundant isoforms has been quantified. De novo sequencing provided sequence information of a so far unidentified peptide, most probably belonging to SuSy2, the second most abundant isoform in M. truncatula root nodules. TiO2-phosphopeptide enrichment led to the identification of not only a phosphorylation site at Ser11 in SuSy1, but also of several novel phosphorylation sites present in other root nodule proteins such as alkaline invertase (AI; EC 3.2.1.26) and an RNA-binding protein. PMID:18772307

  8. Assessment methods for angiogenesis and current approaches for its quantification.

    PubMed

    AlMalki, Waleed Hassan; Shahid, Imran; Mehdi, Abeer Yousaf; Hafeez, Muhammad Hassan

    2014-01-01

    Angiogenesis is a physiological process which describes the development of new blood vessels from the existing vessels. It is a common and the most important process in the formation and development of blood vessels, so it is supportive in the healing of wounds and granulation of tissues. The different assays for the evaluation of angiogenesis have been described with distinct advantages and some limitations. In order to develop angiogenic and antiangiogenic techniques, continuous efforts have been resulted to give animal models for more quantitative analysis of angiogenesis. Most of the studies on angiogenic inducers and inhibitors rely on various models, both in vitro, in vivo and in ova, as indicators of efficacy. The angiogenesis assays are very much helpful to test efficacy of both pro- and anti- angiogenic agents. The development of non-invasive procedures for quantification of angiogenesis will facilitate this process significantly. The main objective of this review article is to focus on the novel and existing methods of angiogenesis and their quantification techniques. These findings will be helpful to establish the most convenient methods for the detection, quantification of angiogenesis and to develop a novel, well tolerated and cost effective anti-angiogenic treatment in the near future. PMID:24987169

  9. Assessment methods for angiogenesis and current approaches for its quantification

    PubMed Central

    AlMalki, Waleed Hassan; Shahid, Imran; Mehdi, Abeer Yousaf; Hafeez, Muhammad Hassan

    2014-01-01

    Angiogenesis is a physiological process which describes the development of new blood vessels from the existing vessels. It is a common and the most important process in the formation and development of blood vessels, so it is supportive in the healing of wounds and granulation of tissues. The different assays for the evaluation of angiogenesis have been described with distinct advantages and some limitations. In order to develop angiogenic and antiangiogenic techniques, continuous efforts have been resulted to give animal models for more quantitative analysis of angiogenesis. Most of the studies on angiogenic inducers and inhibitors rely on various models, both in vitro, in vivo and in ova, as indicators of efficacy. The angiogenesis assays are very much helpful to test efficacy of both pro- and anti- angiogenic agents. The development of non-invasive procedures for quantification of angiogenesis will facilitate this process significantly. The main objective of this review article is to focus on the novel and existing methods of angiogenesis and their quantification techniques. These findings will be helpful to establish the most convenient methods for the detection, quantification of angiogenesis and to develop a novel, well tolerated and cost effective anti-angiogenic treatment in the near future. PMID:24987169

  10. 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. PMID:27396652

  11. New identification method for Hammerstein models based on approximate least absolute deviation

    NASA Astrophysics Data System (ADS)

    Xu, Bao-Chang; Zhang, Ying-Dan

    2016-07-01

    Disorder and peak noises or large disturbances can deteriorate the identification effects of Hammerstein non-linear models when using the least-square (LS) method. The least absolute deviation technique can be used to resolve this problem; however, its absolute value cannot meet the need of differentiability required by most algorithms. To improve robustness and resolve the non-differentiable problem, an approximate least absolute deviation (ALAD) objective function is established by introducing a deterministic function that exhibits the characteristics of absolute value under certain situations. A new identification method for Hammerstein models based on ALAD is thus developed in this paper. The basic idea of this method is to apply the stochastic approximation theory in the process of deriving the recursive equations. After identifying the parameter matrix of the Hammerstein model via the new algorithm, the product terms in the matrix are separated by calculating the average values. Finally, algorithm convergence is proven by applying the ordinary differential equation method. The proposed algorithm has a better robustness as compared to other LS methods, particularly when abnormal points exist in the measured data. Furthermore, the proposed algorithm is easier to apply and converges faster. The simulation results demonstrate the efficacy of the proposed algorithm.

  12. 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.

  13. 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,…

  14. 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.

  15. Absolute quantification of DcR3 and GDF15 from human serum by LC-ESI MS

    PubMed Central

    Lancrajan, Ioana; Schneider-Stock, Regine; Naschberger, Elisabeth; Schellerer, Vera S; Stürzl, Michael; Enz, Ralf

    2015-01-01

    Biomarkers are widely used in clinical diagnosis, prognosis and therapy monitoring. Here, we developed a protocol for the efficient and selective enrichment of small and low concentrated biomarkers from human serum, involving a 95% effective depletion of high-abundant serum proteins by partial denaturation and enrichment of low-abundant biomarkers by size exclusion chromatography. The recovery of low-abundance biomarkers was above 97%. Using this protocol, we quantified the tumour markers DcR3 and growth/differentiation factor (GDF)15 from 100 μl human serum by isotope dilution mass spectrometry, using 15N metabolically labelled and concatamerized fingerprint peptides for the both proteins. Analysis of three different fingerprint peptides for each protein by liquid chromatography electrospray ionization mass spectrometry resulted in comparable concentrations in three healthy human serum samples (DcR3: 27.23 ± 2.49 fmol/ml; GDF15: 98.11 ± 0.49 fmol/ml). In contrast, serum levels were significantly elevated in tumour patients for DcR3 (116.94 ± 57.37 fmol/ml) and GDF15 (164.44 ± 79.31 fmol/ml). Obtained data were in good agreement with ELISA and qPCR measurements, as well as with literature data. In summary, our protocol allows the reliable quantification of biomarkers, shows a higher resolution at low biomarker concentrations than antibody-based strategies, and offers the possibility of multiplexing. Our proof-of-principle studies in patient sera encourage the future analysis of the prognostic value of DcR3 and GDF15 for colon cancer patients in larger patient cohorts. PMID:25823874

  16. [Research on absolute calibration of sun channel of sun photometer using laser raster scanning method].

    PubMed

    Xu, Wen-Bin; Li, Jian-Jun; Zheng, Xiao-Bing

    2013-01-01

    In the present paper, a new calibration method of absolute spectral irradiance responsivity of sun channel of sun photometer was developed. A tunable laser was used as source and a standard tranfer detector, calibrated against cryogenic absolute radiometer, was used to measure laser beam power. By raster scanning of a single collimated laser beam to generate the uniform irradiance field at the plane of effective aperture stop of sun photometer, the absolute irradiance responsivity of center wavelength of the 870 nm unpolarized sun channels of sun photometer was obtained accurately. The relative spectral irradiance responsivity of corresponding channel was obtained by using lamp-monochromator system and then used to acquire the absolute spectral irradiance responsivity in the laboratory. On the basis of the above results, the top-of-the-atmosphere responsive constant V0 was obtained by integration with extraterrestrial solar spectral irradiance data. Comparing the calibration result with that from GSFC, NASA in 2009, the difference is only 3.75%. In the last, the uncertainties of calibration were evaluated and reached to 2.06%. The principle feasibility of the new method was validated. PMID:23586268

  17. Technological and Analytical Methods for Arabinoxylan Quantification from Cereals.

    PubMed

    Döring, Clemens; Jekle, Mario; Becker, Thomas

    2016-04-25

    Arabinoxylan (AX) is the major nonstarch polysaccharide contained in various types of grains. AX consists of a backbone of β1.4D-xylopyranosyl residues with randomly linked αlarabinofuranosyl units. Once isolated and included as food additive, AX affects foodstuff attributes and has positive effects on human health. AX can be classified into waterextractable and waterunextractable AX. For isolating AX out of their natural matrix, a range of methods was developed, adapted, and improved. This review presents a survey of the commonly used extraction methods for AX by the influence of different techniques. It also provides a brief overview of the structural and technological impact of AX as a dough additive. A concluding section summarizes different detection methods for analyzing and quantification AX. PMID:25629383

  18. A surrogate accelerated multicanonical Monte Carlo method for uncertainty quantification

    NASA Astrophysics Data System (ADS)

    Wu, Keyi; Li, Jinglai

    2016-09-01

    In this work we consider a class of uncertainty quantification problems where the system performance or reliability is characterized by a scalar parameter y. The performance parameter y is random due to the presence of various sources of uncertainty in the system, and our goal is to estimate the probability density function (PDF) of y. We propose to use the multicanonical Monte Carlo (MMC) method, a special type of adaptive importance sampling algorithms, to compute the PDF of interest. Moreover, we develop an adaptive algorithm to construct local Gaussian process surrogates to further accelerate the MMC iterations. With numerical examples we demonstrate that the proposed method can achieve several orders of magnitudes of speedup over the standard Monte Carlo methods.

  19. 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

  20. Deployment dynamics of a simplified spinning IKAROS solar sail via absolute coordinate based method

    NASA Astrophysics Data System (ADS)

    Zhao, Jiang; Tian, Qiang; Hu, Hai-Yan

    2013-02-01

    The spinning solar sail of large scale has been well developed in recent years. Such a solar sail can be considered as a rigid-flexible multibody system mainly composed of a spinning central rigid hub, a number of flexible thin tethers, sail membranes, and tip masses. A simplified interplanetary kite-craft accelerated by radiation of the Sun (IKAROS) model is established in this study by using the absolute-coordinate-based (ACB) method that combines the natural coordinate formulation (NCF) describing the central rigid hub and the absolute nodal coordinate formulation (ANCF) describing flexible parts. The initial configuration of the system in the second-stage deployment is determined through both dynamic and static analyses. The huge set of stiff equations of system dynamics is solved by using the generalized-alpha method, and thus the deployment dynamics of the system can be well understood.

  1. 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.

  2. 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. PMID:26873217

  3. Method to calibrate the absolute energy scale of air showers with ultrahigh energy photons.

    PubMed

    Homola, Piotr; Risse, Markus

    2014-04-18

    Calibrating the absolute energy scale of air showers initiated by ultrahigh energy (UHE) cosmic rays is an important experimental issue. Currently, the corresponding systematic uncertainty amounts to 14%-21% using the fluorescence technique. Here, we describe a new, independent method which can be applied if ultrahigh energy photons are observed. While such photon-initiated showers have not yet been identified, the capabilities of present and future cosmic-ray detectors may allow their discovery. The method makes use of the geomagnetic conversion of UHE photons (preshower effect), which significantly affects the subsequent longitudinal shower development. The conversion probability depends on photon energy and can be calculated accurately by QED. The comparison of the observed fraction of converted photon events to the expected one allows the determination of the absolute energy scale of the observed photon air showers and, thus, an energy calibration of the air shower experiment. We provide details of the method and estimate the accuracy that can be reached as a function of the number of observed photon showers. Already a very small number of UHE photons may help to test and fix the absolute energy scale. PMID:24785024

  4. 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...

  5. 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. PMID:25041979

  6. 21 CFR 530.24 - Procedure for announcing analytical methods for drug residue quantification.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Procedure for announcing analytical methods for...-Producing Animals § 530.24 Procedure for announcing analytical methods for drug residue quantification. (a) FDA may issue an order announcing a specific analytical method or methods for the quantification...

  7. Absolute testing of flats in sub-stitching interferometer by rotation-shift method

    NASA Astrophysics Data System (ADS)

    Jia, Xin; Xu, Fuchao; Xie, Weimin; Li, Yun; Xing, Tingwen

    2015-09-01

    Most of the commercial available sub-aperture stitching interferometers measure the surface with a standard lens that produces a reference wavefront, and the precision of the interferometer is generally limited by the standard lens. The test accuracy can be achieved by removing the error of reference surface by the absolute testing method. When the testing accuracy (repeatability and reproducibility) is close to 1nm, in addition to the reference surface, other factors will also affect the measuring accuracy such as environment, zoom magnification, stitching precision, tooling and fixture, the characteristics of optical materials and so on. We establish a stitching system in the thousand level cleanroom. The stitching system is including the Zygo interferometer, the motion system with Bilz active isolation system at level VC-F. We review the traditional absolute flat testing methods and emphasize the method of rotation-shift functions. According to the rotation-shift method we get the profile of the reference lens and the testing lens. The problem of the rotation-shift method is the tilt error. In the motion system, we control the tilt error no more than 4 second to reduce the error. In order to obtain higher testing accuracy, we analyze the influence surface shape measurement accuracy by recording the environment error with the fluke testing equipment.

  8. Evaluation of a new method for stenosis quantification from 3D x-ray angiography images

    NASA Astrophysics Data System (ADS)

    Betting, Fabienne; Moris, Gilles; Knoplioch, Jerome; Trousset, Yves L.; Sureda, Francisco; Launay, Laurent

    2001-05-01

    A new method for stenosis quantification from 3D X-ray angiography images has been evaluated on both phantom and clinical data. On phantoms, for the parts larger or equal to 3 mm, the standard deviation of the measurement error has always found to be less or equal to 0.4 mm, and the maximum measurement error less than 0.17 mm. No clear relationship has been observed between the performances of the quantification method and the acquisition FoV. On clinical data, the 3D quantification method proved to be more robust to vessel bifurcations than its 3D equivalent. On a total of 15 clinical cases, the differences between 2D and 3D quantification were always less than 0.7 mm. The conclusion is that stenosis quantification from 3D X-4ay angiography images is an attractive alternative to quantification from 2D X-ray images.

  9. Rapid method for the quantification of hydroquinone concentration: chemiluminescent analysis.

    PubMed

    Chen, Tung-Sheng; Liou, Show-Yih; Kuo, Wei-Wen; Wu, Hsi-Chin; Jong, Gwo-Ping; Wang, Hsueh-Fang; Shen, Chia-Yao; Padma, V Vijaya; Huang, Chih-Yang; Chang, Yen-Lin

    2015-11-01

    Topical hydroquinone serves as a skin whitener and is usually available in cosmetics or on prescription based on the hydroquinone concentration. Quantification of hydroquinone content therefore becomes an important issue in topical agents. High-performance liquid chromatography (HPLC) is the commonest method for determining hydroquinone content in topical agents, but this method is time-consuming and uses many solvents that can become an environmental issue. We report a rapid method for quantifying hydroquinone content by chemiluminescent analysis. Hydroquinone induces the production of hydrogen peroxide in the presence of basic compounds. Hydrogen peroxide induced by hydroquinone oxidized light-emitting materials such as lucigenin, resulted in the production of ultra-weak chemiluminescence that was detected by a chemiluminescence analyzer. The intensity of the chemiluminescence was found to be proportional to the hydroquinone concentration. We suggest that the rapid (measurement time, 60 s) and virtually solvent-free (solvent volume, <2 mL) chemiluminescent method described here for quantifying hydroquinone content may be an alternative to HPLC analysis. PMID:25693839

  10. A new absolute method for the standardization of radionuclides emitting low-energy radiation.

    PubMed

    Leblanc, E; de, Marcillac P; Coron, N; Leblanc, J; Loidl, M; Metge, J F; Bouchard, J

    2002-01-01

    Microcalorimeters (or bolometers) operated at temperatures below 100 mK allow individual counting of photons and electrons with a very low energy detection threshold. The physics is based on the pulse temperature increase of the target (or absorber) of the detector due to the complete absorption of both electrons and photons. Since this target can be constructed with a perfect 4-pi geometry, a bolometer offers potentially a new method for absolute activity measurements of radionuclides emitting low-energy radiation. In this paper we present our first results of a feasibility study of activity standardization of a 55Fe solution with a prototype 4-pi bolometer. PMID:11839023

  11. A Multiplex Immunoassay Method for Simultaneous Quantification of Iron, Vitamin A and Inflammation Status Markers

    PubMed Central

    Crudder, Christopher; Levin, Carol E.; Garrett, Dean; Lyman, Chris; Boyle, David S.

    2014-01-01

    Deficiencies of vitamin A and iron affect a significant portion of the world's population, and efforts to characterize patterns of these deficiencies are hampered by a lack of measurement tools appropriate for large-scale population-based surveys. Vitamin A and iron are not easily measured directly, so reliable proxy markers for deficiency status have been identified and adopted. Measurement of inflammatory markers is necessary to interpret vitamin A and iron status markers, because circulating levels are altered by inflammation. We developed a multiplex immunoassay method for simultaneous measurement of five markers relevant to assessing inflammation, vitamin A and iron status: α-1-acid glycoprotein, C-reactive protein, retinol binding protein 4, ferritin and soluble transferrin receptor. Serum and plasma specimens were used to optimize the assay protocol. To evaluate assay performance, plasma from 72 volunteers was assayed using the multiplex technique and compared to conventional immunoassay methods for each of the five markers. Results of the new and conventional assay methods were highly correlated (Pearson Correlations of 0.606 to 0.991, p<.0001). Inter-assay imprecision for the multiplex panel varied from 1% to 8%, and all samples fell within the limits of quantification for all assays at a single dilution. Absolute values given by the multiplex and conventional assays differed, indicating a need for further work to devise a new standard curve. This multiplexed micronutrient immunoassay technique has excellent potential as a cost effective tool for use in large-scale deficiency assessment efforts. PMID:25525806

  12. Laser interferometry method for absolute measurement of the acceleration of gravity

    NASA Technical Reports Server (NTRS)

    Hudson, O. K.

    1971-01-01

    Gravimeter permits more accurate and precise absolute measurement of g without reference to Potsdam values as absolute standards. Device is basically Michelson laser beam interferometer in which one arm is mass fitted with corner cube reflector.

  13. 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…

  14. 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…

  15. 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. PMID:25473819

  16. Towards a stable and absolute atmospheric carbon dioxide instrument using spectroscopic null method

    NASA Astrophysics Data System (ADS)

    Xiang, B.; Nelson, D. D.; McManus, J. B.; Zahniser, M. S.; Wofsy, S. C.

    2013-07-01

    We present a novel spectral method to measure atmospheric carbon dioxide (CO2) with high precision and stability without resorting to calibration tanks during long-term operation. This spectral null method improves precision by reducing spectral proportional noise associated with laser emission instabilities. We employ sealed quartz cells with known CO2 column densities to serve as the permanent internal references in the null method, which improve the instrument's stability and accuracy. A prototype instrument - ABsolute Carbon dioxide (ABC) is developed using this new approach. The instrument has a one-second precision of 0.02 ppm, which averages down to 0.007 ppm within one minute. Long-term stability of within 0.1 ppm is achieved without any calibrations for over a one-month period. These results have the potential for eliminating the need for calibration cylinders for high accuracy field measurements of carbon dioxide.

  17. Towards a stable and absolute atmospheric carbon dioxide instrument using spectroscopic null method

    NASA Astrophysics Data System (ADS)

    Xiang, B.; Nelson, D. D.; McManus, J. B.; Zahniser, M. S.; Wofsy, S. C.

    2013-02-01

    We present a novel spectral method to measure atmospheric carbon dioxide (CO2) with high precision and stability without resorting to calibration tanks during long-term operation. This spectral null method improves precision by reducing spectral proportional noise associated with laser emission instabilities. We employ sealed quartz cells with known CO2 column densities to serve as the permanent internal references in the null method, which improve the instrument's stability and accuracy. A prototype instrument - ABsolute Carbon dioxide (ABC) is developed using this new approach. The instrument has one-second precision of 0.02 ppm, which averages down to 0.007 ppm within one minute. Long-term stability of within 0.1 ppm is achieved without any calibrations for over a one-month period. These results have the potential for eliminating the need for calibration cylinders for high accuracy field measurements of carbon dioxide.

  18. Simple method for absolute calibration of geophones, seismometers, and other inertial vibration sensors

    SciTech Connect

    Kann, Frank van; Winterflood, John

    2005-03-01

    A simple but powerful method is presented for calibrating geophones, seismometers, and other inertial vibration sensors, including passive accelerometers. The method requires no cumbersome or expensive fixtures such as shaker platforms and can be performed using a standard instrument commonly available in the field. An absolute calibration is obtained using the reciprocity property of the device, based on the standard mathematical model for such inertial sensors. It requires only simple electrical measurement of the impedance of the sensor as a function of frequency to determine the parameters of the model and hence the sensitivity function. The method is particularly convenient if one of these parameters, namely the suspended mass is known. In this case, no additional mechanical apparatus is required and only a single set of impedance measurements yields the desired calibration function. Moreover, this measurement can be made with the device in situ. However, the novel and most powerful aspect of the method is its ability to accurately determine the effective suspended mass. For this, the impedance measurement is made with the device hanging from a simple spring or flexible cord (depending on the orientation of its sensitive axis). To complete the calibration, the device is weighed to determine its total mass. All the required calibration parameters, including the suspended mass, are then determined from a least-squares fit to the impedance as a function of frequency. A demonstration using both a 4.5 Hz geophone and a 1 Hz seismometer shows that the method can yield accurate absolute calibrations with an error of 0.1% or better, assuming no a priori knowledge of any parameters.

  19. NMR method for accurate quantification of polysorbate 80 copolymer composition.

    PubMed

    Zhang, Qi; Wang, Aifa; Meng, Yang; Ning, Tingting; Yang, Huaxin; Ding, Lixia; Xiao, Xinyue; Li, Xiaodong

    2015-10-01

    (13)C NMR spectroscopic integration employing short relaxation delays and a 30° pulse width was evaluated as a quantitative tool for analyzing the components of polysorbate 80. (13)C NMR analysis revealed that commercial polysorbate 80 formulations are a complex oligomeric mixture of sorbitan polyethoxylate esters and other intermediates, such as isosorbide polyethoxylate esters and poly(ethylene glycol) (PEG) esters. This novel approach facilitates the quantification of the component ratios. In this study, the ratios of the three major oligomers in polysorbate 80 were measured and the PEG series was found to be the major component of commercial polysorbate 80. The degree of polymerization of -CH2CH2O- groups and the ratio of free to bonded -CH2CH2O- end groups, which correlate with the hydrophilic/hydrophobic nature of the polymer, were analyzed, and were suggested to be key factors for assessing the likelihood of adverse biological reactions to polysorbate 80. The (13)C NMR data suggest that the feed ratio of raw materials and reaction conditions in the production of polysorbate 80 are not well controlled. Our results demonstrate that (13)C NMR is a universal, powerful tool for polysorbate analysis. Such analysis is crucial for the synthesis of a high-quality product, and is difficult to obtain by other methods. PMID:26356097

  20. 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.

  1. 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. PMID:25993885

  2. Systematic development of a group quantification method using evaporative light scattering detector for relative quantification of ginsenosides in ginseng products.

    PubMed

    Lee, Gwang Jin; Shin, Byong-Kyu; Yu, Yun-Hyun; Ahn, Jongsung; Kwon, Sung Won; Park, Jeong Hill

    2016-09-01

    The determination for the contents of multi-components in ginseng products has come to the fore by demands of in-depth information, but the associated industries confront the high cost of securing pure standards for the continuous quality evaluation of the products. This study aimed to develop a prospective high-performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD) method for relative quantification of ginsenosides in ginseng products without a considerable change from the conventional gradient analysis. We investigated the effects of mobile phase composition and elution bandwidth, which are potential variables affecting the ELSD response in the gradient analysis. Similar ELSD response curves of nine major ginsenosides were obtained under the identical flow injection conditions, and the response increased as the percentage of organic solvent increased. The nine ginsenosides were divided into three groups to confirm the effect of elution bandwidth. The ELSD response significantly decreased in case of the late eluted ginsenoside in the individual groups under the isocratic conditions. With the consideration of the two important effects, stepwise changes of the gradient condition were carried out to reach a group quantification method. The inconsistent responses of the nine ginsenosides were reconstituted to three normalized responses by the stepwise changes of the gradient condition, and this result actualized relative quantification in the individual groups. The availability was confirmed by comparing the ginsenoside contents in a base material of ginseng products determined by the direct and group quantification method. The largest difference in the determination results from the two methods was 8.26%, and the difference of total contents was only 0.91%. PMID:27262109

  3. Absolute protein quantification of clinically relevant cytochrome P450 enzymes and UDP-glucuronosyltransferases by mass spectrometry-based targeted proteomics.

    PubMed

    Gröer, C; Busch, D; Patrzyk, M; Beyer, K; Busemann, A; Heidecke, C D; Drozdzik, M; Siegmund, W; Oswald, S

    2014-11-01

    Cytochrome P450 (CYP) enzymes and UDP-glucuronosyltransferases (UGT) are major determinants in the pharmacokinetics of most drugs on the market. To investigate their impact on intestinal and hepatic drug metabolism, we developed and validated quantification methods for nine CYP (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4 and CYP3A5) and four UGT enzymes (UGT1A1, UGT1A3, UGT2B7 and UGT2B15) that have been shown to be of clinical relevance in human drug metabolism. Protein quantification was performed by targeted proteomics using liquid chromatography with tandem mass spectrometry (LC-MS/MS)-based determination of enzyme specific peptides after tryptic digestion using in each case stable isotope labelled peptides as internal standard. The chromatography of the respective peptides was performed with gradient elution using a reversed phase (C18) column (Ascentis(®) Express Peptide ES-C18, 100mm×2.1mm, 2.7μm) and 0.1% formic acid (FA) as well as acetonitrile with 0.1% FA as mobile phases at a flow rate of 300μl/min. The MS/MS detection of all peptides was done simultaneously with a scheduled multiple reaction monitoring (MRM) method in the positive mode by monitoring in each case three mass transitions per proteospecific peptide and the internal standard. The assays were validated according to current bioanalytical guidelines with respect to specificity, linearity (0.25-50nM), within-day and between-day accuracy and precision, digestion efficiency as well as stability. Finally, the developed method was successfully applied to determine the CYP and UGT protein amount in human liver and intestinal microsomes. The method was shown to possess sufficient specificity, sensitivity, accuracy, precision and stability to quantify clinically relevant human CYP and UGT enzymes. PMID:25218440

  4. A new method for the absolute radiance calibration for UV-vis measurements of scattered sunlight

    NASA Astrophysics Data System (ADS)

    Wagner, T.; Beirle, S.; Dörner, S.; Penning de Vries, M.; Remmers, J.; Rozanov, A.; Shaiganfar, R.

    2015-10-01

    Absolute radiometric calibrations are important for measurements of the atmospheric spectral radiance. Such measurements can be used to determine actinic fluxes, the properties of aerosols and clouds, and the shortwave energy budget. Conventional calibration methods in the laboratory are based on calibrated light sources and reflectors and are expensive, time consuming and subject to relatively large uncertainties. Also, the calibrated instruments might change during transport from the laboratory to the measurement sites. Here we present a new calibration method for UV-vis instruments that measure the spectrally resolved sky radiance, for example zenith sky differential optical absorption spectroscopy (DOAS) instruments or multi-axis (MAX)-DOAS instruments. Our method is based on the comparison of the solar zenith angle dependence of the measured zenith sky radiance with radiative transfer simulations. For the application of our method, clear-sky measurements during periods with almost constant aerosol optical depth are needed. The radiative transfer simulations have to take polarisation into account. We show that the calibration results are almost independent from the knowledge of the aerosol optical properties and surface albedo, which causes a rather small uncertainty of about < 7 %. For wavelengths below about 330 nm it is essential that the ozone column density during the measurements be constant and known.

  5. Probing carbohydrate product expulsion from a processive cellulase with multiple absolute binding free energy methods.

    PubMed

    Bu, Lintao; Beckham, Gregg T; Shirts, Michael R; Nimlos, Mark R; Adney, William S; Himmel, Michael E; Crowley, Michael F

    2011-05-20

    Understanding the enzymatic mechanism that cellulases employ to degrade cellulose is critical to efforts to efficiently utilize plant biomass as a sustainable energy resource. A key component of cellulase action on cellulose is product inhibition from monosaccharide and disaccharides in the product site of cellulase tunnel. The absolute binding free energy of cellobiose and glucose to the product site of the catalytic tunnel of the Family 7 cellobiohydrolase (Cel7A) of Trichoderma reesei (Hypocrea jecorina) was calculated using two different approaches: steered molecular dynamics (SMD) simulations and alchemical free energy perturbation molecular dynamics (FEP/MD) simulations. For the SMD approach, three methods based on Jarzynski's equality were used to construct the potential of mean force from multiple pulling trajectories. The calculated binding free energies, -14.4 kcal/mol using SMD and -11.2 kcal/mol using FEP/MD, are in good qualitative agreement. Analysis of the SMD pulling trajectories suggests that several protein residues (Arg-251, Asp-259, Asp-262, Trp-376, and Tyr-381) play key roles in cellobiose and glucose binding to the catalytic tunnel. Five mutations (R251A, D259A, D262A, W376A, and Y381A) were made computationally to measure the changes in free energy during the product expulsion process. The absolute binding free energies of cellobiose to the catalytic tunnel of these five mutants are -13.1, -6.0, -11.5, -7.5, and -8.8 kcal/mol, respectively. The results demonstrated that all of the mutants tested can lower the binding free energy of cellobiose, which provides potential applications in engineering the enzyme to accelerate the product expulsion process and improve the efficiency of biomass conversion. PMID:21454590

  6. A simplified confinement method for calculating absolute free energies and free energy and entropy differences.

    PubMed

    Ovchinnikov, Victor; Cecchini, Marco; Karplus, Martin

    2013-01-24

    A simple and robust formulation of the path-independent confinement method for the calculation of free energies is presented. The simplified confinement method (SCM) does not require matrix diagonalization or switching off the molecular force field, and has a simple convergence criterion. The method can be readily implemented in molecular dynamics programs with minimal or no code modifications. Because the confinement method is a special case of thermodynamic integration, it is trivially parallel over the integration variable. The accuracy of the method is demonstrated using a model diatomic molecule, for which exact results can be computed analytically. The method is then applied to the alanine dipeptide in vacuum, and to the α-helix ↔ β-sheet transition in a 16-residue peptide modeled in implicit solvent. The SCM requires less effort for the calculation of free energy differences than previous formulations because it does not require computing normal modes. The SCM has a diminished advantage for determining absolute free energy values, because it requires decreasing the MD integration step to obtain accurate results. An approximate confinement procedure is introduced, which can be used to estimate directly the configurational entropy difference between two macrostates, without the need for additional computation of the difference in the free energy or enthalpy. The approximation has convergence properties similar to those of the standard confinement method for the calculation of free energies. The use of the approximation requires about 5 times less wall-clock simulation time than that needed to compute enthalpy differences to similar precision from an MD trajectory. For the biomolecular systems considered in this study, the errors in the entropy approximation are under 10%. Practical applications of the methods to proteins are currently limited to implicit solvent simulations. PMID:23268557

  7. Comparison of methods and achievable uncertainties for the relative and absolute measurement of photoluminescence quantum yields.

    PubMed

    Würth, Christian; Grabolle, Markus; Pauli, Jutta; Spieles, Monika; Resch-Genger, Ute

    2011-05-01

    The photoluminescence quantum yield (Φ(f)) that presents a direct measure for the efficiency of the conversion of absorbed photons into emitted photons is one of the spectroscopic key parameters of functional fluorophores. It determines the suitability of such materials for applications in, for example, (bio)analysis, biosensing, and fluorescence imaging as well as as active components in optical devices. The reborn interest in accurate Φ(f) measurements in conjunction with the controversial reliability of reported Φ(f) values of many common organic dyes encouraged us to compare two relative and one absolute fluorometric method for the determination of the fluorescence quantum yields of quinine sulfate dihydrate, coumarin 153, fluorescein, rhodamine 6G, and rhodamine 101. The relative methods include the use of a chain of Φ(f) transfer standards consisting of several "standard dye" versus "reference dye" pairs linked to a golden Φ(f) standard that covers the ultraviolet and visible spectral region, and the use of different excitation wavelengths for standard and sample, respectively. Based upon these measurements and the calibration of the instruments employed, complete uncertainty budgets for the resulting Φ(f) values are derived for each method, thereby providing evaluated standard operation procedures for Φ(f) measurements and, simultaneously, a set of assessed Φ(f) standards. PMID:21473570

  8. Methods for external event screening quantification: Risk Methods Integration and Evaluation Program (RMIEP) methods development

    SciTech Connect

    Ravindra, M.K.; Banon, H.

    1992-07-01

    In this report, the scoping quantification procedures for external events in probabilistic risk assessments of nuclear power plants are described. External event analysis in a PRA has three important goals; (1) the analysis should be complete in that all events are considered; (2) by following some selected screening criteria, the more significant events are identified for detailed analysis; (3) the selected events are analyzed in depth by taking into account the unique features of the events: hazard, fragility of structures and equipment, external-event initiated accident sequences, etc. Based on the above goals, external event analysis may be considered as a three-stage process: Stage I: Identification and Initial Screening of External Events; Stage II: Bounding Analysis; Stage III: Detailed Risk Analysis. In the present report, first, a review of published PRAs is given to focus on the significance and treatment of external events in full-scope PRAs. Except for seismic, flooding, fire, and extreme wind events, the contributions of other external events to plant risk have been found to be negligible. Second, scoping methods for external events not covered in detail in the NRC`s PRA Procedures Guide are provided. For this purpose, bounding analyses for transportation accidents, extreme winds and tornadoes, aircraft impacts, turbine missiles, and chemical release are described.

  9. Methods for external event screening quantification: Risk Methods Integration and Evaluation Program (RMIEP) methods development

    SciTech Connect

    Ravindra, M.K.; Banon, H. )

    1992-07-01

    In this report, the scoping quantification procedures for external events in probabilistic risk assessments of nuclear power plants are described. External event analysis in a PRA has three important goals; (1) the analysis should be complete in that all events are considered; (2) by following some selected screening criteria, the more significant events are identified for detailed analysis; (3) the selected events are analyzed in depth by taking into account the unique features of the events: hazard, fragility of structures and equipment, external-event initiated accident sequences, etc. Based on the above goals, external event analysis may be considered as a three-stage process: Stage I: Identification and Initial Screening of External Events; Stage II: Bounding Analysis; Stage III: Detailed Risk Analysis. In the present report, first, a review of published PRAs is given to focus on the significance and treatment of external events in full-scope PRAs. Except for seismic, flooding, fire, and extreme wind events, the contributions of other external events to plant risk have been found to be negligible. Second, scoping methods for external events not covered in detail in the NRC's PRA Procedures Guide are provided. For this purpose, bounding analyses for transportation accidents, extreme winds and tornadoes, aircraft impacts, turbine missiles, and chemical release are described.

  10. Comparison of microvolume DNA quantification methods for use with volume-sensitive environmental DNA extracts

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Accurate DNA concentration estimates from environmental samples using minimal sample volumes are essential for most downstream applications. To compare the efficacy of microvolume quantification methods, DNA was extracted from soil, compost, and pure culture samples, and quantified using two absorba...

  11. SIMPLE METHOD FOR THE REPRESENTATION, QUANTIFICATION, AND COMPARISON OF THE VOLUMES AND SHAPES OF CHEMICAL COMPOUNDS

    EPA Science Inventory

    A conceptually and computationally simple method for the definition, display, quantification, and comparison of the shapes of three-dimensional mathematical molecular models is presented. Molecular or solvent-accessible volume and surface area can also be calculated. Algorithms, ...

  12. Absolute flatness measurements of silicon mirrors by a three-intersection method by near-infrared interferometry.

    PubMed

    Uchikoshi, Junichi; Hayashi, Yoshinori; Ajari, Noritaka; Kawai, Kentaro; Arima, Kenta; Morita, Mizuho

    2013-01-01

    Absolute flatness of three silicon plane mirrors have been measured by a three-intersection method based on the three-flat method using a near-infrared interferometer. The interferometer was constructed using a near-infrared laser diode with a 1,310-nm wavelength light where the silicon plane mirror is transparent. The height differences at the coordinate values between the absolute line profiles by the three-intersection method have been evaluated. The height differences of the three flats were 4.5 nm or less. The three-intersection method using the near-infrared interferometer was useful for measuring the absolute flatness of the silicon plane mirrors. PMID:23758916

  13. 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.

  14. 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.

  15. 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

  16. 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…

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

    PubMed

    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

  18. 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

  19. Prospective Comparison of Liver Stiffness Measurements between Two Point Shear Wave Elastography Methods: Virtual Touch Quantification and Elastography Point Quantification

    PubMed Central

    Yoo, Hyunsuk; Yoon, Jeong Hee; Lee, Dong Ho; Chang, Won; Han, Joon Koo

    2016-01-01

    Objective To prospectively compare technical success rate and reliable measurements of virtual touch quantification (VTQ) elastography and elastography point quantification (ElastPQ), and to correlate liver stiffness (LS) measurements obtained by the two elastography techniques. Materials and Methods Our study included 85 patients, 80 of whom were previously diagnosed with chronic liver disease. The technical success rate and reliable measurements of the two kinds of point shear wave elastography (pSWE) techniques were compared by χ2 analysis. LS values measured using the two techniques were compared and correlated via Wilcoxon signed-rank test, Spearman correlation coefficient, and 95% Bland-Altman limit of agreement. The intraobserver reproducibility of ElastPQ was determined by 95% Bland-Altman limit of agreement and intraclass correlation coefficient (ICC). Results The two pSWE techniques showed similar technical success rate (98.8% for VTQ vs. 95.3% for ElastPQ, p = 0.823) and reliable LS measurements (95.3% for VTQ vs. 90.6% for ElastPQ, p = 0.509). The mean LS measurements obtained by VTQ (1.71 ± 0.47 m/s) and ElastPQ (1.66 ± 0.41 m/s) were not significantly different (p = 0.209). The LS measurements obtained by the two techniques showed strong correlation (r = 0.820); in addition, the 95% limit of agreement of the two methods was 27.5% of the mean. Finally, the ICC of repeat ElastPQ measurements was 0.991. Conclusion Virtual touch quantification and ElastPQ showed similar technical success rate and reliable measurements, with strongly correlated LS measurements. However, the two methods are not interchangeable due to the large limit of agreement. PMID:27587964

  20. 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. PMID:25599237

  1. Creating standards for absolute quantification of Coxiella burnetii in real-time PCR--a comparative study based on transmission electron microscopy.

    PubMed

    Sting, Reinhard; Molz, Kerstin; Hoferer, Marc

    2015-01-01

    Quantitative standards are a prerequisite for quality control and quantification of pathogens. In this study the creation of quantitative standards for use in qPCR is described using the pathogen Coxiella burnetii. Quantification of Coxiella burnetii particles by transmission electron microscopy (TEM) was used as primary standard and compared with data obtained by light microscopy as well as genome equivalents (GE) and plasmid units (recombinant plasmid). Based on pathogen quantification using TEM and light microscopy, pathogen detection limits of 6 and 2 C. burnetii particles could be determined per com1 qPCR reaction, respectively. In comparison, the detection limits were 17 and 13 pathogen units using GE and plasmid units, respectively. The standard generated by TEM can be used as gold standard for universal application due to high accuracy, quantitative control of the producing process and supplying intact pathogen particles. PMID:25465354

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

    SciTech Connect

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

    2012-08-15

    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 {mu}m) 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{sup 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.

  3. 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.

  4. 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.

  5. 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…

  6. Pixel-to-pixel correspondence alignment method of a 2CCD camera by using absolute phase map

    NASA Astrophysics Data System (ADS)

    Huang, Shujun; Liu, Yue; Bai, Xuefei; Wang, Zhangying; Zhang, Zonghua

    2015-06-01

    An alignment method of a 2CCD camera to build pixel-to-pixel correspondence between the infrared (IR) CCD sensor and the visible CCD sensor by using the absolute phase data is presented. Vertical and horizontal sinusoidal fringe patterns are generated by software and displayed on a liquid crystal display screen. The displayed fringe patterns are captured simultaneously by the IR sensor and the visible sensor of the 2CCD camera. The absolute phase values of each pixel at IR and visible channels are calculated from the captured fringe pattern images by using Fourier transform and the optimum three-fringe number selection method. The accurate pixel corresponding relationship between the two sensors can be determined along the vertical and the horizontal directions by comparing the obtained absolute phase data in IR and visible channels. Experimental results show the high accuracy, effectiveness, and validity of the proposed 2CCD alignment method. By using the continuous absolute phase information, this method can determine the pixel-to-pixel correspondence with high resolution.

  7. Ferromagnetic particles as a rapid and robust sample preparation for the absolute quantification of seven eicosanoids in human plasma by UHPLC-MS/MS.

    PubMed

    Suhr, Anna Catharina; Bruegel, Mathias; Maier, Barbara; Holdt, Lesca Miriam; Kleinhempel, Alisa; Teupser, Daniel; Grimm, Stefanie H; Vogeser, Michael

    2016-06-01

    We used ferromagnetic particles as a novel technique to deproteinize plasma samples prior to quantitative UHPLC-MS/MS analysis of seven eicosanoids [thromboxane B2 (TXB2), prostaglandin E2 (PGE2), PGD2, 5-hydroxyeicosatetraenoic acid (5-HETE), 11-HETE, 12-HETE, arachidonic acid (AA)]. A combination of ferromagnetic particle enhanced deproteination and subsequent on-line solid phase extraction (on-line SPE) realized quick and convenient semi-automated sample preparation-in contrast to widely used manual SPE techniques which are rather laborious and therefore impede the investigation of AA metabolism in larger patient cohorts. Method evaluation was performed according to a protocol based on the EMA guideline for bioanalytical method validation, modified for endogenous compounds. Calibrators were prepared in ethanol. The calibration curves were found to be linear in a range of 0.1-80ngmL(-1) (TXB2, PGE2, PGD2), 0.05-40ngmL(-1) (5-HETE, 11-HETE), 0.5-400ngmL(-1) (12-HETE) and 25-9800ngmL(-1) (AA). Regarding all analytes and all quality controls, the resulting precision data (inter-assay 2.6 %-15.5 %; intra-assay 2.5 %-15.1 %, expressed as variation coefficient) as well as the accuracy results (inter-assay 93.3 %-125 %; intra-assay 91.7 %-114 %) were adequate. Further experiments addressing matrix effect, recovery and robustness, yielded also very satisfying results. As a proof of principle, the newly developed LC-MS/MS assay was employed to determine the capacity of AA metabolite release after whole blood stimulation in healthy blood donors. For this purpose, whole blood specimens of 5 healthy blood donors were analyzed at baseline and after a lipopolysaccharide (LPS) induced blood cell activation. In several baseline samples some eicosanoids levels were below the Lower Limit of Quantification. However, in the stimulated samples all chosen eicosanoids (except PGD2) could be quantified. These results, in context with those obtained in validation, demonstrate the

  8. Methods for the efficient quantification of fruit provitamin A contents.

    PubMed

    Davey, Mark W; Keulemans, Johan; Swennen, Rony

    2006-12-15

    As part of a screening program to identify micronutrient-rich banana and plantain (Musa) varieties, a simple, robust, and comparatively rapid protocol for the quantification of the provitamin A carotenoids contents of fruit pulp and peel tissues by HPLC and by spectrophotometry has been developed. Major points to note include the use lyophilisation and extensive tissue disruption procedures to ensure quantitative recoveries, and the avoidance of saponification and/or concentration steps which lead to significant losses of provitamin A carotenoids. The protocol showed excellent reproducibility between replicate extractions, without the need for an internal standard. Application of the methodology demonstrated that Musa fruit pulp has a relatively simple provitamin A carotenoids content, quite different from the overlying peel, and that the proportions of alpha- and beta-carotene are characteristic for each genotype. The protocol was also used to profile the provitamin A carotenoids of several other fruits. PMID:17049540

  9. 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

  10. 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. PMID:26525253

  11. 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.

  12. 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…

  13. 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...

  14. Laser induced deflection (LID) method for absolute absorption measurements of optical materials and thin films

    NASA Astrophysics Data System (ADS)

    Mühlig, Christian; Bublitz, Simon; Paa, Wolfgang

    2011-05-01

    We use optimized concepts to measure directly low absorption in optical materials and thin films at various laser wavelengths by the laser induced deflection (LID) technique. An independent absolute calibration, using electrical heaters, is applied to obtain absolute absorption data without the actual knowledge of the photo-thermal material properties. Verification of the absolute calibration is obtained by measuring different silicon samples at 633 nm where all laser light, apart from the measured reflection/scattering, is absorbed. Various experimental results for bulk materials and thin films are presented including measurements of fused silica and CaF2 at 193 nm, nonlinear crystals (LBO) for frequency conversion and AR coated fused silica for high power material processing at 1030 nm and Yb-doped silica raw materials for high power fiber lasers at 1550 nm. In particular for LBO the need of an independent calibration is demonstrated since thermal lens generation is dominated by stress-induced refractive index change which is in contrast to most of the common optical materials. The measured results are proven by numerical simulations and their influence on the measurement strategy and the obtained accuracy are shown.

  15. 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.

  16. 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.

  17. RNA-Skim: a rapid method for RNA-Seq quantification at transcript level

    PubMed Central

    Zhang, Zhaojun; Wang, Wei

    2014-01-01

    Motivation: RNA-Seq technique has been demonstrated as a revolutionary means for exploring transcriptome because it provides deep coverage and base pair-level resolution. RNA-Seq quantification is proven to be an efficient alternative to Microarray technique in gene expression study, and it is a critical component in RNA-Seq differential expression analysis. Most existing RNA-Seq quantification tools require the alignments of fragments to either a genome or a transcriptome, entailing a time-consuming and intricate alignment step. To improve the performance of RNA-Seq quantification, an alignment-free method, Sailfish, has been recently proposed to quantify transcript abundances using all k-mers in the transcriptome, demonstrating the feasibility of designing an efficient alignment-free method for transcriptome quantification. Even though Sailfish is substantially faster than alternative alignment-dependent methods such as Cufflinks, using all k-mers in the transcriptome quantification impedes the scalability of the method. Results: We propose a novel RNA-Seq quantification method, RNA-Skim, which partitions the transcriptome into disjoint transcript clusters based on sequence similarity, and introduces the notion of sig-mers, which are a special type of k-mers uniquely associated with each cluster. We demonstrate that the sig-mer counts within a cluster are sufficient for estimating transcript abundances with accuracy comparable with any state-of-the-art method. This enables RNA-Skim to perform transcript quantification on each cluster independently, reducing a complex optimization problem into smaller optimization tasks that can be run in parallel. As a result, RNA-Skim uses <4% of the k-mers and <10% of the CPU time required by Sailfish. It is able to finish transcriptome quantification in <10 min per sample by using just a single thread on a commodity computer, which represents >100 speedup over the state-of-the-art alignment-based methods, while delivering

  18. Streptavidin conjugation and quantification-a method evaluation for nanoparticles.

    PubMed

    Quevedo, Pablo Darío; Behnke, Thomas; Resch-Genger, Ute

    2016-06-01

    Aiming at the development of validated protocols for protein conjugation of nanomaterials and the determination of protein labeling densities, we systematically assessed the conjugation of the model protein streptavidin (SAv) to 100-, 500-, and 1000-nm-sized polystyrene and silica nanoparticles and dye-encoded polymer particles with two established conjugation chemistries, based upon achievable coupling efficiencies and labeling densities. Bioconjugation reactions compared included EDC/sulfo NHS ester chemistry for direct binding of the SAv to carboxyl groups at the particle surface and maleimide-thiol chemistry in conjunction with heterobifunctional PEG linkers and aminated nanoparticles (NPs). Quantification of the total and functional amounts of SAv on these nanomaterials and unreacted SAv in solution was performed with the BCA assay and the biotin-FITC (BF) titration, relying on different signal generation principles, which are thus prone to different interferences. Our results revealed a clear influence of the conjugation chemistry on the amount of NP crosslinking, yet under optimized reaction conditions, EDC/sulfo NHS ester chemistry and the attachment via heterobifunctional PEG linkers led to comparably efficient SAv coupling and good labeling densities. Particle size can obviously affect protein labeling densities and particularly protein functionality, especially for larger particles. For unstained nanoparticles, direct bioconjugation seems to be the most efficient strategy, whereas for dye-encoded nanoparticles, PEG linkers are to be favored for the prevention of dye-protein interactions which can affect protein functionality specifically in the case of direct SAv binding. Moreover, an influence of particle size on achievable protein labeling densities and protein functionality could be demonstrated. PMID:27038055

  19. Comparison of isolation and quantification methods to measure humic-like substances (HULIS) in atmospheric particles

    NASA Astrophysics Data System (ADS)

    Fan, Xingjun; Song, Jianzhong; Peng, Ping'an

    2012-12-01

    Humic-like Substances (HULIS) comprise a significant fraction of the water-soluble organic aerosol mass and influence the cloud microphysical properties and climate effects of aerosols in the atmosphere. In this work, the most frequently used HULIS isolation and quantification methods including ENVI-18, HLB, XAD-8 and DEAE were comparatively characterized with two model standards, ten interfering compounds, and five ambient aerosol samples. Quantification of HULIS is performed with a TOC analyzer, complemented by an investigation of the chemical structure of the extracted fractions by UV-Vis spectroscopy. The results show that the four isolation methods were all characterized by high reliability, high reproducibility, and low limit of detection (LOD), indicating that each method can be used to efficiently recover Suwannee River Fulvic Acid (SRFA) and be applied to the quantification of the lower amount of HULIS in atmospheric particles. The analytical results of the UV-Vis spectra of HULIS fractions isolated also indicate that they are all favorable for extraction of compounds of high UV absorbance, high MW, and high aromaticity and that the DEAE protocol is the most significant one. Compared with the DEAE method that favors extraction of highly UV-absorbing and more aromatic compounds, SRFA isolated by the ENVI-18, HLB, and XAD-8 protocols were more representative of the global matrix. Each method has its own advantages and disadvantages and is suitable for a particular application. No single method is ideal for both isolation and quantification of HULIS in atmospheric samples.

  20. Development of an absolute method for efficiency calibration of a coaxial HPGe detector for large volume sources

    NASA Astrophysics Data System (ADS)

    Ortiz-Ramírez, Pablo C.

    2015-09-01

    In this work an absolute method for the determination of the full energy peak efficiency of a gamma spectroscopy system for voluminous sources is presented. The method was tested for a high-resolution coaxial HPGe detector and cylindrical homogeneous volume source. The volume source is represented by a set of point sources filling its volume. We found that the absolute efficiency of a volume source can be determined as the average over its volume of the absolute efficiency of each point source. Experimentally, we measure the intrinsic efficiency as a function upon source-detector position. Then, considering the solid angle and the attenuations of the gamma rays emitted to the detector by each point source, considered as embedded in the source matrix, the absolute efficiency for each point source inside of the volume was determined. The factor associate with the solid angle and the self-attenuation of photons in the sample was deduced from first principles without any mathematical approximation. The method was tested by determining the specific activity of 137Cs in cylindrical homogeneous sources, using IAEA reference materials with specific activities between 14.2 Bq/kg and 9640 Bq/kg at the moment of the experimentation. The results obtained shown a good agreement with the expected values. The relative difference was less than 7% in most of the cases. The main advantage of this method is that it does not require of the use of expensive and hard to produce standard materials. In addition it does not require of matrix effect corrections, which are the main cause of error in this type of measurements, and it is easy to implement in any nuclear physics laboratory.

  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. Localized 2D COSY sequences: Method and experimental evaluation for a whole metabolite quantification approach

    NASA Astrophysics Data System (ADS)

    Martel, Dimitri; Tse Ve Koon, K.; Le Fur, Yann; Ratiney, Hélène

    2015-11-01

    Two-dimensional spectroscopy offers the possibility to unambiguously distinguish metabolites by spreading out the multiplet structure of J-coupled spin systems into a second dimension. Quantification methods that perform parametric fitting of the 2D MRS signal have recently been proposed for resolved PRESS (JPRESS) but not explicitly for Localized Correlation Spectroscopy (LCOSY). Here, through a whole metabolite quantification approach, correlation spectroscopy quantification performances are studied. The ability to quantify metabolite relaxation constant times is studied for three localized 2D MRS sequences (LCOSY, LCTCOSY and the JPRESS) in vitro on preclinical MR systems. The issues encountered during implementation and quantification strategies are discussed with the help of the Fisher matrix formalism. The described parameterized models enable the computation of the lower bound for error variance - generally known as the Cramér Rao bounds (CRBs), a standard of precision - on the parameters estimated from these 2D MRS signal fittings. LCOSY has a theoretical net signal loss of two per unit of acquisition time compared to JPRESS. A rapid analysis could point that the relative CRBs of LCOSY compared to JPRESS (expressed as a percentage of the concentration values) should be doubled but we show that this is not necessarily true. Finally, the LCOSY quantification procedure has been applied on data acquired in vivo on a mouse brain.

  3. 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. PMID:22711674

  4. Absolute nuclear material assay

    DOEpatents

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

    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.

  5. 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.

  6. Development of a new method for d-xylose detection and quantification in urine, based on the use of recombinant xylose dehydrogenase from Caulobacter crescentus.

    PubMed

    Sánchez-Moreno, Israel; García-Junceda, Eduardo; Hermida, Carmen; Fernández-Mayoralas, Alfonso

    2016-09-20

    The gene xylB from Caulobacter crescentus has been cloned and expressed in Escherichia coli providing a high yield of xylose dehydrogenase (XylB) production and excellent purity (97%). Purified recombinant XylB showed an absolute dependence on the cofactor NAD(+) and a strong preference for d-xylose against other assayed mono and disaccharides. Additionally, XylB showed strong stability when stored as freeze-dried powder at least 250days both at 4°C and room temperature. In addition, more than 80% of the initial activity of rehydrated freeze-dried enzyme remained after 150days of incubation at 4°C. Based on these characteristics, the capability of XylB in d-xylose detection and quantification was studied. The linearity of the method was maintained up to concentrations of d-xylose of 10mg/dL and the calculated limits of detection (LoD) and quantification (LoQ) of xylose in buffer were 0.568mg/dL and 1.89mg/dL respectively. Thus, enzymatic detection was found to be an excellent method for quantification of d-xylose in both buffer and urine samples. This method can easily be incorporated in a new test for the diagnosis of hypolactasia through the measurement of intestinal lactase activity. PMID:27480343

  7. 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. PMID:26994331

  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. Comparison of biochemical and microscopic methods for quantification of mycorrhizal fungi in soil and roots

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Arbuscular mycorrhizal fungi (AMF) are well-known plant symbionts which provide enhanced phosphorus uptake as well as other benefits to their host plants. Quantification of mycorrhizal biomass and root colonization has traditionally been performed by root staining and microscopic examination methods...

  10. 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 ...

  11. 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 ...

  12. 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 ...

  13. 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 ...

  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

    2016-02-01

    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. PMID:26880368

  15. A new spectrophotometric method for quantification of potassium solubilized by bacterial cultures.

    PubMed

    Rajawat, Mahendra Vikram Singh; Singh, Surender; Saxena, Anil Kumar

    2014-03-01

    A new spectrophotometric method was developed for the quantification of potassium in the culture broth supernatant of K-solubilizing bacteria. The standard curve of potassium with the new method, which is based on the measurement of cobalt, showed a regression coefficient (R2) of 0.998. The quantification values of potassium obtained with flame photometric method and the newly developed method showed a significant correlation (r) of 0.978. The new method depends on the precipitation of sodium cobaltinitrite with solubilized potassium in liquid medium as potassium sodium cobaltinitrite, which develops bluish green colour by the addition of conc. HCl. The intensity of developed colour can be recorded at 623 nm. This method involves less number of steps, is easy and time saving, and can be used for the reliable estimation of available potassium in culture broth supernatant of K-solubilizing bacteria. PMID:24669669

  16. Pitfalls and advantages of different strategies for the absolute quantification of N-acetyl aspartate, creatine and choline in white and grey matter by 1H-MRS.

    PubMed

    Malucelli, E; Manners, D N; Testa, C; Tonon, C; Lodi, R; Barbiroli, B; Iotti, S

    2009-12-01

    This study extensively investigates different strategies for the absolute quantitation of N-acetyl aspartate, creatine and choline in white and grey matter by (1)H-MRS at 1.5 T. The main focus of this study was to reliably estimate metabolite concentrations while reducing the scan time, which remains as one of the main problems in clinical MRS. Absolute quantitation was based on the water-unsuppressed concentration as the internal standard. We compared strategies based on various experimental protocols and post-processing strategies. Data were obtained from 30 control subjects using a PRESS sequence at several TE to estimate the transverse relaxation time, T(2), of the metabolites. Quantitation was performed with the algorithm QUEST using two different metabolite signal basis sets: a whole-metabolite basis set (WhoM) and a basis set in which the singlet signals were split from the coupled signals (MSM). The basis sets were simulated in vivo for each TE used. Metabolites' T(2)s were then determined by fitting the estimated signal amplitudes of the metabolites obtained at different TEs. Then the absolute concentrations (mM) of the metabolites were assessed for each subject using the estimated signal amplitudes and either the mean estimated relaxation times of all subjects (mean protocol, MP) or the T(2) estimated from the spectra derived from the same subject (individual protocol, IP). Results showed that MP represents a less time-consuming alternative to IP in the quantitation of brain metabolites by (1)H-MRS in both grey and white matter, with a comparable accuracy when performed by MSM. It was also shown that the acquisition time might be further reduced by using a variant of MP, although with reduced accuracy. In this variant, only one water-suppressed and one water-unsuppressed spectra were acquired, drastically reducing the duration of the entire MRS examination. However, statistical analysis highlights the reduced accuracy of MP when performed using Who

  17. 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. PMID:26319303

  18. 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.

  19. Critical points of DNA quantification by real-time PCR – effects of DNA extraction method and sample matrix on quantification of genetically modified organisms

    PubMed Central

    Cankar, Katarina; Štebih, Dejan; Dreo, Tanja; Žel, Jana; Gruden, Kristina

    2006-01-01

    Background Real-time PCR is the technique of choice for nucleic acid quantification. In the field of detection of genetically modified organisms (GMOs) quantification of biotech products may be required to fulfil legislative requirements. However, successful quantification depends crucially on the quality of the sample DNA analyzed. Methods for GMO detection are generally validated on certified reference materials that are in the form of powdered grain material, while detection in routine laboratories must be performed on a wide variety of sample matrixes. Due to food processing, the DNA in sample matrixes can be present in low amounts and also degraded. In addition, molecules of plant origin or from other sources that affect PCR amplification of samples will influence the reliability of the quantification. Further, the wide variety of sample matrixes presents a challenge for detection laboratories. The extraction method must ensure high yield and quality of the DNA obtained and must be carefully selected, since even components of DNA extraction solutions can influence PCR reactions. GMO quantification is based on a standard curve, therefore similarity of PCR efficiency for the sample and standard reference material is a prerequisite for exact quantification. Little information on the performance of real-time PCR on samples of different matrixes is available. Results Five commonly used DNA extraction techniques were compared and their suitability for quantitative analysis was assessed. The effect of sample matrix on nucleic acid quantification was assessed by comparing 4 maize and 4 soybean matrixes. In addition 205 maize and soybean samples from routine analysis were analyzed for PCR efficiency to assess variability of PCR performance within each sample matrix. Together with the amount of DNA needed for reliable quantification, PCR efficiency is the crucial parameter determining the reliability of quantitative results, therefore it was chosen as the primary

  20. Is biofilm removal properly assessed? Comparison of different quantification methods in a 96-well plate system.

    PubMed

    Stiefel, Philipp; Rosenberg, Urs; Schneider, Jana; Mauerhofer, Stefan; Maniura-Weber, Katharina; Ren, Qun

    2016-05-01

    Various methods have been reported to quantify total biofilm or different components of biofilm; however, these methods are often confusedly used, leading to discrepancies and misleading results. In this study, different methods for quantification of biofilm, including those for total biomass, total amount of bacterial cells, viable cell number, and amount of extracellular polymeric substances, were systematically compared in microtiter plates. To evaluate which method is suitable for assessment of biofilm removal and for bacterial killing, biofilm samples were treated with various cleaners possessing removing and/or killing capacities. It was found that most of the methods tested in this study in general exhibited high reproducibility and repeatability. Crystal Violet staining was a simple but reliable method for total biomass quantification. Total bacteria cell numbers could be reliably quantified by the fluorescent DNA-binding dye Acridine Orange. Viable cells could be quantified by either an ATP-based assay or a proliferation assay. Both of these viability methods showed a broad detection range and led to precise measurement. For quantification of proteins in the biofilm, staining with fluorescein isothiocyanate was most suitable. Furthermore, it was revealed that a combination of different methods is required to determine if a cleaner kills or removes biofilm. PMID:26923144

  1. An Innovative Method for Exosome Quantification and Size Measurement

    PubMed Central

    Mehdiani, Arash; Maier, Anatol; Pinto, Antonio; Barth, Mareike; Akhyari, Payam; Lichtenberg, Artur

    2015-01-01

    Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about their complex pathways, their bioavailability and their diverse functions in health and disease. Current work focuses on the presence and the behavior of exosomes (in vitro as well as in vivo) in the context of different human disorders, especially in the fields of oncology, gynecology and cardiology. Unfortunately, neither a consensus regarding a gold standard for exosome isolation exists, nor is there an agreement on such a method for their quantitative analysis. As there are many methods for the purification of exosomes and also many possibilities for their quantitative and qualitative analysis, it is difficult to determine a combination of methods for the ideal approach. Here, we demonstrate nanoparticle tracking analysis (NTA), a semi-automated method for the characterization of exosomes after isolation from human plasma by ultracentrifugation. The presented results show that this approach for isolation, as well as the determination of the average number and size of exosomes, delivers reproducible and valid data, as confirmed by other methods, such as scanning electron microscopy (SEM). PMID:25650897

  2. Software-assisted serum metabolite quantification using NMR.

    PubMed

    Jung, Young-Sang; Hyeon, Jin-Seong; Hwang, Geum-Sook

    2016-08-31

    The goal of metabolomics is to analyze a whole metabolome under a given set of conditions, and accurate and reliable quantitation of metabolites is crucial. Absolute concentration is more valuable than relative concentration; however, the most commonly used method in NMR-based serum metabolic profiling, bin-based and full data point peak quantification, provides relative concentration levels of metabolites and are not reliable when metabolite peaks overlap in a spectrum. In this study, we present the software-assisted serum metabolite quantification (SASMeQ) method, which allows us to identify and quantify metabolites in NMR spectra using Chenomx software. This software uses the ERETIC2 utility from TopSpin to add a digitally synthesized peak to a spectrum. The SASMeQ method will advance NMR-based serum metabolic profiling by providing an accurate and reliable method for absolute quantification that is superior to bin-based quantification. PMID:27506360

  3. Gallic Acid: Review of the Methods of Determination and Quantification.

    PubMed

    Fernandes, Felipe Hugo Alencar; Salgado, Hérida Regina Nunes

    2016-05-01

    Gallic acid (3,4,5 trihydroxybenzoic acid) is a secondary metabolite present in most plants. This metabolite is known to exhibit a range of bioactivities including antioxidant, antimicrobial, anti-inflammatory, and anticancer. There are various methods to analyze gallic acid including spectrometry, chromatography, and capillary electrophoresis, among others. They have been developed to identify and quantify this active ingredient in most biological matrices. The aim of this article is to review the available information on analytical methods for gallic acid, as well as presenting the advantages and limitations of each technique. PMID:26440222

  4. Absolute and relative quantification and calibration for sectioning fluorescence microscopy using standardized uniform fluorescent layers and SIPchart-based correction procedures

    NASA Astrophysics Data System (ADS)

    Zwier, J. M.; Oomen, L.; Brocks, L.; Jalink, K.; Brakenhoff, G. J.

    2007-02-01

    The total or integrated fluorescence intensity of a through-focus series of a thin standardized uniform fluorescent or calibration layer is shown to be suitable for image intensity correction and calibration in sectioning microscopy. This integrated intensity can be derived from the earlier introduced SectionedImagingProperty or SIPcharts, derived from the 3D layer datasets. By correcting the 3D image of an object with the 3D image of the standardized uniform fluorescent layer obtained under identical conditions one is able to express the object fluorescence in units fluorescence of the calibration layer. With object fluorescence intensities in fluorescence layer unit's or FLU's the object image intensities becomes independent of microscope system and imaging conditions. A direct result is that the often-appreciable lateral intensity variations present in confocal microscopy are eliminated (shading correction). Of more general value is that images obtained with different objectives, magnifications or from different microscope systems can be quantitatively related to each other. The effectiveness of shading correction and relating images obtained under various microscope conditions is demonstrated on images of standard fluorocent beads. Expressing the object fluorescence in FLU units seems to be a promising approach for general quantification of sectioning imaging enabling cross-correlation of imaging results over time and between imaging systems.

  5. 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. PMID:20544913

  6. QUANTIFICATION OF MUNICIPAL DISPOSAL METHODS FOR INDUSTRIALLY GENERATED HAZARDOUS WASTES

    EPA Science Inventory

    Estimations of the amounts of industrial hazardous wastes being disposed of according to various methods of disposal were generated for significant portions of the five following SIC codes: 28, Chemical and Allied Products; 29, Petroleum Refining and Related Industries; 30, Rubbe...

  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. Absolute calibration method for laser megajoule neutron yield measurement by activation diagnostics.

    PubMed

    Landoas, Olivier; Glebov, Vladimir Yu; Rossé, Bertrand; Briat, Michelle; Disdier, Laurent; Sangster, Thomas C; Duffy, Tim; Marmouget, Jean Gabriel; Varignon, Cyril; Ledoux, Xavier; Caillaud, Tony; Thfoin, Isabelle; Bourgade, Jean-Luc

    2011-07-01

    The laser megajoule (LMJ) and the National Ignition Facility (NIF) plan to demonstrate thermonuclear ignition using inertial confinement fusion (ICF). The neutron yield is one of the most important parameters to characterize ICF experiment performance. For decades, the activation diagnostic was chosen as a reference at ICF facilities and is now planned to be the first nuclear diagnostic on LMJ, measuring both 2.45 MeV and 14.1 MeV neutron yields. Challenges for the activation diagnostic development are absolute calibration, accuracy, range requirement, and harsh environment. At this time, copper and zirconium material are identified for 14.1 MeV neutron yield measurement and indium material for 2.45 MeV neutrons. A series of calibrations were performed at Commissariat à l'Energie Atomique (CEA) on a Van de Graff facility to determine activation diagnostics efficiencies and to compare them with results from calculations. The CEA copper activation diagnostic was tested on the OMEGA facility during DT implosion. Experiments showed that CEA and Laboratory for Laser Energetics (LLE) diagnostics agree to better than 1% on the neutron yield measurement, with an independent calibration for each system. Also, experimental sensitivities are in good agreement with simulations and allow us to scale activation diagnostics for the LMJ measurement range. PMID:21806179

  9. Absolutely referenced distance measurement by combination of time-of-flight and digital holographic methods

    NASA Astrophysics Data System (ADS)

    Fratz, Markus; Weimann, Claudius; Wölfelschneider, Harald; Koos, Christian; Höfler, Heinrich

    2014-03-01

    We present a novel optical system for distance measurement based on the combination of optical time-of-flight metrology and digital holography. In addition absolute calibration of the measurement results is performed by a sideband modulation technique. For the time-of-flight technique a diode laser (1470 nm) is modulated sinusoidally (128 MHz). The light reflected and scattered by an object is detected by an avalanche-photo-diode. The phase difference between the sent and detected modulation is a measure for the distance between the sensor and the object. This allows for distance measurements up to 1.17 m with resolutions of ~2 mm. The interferometric setup uses 4 whispering-gallery-mode lasers to perform multiwavelengths-holographic distance measurements. The four wavelengths span the range from 1547 nm to 1554 nm. The unambiguous measurement measurement-range of the interferometric setup is approx. 7 mm while resolutions of 0.6 μm are observed. Both setups are integrated into one setup and perform measurements synchronously. Exact knowledge of the frequency differences of hundreds of GHz between the four lasers is crucial for the interferometric fine scale measurement. For this aim the light of the lasers is phase-modulated with frequencies of 36 GHz and 40 GHz to produce optical sidebands of higher order, thus generating beat signals in the hundreds-of-MHz regime, which can be measured electronically. The setup shows a way to measure distances in the meter range with sub-micron resolution.

  10. 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).

  11. 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.

  12. Visualization and Quantification of Fingering Flow Using Light Transmission Method

    NASA Astrophysics Data System (ADS)

    Rezanezhad, F.; Roth, K.

    2007-12-01

    With the aim of studying the physical process concerning the unstable fingering phenomena in two dimensions, experiments of vertical infiltration through layered sand were carried out in the laboratory using Hele-Shaw cells. We developed a light transmission method to measure the dynamics of water saturation within flow fingers in great detail with high spatial and temporal resolution. The method was calibrated using X-ray absorption. We improved the measured light transmission with correction for scattering effects through deconvolution with a point spread function which allows us to obtain quantitative high spatial resolution measurements. After fingers had fully developed, we added a dye tracer in order to distinguish mobile and immobile water fractions. Fully developed fingers consist of a tip, a core with mobile water, and a hull with immobile water. We analyzed the dynamics of water saturation within the finger tip, along the finger core behind the tip, and within the fringe of the fingers during radial growth. Our results confirm previous findings of saturation overshoot in the finger tips and revealed a saturation minimum behind the tip as a new feature. The finger development was characterized by a gradual increase in water content within the core of the finger behind this minimum and a gradual widening of the fingers to a quasi-stable state which evolves at time scales that are orders of magnitude longer than those of fingers' evolution. In this state, a sharp separation into a core with fast convective flow and a fringe with exceedingly slow flow was detected. All observed phenomena, with the exception of saturation overshoot, could be consistently explained based on the hysteretic behavior of the soil-water characteristic.

  13. 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. PMID:26698959

  14. Radar prediction of absolute rain fade distributions for earth-satellite paths and general methods for extrapolation of fade statistics to other locations

    NASA Technical Reports Server (NTRS)

    Goldhirsh, J.

    1982-01-01

    The first absolute rain fade distribution method described establishes absolute fade statistics at a given site by means of a sampled radar data base. The second method extrapolates absolute fade statistics from one location to another, given simultaneously measured fade and rain rate statistics at the former. Both methods employ similar conditional fade statistic concepts and long term rain rate distributions. Probability deviations in the 2-19% range, with an 11% average, were obtained upon comparison of measured and predicted levels at given attenuations. The extrapolation of fade distributions to other locations at 28 GHz showed very good agreement with measured data at three sites located in the continental temperate region.

  15. MRI-based methods for quantification of the cerebral metabolic rate of oxygen.

    PubMed

    Rodgers, Zachary B; Detre, John A; Wehrli, Felix W

    2016-07-01

    The brain depends almost entirely on oxidative metabolism to meet its significant energy requirements. As such, the cerebral metabolic rate of oxygen (CMRO2) represents a key measure of brain function. Quantification of CMRO2 has helped elucidate brain functional physiology and holds potential as a clinical tool for evaluating neurological disorders including stroke, brain tumors, Alzheimer's disease, and obstructive sleep apnea. In recent years, a variety of magnetic resonance imaging (MRI)-based CMRO2 quantification methods have emerged. Unlike positron emission tomography - the current "gold standard" for measurement and mapping of CMRO2 - MRI is non-invasive, relatively inexpensive, and ubiquitously available in modern medical centers. All MRI-based CMRO2 methods are based on modeling the effect of paramagnetic deoxyhemoglobin on the magnetic resonance signal. The various methods can be classified in terms of the MRI contrast mechanism used to quantify CMRO2: T2*, T2', T2, or magnetic susceptibility. This review article provides an overview of MRI-based CMRO2 quantification techniques. After a brief historical discussion motivating the need for improved CMRO2 methodology, current state-of-the-art MRI-based methods are critically appraised in terms of their respective tradeoffs between spatial resolution, temporal resolution, and robustness, all of critical importance given the spatially heterogeneous and temporally dynamic nature of brain energy requirements. PMID:27089912

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

    PubMed

    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

  17. 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

  18. Absolute optical oscillator strengths for the electronic excitation of atoms at high resolution: Experimental methods and measurements for helium

    SciTech Connect

    Chan, W.F.; Cooper, G.; Brion, C.E. )

    1991-07-01

    An alternative method is described for the measurement of absolute optical oscillator strengths (cross sections) for electronic excitation of free atoms and molecules throughout the discrete region of the valence-shell spectrum at high energy resolution (full width at half maximum of 0.048 eV). The technique, utilizing the virtual-photon field of a fast electron inelastically scattered at negligible momentum transfer, avoids many of the difficulties associated with the various direct optical techniques that have traditionally been used for absolute optical oscillator strength measurements. The method is also free of the bandwidth (line saturation) effects that can seriously limit the accuracy of photoabsorption cross-section measurements for discrete transitions of narrow linewidth obtained using the Beer-Lambert law ({ital I}{sub 0}/{ital I}=exp({ital nl}{sigma}{sub {ital p}})). Since the line-saturation effects are not widely appreciated and are only usually considered in the context of peak heights, a detailed analysis of this problem is presented, with consideration of the integrated cross section (oscillator strength) over the profile of each discrete peak.

  19. Absolute radiometric calibration of the RapidEye multispectral imager using the reflectance-based vicarious calibration method

    NASA Astrophysics Data System (ADS)

    Naughton, Denis; Brunn, Andreas; Czapla-Myers, Jeff; Douglass, Scott; Thiele, Michael; Weichelt, Horst; Oxfort, Michael

    2011-01-01

    RapidEye AG is a commercial provider of geospatial information products and customized solutions derived from Earth observation image data. The source of the data is the RapidEye constellation consisting of five low-earth-orbit imaging satellites. We describe the rationale, methods, and results of a reflectance-based vicarious calibration campaign that was conducted between April 2009 and May 2010 at Railroad Valley Playa and Ivanpah Playa to determine the on-orbit radiometric accuracy of the RapidEye sensor. In situ surface spectral reflectance measurements of known ground targets and an assessment of the atmospheric conditions above the sites were taken during spacecraft overpasses. The ground data are used as input to a radiative transfer code to compute a band-specific top-of-atmosphere spectral radiance. A comparison of these predicted values based on absolute physical data to the measured at-sensor spectral radiance provide the absolute calibration of the sensor. Initial assessments show that the RapidEye sensor response is within 8% of the predicted values. Outcomes from this campaign are then used to update the calibration parameters in the ground segment processing system. Subsequent verification events confirmed that the measured RapidEye response improved to within 4% of the predictions based on the vicarious calibration method.

  20. Comparison of methods for generation of absolute reflectance-factor values for bidirectional reflectance-distribution function studies.

    PubMed

    Feng, X; Schott, J R; Gallagher, T

    1993-03-01

    Currently, spectrophotometric standard reference materials are calibrated only by using the illumination and viewing geometries recommended by the Commission Internationale de l'Eclairage, and for some geometries the spectral range is limited to the visible wavelengths. A need exists for procedures that calibrate standards at many other geometries and for a broader spectral range. Two methods for calibrating the spectral bidirectional reflectance factor are described. The absolute bidirectional reflectance factor of a sintered polytetrafluoroethylene (PTFE) sample is determined for nearly all the possible illumination and viewing geometries from 400 nm to 2500 nm. The references are a 45/0 reflectance standard calibrated by the National Institute of Standards and Technology and a sintered PTFE sample with a directional, hemispherical reflectance factor traceable to the Institute. The results of the two methods agree to within 0.01 in reflectance factor values. With this PTFE sample as a transfer standard, the instrument described can also be used to measure the absolute bidirectional reflectance factor at nearly all the illumination and viewing geometries from 400 nm to 2500 nm. PMID:20820258

  1. 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.

  2. 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

  3. A Quantification Method for Breast Tissue Thickness and Iodine Concentration Using Photon-Counting Detector.

    PubMed

    Han, Seokmin

    2015-10-01

    The purpose of contrast-enhanced digital mammography (CEDM) is to facilitate detection and characterization of the lesions in the breast using intravenous injection of an iodinated contrast agent. CEDM produces iodine images with gray levels proportional to iodine concentration at each pixel, which can be considered as quantification of iodine. While dual-energy CEDM requires an accurate knowledge of the thickness of compressed breast for the quantification, it is known that the accuracy of the built-in thickness measurement is not satisfactory. Triple-energy CEDM, which can provide a third image, can alleviate the limitation of dual-energy CEDM. If triple exposure technique is applied, it can lead to increased risk of motion artifact. An energy-resolving photon-counting detector (PCD) that can acquire multispectral X-ray images can reduce the risk of motion artifact. In this research, an easily implementable method for iodine quantification in breast imaging was suggested, and it was applied to the images of breast phantom with various iodine concentrations. The iodine concentrations in breast phantom simulate lesions filled with different iodine concentrations in the breast. The result shows that the proposed method can quantify the iodine concentrations in breast phantom accurately. PMID:25708894

  4. 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. PMID:26760242

  5. 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

  6. A method for establishing absolute full-energy peak efficiency and its confidence interval for HPGe detectors

    NASA Astrophysics Data System (ADS)

    Rizwan, U.; Chester, A.; Domingo, T.; Starosta, K.; Williams, J.; Voss, P.

    2015-12-01

    A method is proposed for establishing the absolute efficiency calibration of a HPGe detector including the confidence interval in the energy range of 79.6-3451.2 keV. The calibrations were accomplished with the 133Ba, 60Co, 56Co and 152Eu point-like radioactive sources with only the 60Co source being activity calibrated to an accuracy of 2% at the 90% confidence level. All data sets measured from activity calibrated and uncalibrated sources were fit simultaneously using the linearized least squares method. The proposed fit function accounts for scaling of the data taken with activity uncalibrated sources to the data taken with the high accuracy activity calibrated source. The confidence interval for the fit was found analytically using the covariance matrix. Accuracy of the fit was below 3.5% at the 90% confidence level in the 79.6-3451.2 keV energy range.

  7. 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.

  8. Determination of the specific heat capacity of a graphite sample using absolute and differential methods

    NASA Astrophysics Data System (ADS)

    Picard, Susanne; Burns, David T.; Roger, Philippe

    2007-10-01

    An experimental assembly has been constructed to measure the specific heat capacity of macroscopic graphite samples at room temperature. The same batch of graphite constitutes the core of a graphite calorimeter, which is currently being realized to measure the absorbed dose due to ionizing radiation. Two different experimental procedures have been applied. In the first method the specific heat capacity of graphite was measured directly, where its value is corrected for the influence of impurities. The second method, to our knowledge not previously applied to macroscopic samples, is based on a series of differential measurements where no correction for added impurities is needed. By its nature, the second method reduces systematic effects. The specific heat capacity of a particular graphite sample is determined to be 706.9 J K-1 kg-1 with a combined relative standard uncertainty of 9 parts in 104 at 295.15 K. The specific heat capacity of cyanoacrylate has also been determined.

  9. 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. PMID:22468648

  10. 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

  11. Ultra-trace quantification method for chlordecone in human fluids and tissues.

    PubMed

    Bichon, Emmanuelle; Guiffard, Ingrid; Vénisseau, Anaïs; Marchand, Philippe; Antignac, Jean-Philippe; Le Bizec, Bruno

    2015-08-21

    Chlordecone is an organochlorine pesticide (OCP) considered as a Persistent Organic Pollutant (POP) as it persists in the environment, bio-accumulates through the food web, causes adverse effects to human health and the environment and transports across international boundaries far from its sources. The atypical physico-chemical properties of chlordecone make its inclusion in classical analytical approaches non applicable. The aim of our work was to include chlordecone in a multi organochlorine residue method preventing any degradation during the analytical process and thus allowing quantification at ppt (ngkg(-1) or ngL(-1)) levels for a wide range of OCPs in breast milk, human serum and adipose tissue. After GC-HRMS vs. MS/MS and EI vs. APCI comparisons, the major improvement in terms of sensitivity was found in decreasing the length and film thickness of the gas chromatography column. Thanks to a linear correlation between relative response and quantity of chlordecone injected, LC-(ESI-)-MS/MS was finally preferred. An acetonitrile based gradient optimized on a C30 coreshell HPLC column has led to reaching limits of quantification as low as 8ngL(-1), 25pgmL(-1) and 0.2ngg(-1) fat for breast milk, serum and adipose tissue, respectively, allowing multiresidue OCP quantification at concentration levels compatible with biomonitoring purposes and pre-requisites. PMID:26184709

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

    PubMed

    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

  13. 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

  14. Improved quantification of pyrogenic carbon in soils and sediments by a HPLC-DAD method

    NASA Astrophysics Data System (ADS)

    Wiedemeier, D. B.; Hilf, M. D.; Smittenberg, R. H.; Schmidt, M. W. I.

    2012-04-01

    Fire-derived (pyrogenic) carbon (PyC) is produced by the incomplete combustion of biomass, for example during wildfires. It can persist in the environment for a long time due to its relative resistance against biological and chemical breakdown. Its accurate quantification in soils and sediments is of great interest because the slow turn-over of PyC has implications for the global carbon cycle and carbon budget calculations. Moreover, PyC in pedological and sedimentological records can be used to reconstruct wildfire history or to investigate historical periods like the industrialization. A whole suite of PyC quantification methods exists because PyC is not a defined chemical structure but rather a continuum of thermally altered biomass. The benzene polycarboxylic acids (BPCA) analysis is a molecular marker method that was shown to give conservative estimates of PyC quantity in soils. In addition, it yields qualitative information about the degree of aromaticity and condensation of PyC. The commonly used BPCA method consists in digesting samples with nitric acid that breaks down the PyC into a suite of BPCAs, which are cleaned, derivatized and finally analyzed by gas chromatography-flame ionization detection (GC-FID). Here, we present a modified BPCA method for soils and sediments that uses a high performance liquid chromatography system coupled to diode array detection (HPLC-DAD). We demonstrate that this method greatly enhances the reproducibility of PyC quantification in soil and sediment samples while significantly reducing analysis time. Moreover, much less sample material is needed for precise PyC quantification and we show that the HPLC-DAD method yields consistently higher PyC contents than the GC-FID method. Additionally, the modified method also facilitates δ13C and 14C measurements of the PyC fraction in these complex matrix samples. The isotopic information further improves the assessment of PyC budgets in the environment and the reconstruction of past

  15. 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

  16. 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/. PMID:26935399

  17. 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.

  18. A new method for measuring absolute total electron-impact cross sections with forward scattering corrections

    SciTech Connect

    Ma, C.; Liescheski, P.B.; Bonham, R.A. )

    1989-12-01

    In this article we describe an experimental technique to measure the total electron-impact cross section by measurement of the attenuation of an electron beam passing through a gas at constant pressure with the unwanted forward scattering contribution removed. The technique is based on the different spatial propagation properties of scattered and unscattered electrons. The correction is accomplished by measuring the electron beam attenuation dependence on both the target gas pressure (number density) and transmission length. Two extended forms of the Beer--Lambert law which approximately include the contributions for forward scattering and for forward scattering plus multiple scattering from the gas outside the electron beam were developed. It is argued that the dependence of the forward scattering on the path length through the gas is approximately independent of the model used to describe it. The proposed methods were used to determine the total cross section and forward scattering contribution from argon (Ar) with 300-eV electrons. Our results are compared with those in the literature and the predictions of theory and experiment for the forward scattering and multiple scattering contributions. In addition, Monte Carlo simulations were performed as a further test of the method.

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

    PubMed

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

    2016-07-01

    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. PMID:27279427

  20. Absolute calibration method for fast-streaked, fiber optic light collection, spectroscopy systems.

    SciTech Connect

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

    2010-04-01

    This report outlines a convenient method to calibrate fast (<1ns resolution) streaked, fiber optic light collection, spectroscopy systems. Such a system is used to collect spectral data on plasmas generated in the A-K gap of electron beam diodes fielded on the RITS-6 accelerator (8-12MV, 140-200kA). On RITS, light is collected through a small diameter (200 micron) optical fiber and recorded on a fast streak camera at the output of 1 meter Czerny-Turner monochromator (F/7 optics). To calibrate such a system, it is necessary to efficiently couple light from a spectral lamp into a 200 micron diameter fiber, split it into its spectral components, with 10 Angstroms or less resolution, and record it on a streak camera with 1ns or less temporal resolution.

  1. Absolute conductivity reconstruction in magnetic induction tomography using a nonlinear method.

    PubMed

    Soleimani, Manuchehr; Lionheart, William R B

    2006-12-01

    Magnetic induction tomography (MIT) attempts to image the electrical and magnetic characteristics of a target using impedance measurement data from pairs of excitation and detection coils. This inverse eddy current problem is nonlinear and also severely ill posed so regularization is required for a stable solution. A regularized Gauss-Newton algorithm has been implemented as a nonlinear, iterative inverse solver. In this algorithm, one needs to solve the forward problem and recalculate the Jacobian matrix for each iteration. The forward problem has been solved using an edge based finite element method for magnetic vector potential A and electrical scalar potential V, a so called A, A - V formulation. A theoretical study of the general inverse eddy current problem and a derivation, paying special attention to the boundary conditions, of an adjoint field formula for the Jacobian is given. This efficient formula calculates the change in measured induced voltage due to a small perturbation of the conductivity in a region. This has the advantage that it involves only the inner product of the electric fields when two different coils are excited, and these are convenient computationally. This paper also shows that the sensitivity maps change significantly when the conductivity distribution changes, demonstrating the necessity for a nonlinear reconstruction algorithm. The performance of the inverse solver has been examined and results presented from simulated data with added noise. PMID:17167989

  2. 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

  3. 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. .

  4. A new method for the absolute radiance calibration for UV/vis measurements of scattered sun light

    NASA Astrophysics Data System (ADS)

    Wagner, T.; Beirle, S.; Dörner, S.; Penning de Vries, M.; Remmers, J.; Rozanov, A.; Shaiganfar, R.

    2015-05-01

    Absolute radiometric calibrations are important for measurements of the atmospheric spectral radiance. Such measurements can be used to determine actinic fluxes, the properties of aerosols and clouds and the short wave energy budget. Conventional calibration methods in the laboratory are based on calibrated light sources and reflectors and are expensive, time consuming and subject to relatively large uncertainties. Also, the calibrated instruments might change during transport from the laboratory to the measurement sites. Here we present a new calibration method for UV/vis instruments that measure the spectrally resolved sky radiance, like for example zenith sky Differential Optical Absorption Spectroscopy (DOAS-) instruments or Multi-AXis (MAX-) DOAS instruments. Our method is based on the comparison of the solar zenith angle dependence of the measured zenith sky radiance with radiative transfer simulations. For the application of our method clear sky measurements during periods with almost constant aerosol optical depth are needed. The radiative transfer simulations have to take polarisation into account. We show that the calibration results are almost independent from the knowledge of the aerosol optical properties and surface albedo, which causes a rather small uncertainty of about <7%. For wavelengths below about 330 nm it is essential that the ozone column density during the measurements is constant and known.

  5. A non-invasive diffuse reflectance calibration-free method for absolute determination of exogenous biochemicals concentration in biological tissues

    NASA Astrophysics Data System (ADS)

    Lappa, Alexander V.; Kulikovskiy, Artem N.; Busarov, Oleg G.

    2014-03-01

    The paper presents a new method for distant non-destructive determination of concentration of light absorbing admixtures in turbid media. In particular, it is intended for non-invasive in vivo control of accumulation in patient tissues of various biochemicals introduced to the patients for chemotherapy, photodynamic therapy or diagnostics. It is require that the admixture absorption spectrum should have a clearly marked peak in the wavelength region where the pure medium one varies regularly. Fluorescence of admixtures is not required. The method uses the local diffuse reflectance spectroscopy with optical fiber probe including one emitting and two reading There are several features in the method: the value to be determined is absolute concentration of admixtures; the method needs no calibration measurements on phantoms; it needs no reference measurements on sample with zero admixture concentration; it uses a two parametric kinetic light propagation model and original algorithms to resolve direct and inverse tasks of radiation transport theory. Experimental testing passed with tissue equivalent phantoms and different admixtures, including a chlorine photosensitizer, showed accuracy under 10% in all cases.

  6. Estimating absolute methylation levels at single-CpG resolution from methylation enrichment and restriction enzyme sequencing methods

    PubMed Central

    Stevens, Michael; Cheng, Jeffrey B.; Li, Daofeng; Xie, Mingchao; Hong, Chibo; Maire, Cécile L.; Ligon, Keith L.; Hirst, Martin; Marra, Marco A.; Costello, Joseph F.; Wang, Ting

    2013-01-01

    Recent advancements in sequencing-based DNA methylation profiling methods provide an unprecedented opportunity to map complete DNA methylomes. These include whole-genome bisulfite sequencing (WGBS, MethylC-seq, or BS-seq), reduced-representation bisulfite sequencing (RRBS), and enrichment-based methods such as MeDIP-seq, MBD-seq, and MRE-seq. These methods yield largely comparable results but differ significantly in extent of genomic CpG coverage, resolution, quantitative accuracy, and cost, at least while using current algorithms to interrogate the data. None of these existing methods provides single-CpG resolution, comprehensive genome-wide coverage, and cost feasibility for a typical laboratory. We introduce methylCRF, a novel conditional random fields–based algorithm that integrates methylated DNA immunoprecipitation (MeDIP-seq) and methylation-sensitive restriction enzyme (MRE-seq) sequencing data to predict DNA methylation levels at single-CpG resolution. Our method is a combined computational and experimental strategy to produce DNA methylomes of all 28 million CpGs in the human genome for a fraction (<10%) of the cost of whole-genome bisulfite sequencing methods. methylCRF was benchmarked for accuracy against Infinium arrays, RRBS, WGBS sequencing, and locus-specific bisulfite sequencing performed on the same human embryonic stem cell line. methylCRF transformation of MeDIP-seq/MRE-seq was equivalent to a biological replicate of WGBS in quantification, coverage, and resolution. We used conventional bisulfite conversion, PCR, cloning, and sequencing to validate loci where our predictions do not agree with whole-genome bisulfite data, and in 11 out of 12 cases, methylCRF predictions of methylation level agree better with validated results than does whole-genome bisulfite sequencing. Therefore, methylCRF transformation of MeDIP-seq/MRE-seq data provides an accurate, inexpensive, and widely accessible strategy to create full DNA methylomes. PMID:23804401

  7. Estimating absolute methylation levels at single-CpG resolution from methylation enrichment and restriction enzyme sequencing methods.

    PubMed

    Stevens, Michael; Cheng, Jeffrey B; Li, Daofeng; Xie, Mingchao; Hong, Chibo; Maire, Cécile L; Ligon, Keith L; Hirst, Martin; Marra, Marco A; Costello, Joseph F; Wang, Ting

    2013-09-01

    Recent advancements in sequencing-based DNA methylation profiling methods provide an unprecedented opportunity to map complete DNA methylomes. These include whole-genome bisulfite sequencing (WGBS, MethylC-seq, or BS-seq), reduced-representation bisulfite sequencing (RRBS), and enrichment-based methods such as MeDIP-seq, MBD-seq, and MRE-seq. These methods yield largely comparable results but differ significantly in extent of genomic CpG coverage, resolution, quantitative accuracy, and cost, at least while using current algorithms to interrogate the data. None of these existing methods provides single-CpG resolution, comprehensive genome-wide coverage, and cost feasibility for a typical laboratory. We introduce methylCRF, a novel conditional random fields-based algorithm that integrates methylated DNA immunoprecipitation (MeDIP-seq) and methylation-sensitive restriction enzyme (MRE-seq) sequencing data to predict DNA methylation levels at single-CpG resolution. Our method is a combined computational and experimental strategy to produce DNA methylomes of all 28 million CpGs in the human genome for a fraction (<10%) of the cost of whole-genome bisulfite sequencing methods. methylCRF was benchmarked for accuracy against Infinium arrays, RRBS, WGBS sequencing, and locus-specific bisulfite sequencing performed on the same human embryonic stem cell line. methylCRF transformation of MeDIP-seq/MRE-seq was equivalent to a biological replicate of WGBS in quantification, coverage, and resolution. We used conventional bisulfite conversion, PCR, cloning, and sequencing to validate loci where our predictions do not agree with whole-genome bisulfite data, and in 11 out of 12 cases, methylCRF predictions of methylation level agree better with validated results than does whole-genome bisulfite sequencing. Therefore, methylCRF transformation of MeDIP-seq/MRE-seq data provides an accurate, inexpensive, and widely accessible strategy to create full DNA methylomes. PMID:23804401

  8. 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. PMID:26875074

  9. Methods for Calculating the Absolute Entropy and free energy of biological systems based on ideas from Polymer Physics

    PubMed Central

    Meirovitch, Hagai

    2009-01-01

    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, PiB while the value of PiB is not provided directly; therefore, it is difficult to obtain the absolute entropy, S ~ -ln PiB, 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, 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 α-amylase and acetylcholineesterase in explicit water, where the difference of F between the bound and free states of the loop was calculated. Currently HSMD is being extended for

  10. 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

  11. 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

  12. 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.

  13. A novel method for the quantification of quinic acid in food using stable isotope dilution analysis.

    PubMed

    Erk, Thomas; Bergmann, Hannah; Richling, Elke

    2009-01-01

    Organic acids play an important role in the flavor and taste of plant-derived foods. Quinic acid (QA) is one of the major acids. In the past, several methods like HPLC/UV, GC, and capillary electrophoresis were used for identification and quantification of QA. For the first time, a novel, sensitive, and selective method for the quantification of QA in food using stable isotope dilution analysis with HPLC/MS/MS has been established. Uniformly labeled 13C-QA was used as a standard to reduce sample preparations and to overcome matrix and ionization effects. The method was used to determine the QA content of red wines, instant coffees, and cloudy apple juices. QA contents of instant coffees were 64.4 and 63.6 g/kg powder. The concentrations in red wines were 24.0 and 25.1 mg/L, and 1493.3 and 1705.2 mg/L in cloudy apple juices. PMID:19610361

  14. 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.

  15. 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). PMID:24463407

  16. 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.

  17. 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)

  18. 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.

  19. 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.

  20. Development and validation of a specific and sensitive HPLC-ESI-MS method for quantification of lysophosphatidylinositols and evaluation of their levels in mice tissues.

    PubMed

    Masquelier, Julien; Muccioli, Giulio G

    2016-07-15

    Increasing evidence suggests that lysophosphatidylinositols (LPIs), a subspecies of lysophospholipids, are important endogenous mediators. Although LPIs long remained among the less studied lysophospholipids, the identification of GPR55 as their molecular target sparked a renewed interest in the study of these bioactive lipids. Furthermore, increasing evidence points towards a role for LPIs in cancer development. However, a better understanding of the role and functions of LPIs in physiology and disease requires methods that allow for the quantification of LPI levels in cells and tissues. Because dedicated efficient methods for quantifying LPIs were missing, we decided to develop and validate an HPLC-ESI-MS method for the quantification of LPI species from tissues. LPIs are extracted from tissues by liquid/liquid extraction, pre-purified by solid-phase extraction, and finally analyzed by HPLC-ESI-MS. We determined the method's specificity and selectivity, we established calibration curves, determined the carry over (< 2%), LOD and LLOQ (between 0.116-7.82 and 4.62-92.5pmol on column, respectively), linearity (0.988 80%), intermediate precision (CV<20%) as well as the recovery from tissues. We then applied the method to determine the relative abundance of the LPI species in 15 different mouse tissues. Finally, we quantified the absolute LPI levels in six different mouse tissues. We found that while 18:0 LPI represents more than 60% of all the LPI species in the periphery (e.g. liver, gastrointestinal tract, lungs, spleen) it is much less abundant in the central nervous system where the levels of 20:4 LPI are significantly higher. Thus this validated HPLC-ESI-MS method for quantifying LPIs represents a powerful tool that will facilitate the comprehension of the pathophysiological roles of LPIs. PMID:27208623

  1. Flow cytometry for intracellular SPION quantification: specificity and sensitivity in comparison with spectroscopic methods

    PubMed Central

    Friedrich, Ralf P; Janko, Christina; Poettler, Marina; Tripal, Philipp; Zaloga, Jan; Cicha, Iwona; Dürr, Stephan; Nowak, Johannes; Odenbach, Stefan; Slabu, Ioana; Liebl, Maik; Trahms, Lutz; Stapf, Marcus; Hilger, Ingrid; Lyer, Stefan; Alexiou, Christoph

    2015-01-01

    Due to their special physicochemical properties, iron nanoparticles offer new promising possibilities for biomedical applications. For bench to bedside translation of super-paramagnetic iron oxide nanoparticles (SPIONs), safety issues have to be comprehensively clarified. To understand concentration-dependent nanoparticle-mediated toxicity, the exact quantification of intracellular SPIONs by reliable methods is of great importance. In the present study, we compared three different SPION quantification methods (ultraviolet spectrophotometry, magnetic particle spectroscopy, atomic adsorption spectroscopy) and discussed the shortcomings and advantages of each method. Moreover, we used those results to evaluate the possibility to use flow cytometric technique to determine the cellular SPION content. For this purpose, we correlated the side scatter data received from flow cytometry with the actual cellular SPION amount. We showed that flow cytometry provides a rapid and reliable method to assess the cellular SPION content. Our data also demonstrate that internalization of iron oxide nanoparticles in human umbilical vein endothelial cells is strongly dependent to the SPION type and results in a dose-dependent increase of toxicity. Thus, treatment with lauric acid-coated SPIONs (SEONLA) resulted in a significant increase in the intensity of side scatter and toxicity, whereas SEONLA with an additional protein corona formed by bovine serum albumin (SEONLA-BSA) and commercially available Rienso® particles showed only a minimal increase in both side scatter intensity and cellular toxicity. The increase in side scatter was in accordance with the measurements for SPION content by the atomic adsorption spectroscopy reference method. In summary, our data show that flow cytometry analysis can be used for estimation of uptake of SPIONs by mammalian cells and provides a fast tool for scientists to evaluate the safety of nanoparticle products. PMID:26170658

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

    PubMed

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

    2016-01-01

    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 78Gy. 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 1cm above the seminal vesicles to 1cm 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 4mm. 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 V70 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 5.5cc for Sh-Rw, Lg-Rw, Sh-Rec, and Lg-Rec, respectively). As for R-DVHs, the parameters of V70 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 regardless of

  3. Longitudinal quantification of incipient carious lesions in postorthodontic patients using a fluorescence method.

    PubMed

    Aljehani, Abdulaziz; Yousif, Mirgani A; Angmar-Månsson, Birgit; Shi, Xie-Qi

    2006-10-01

    The aims of this study were to evaluate the effect of two caries-preventive programs, and to apply the laser fluorescence method, DIAGNOdent, for longitudinal quantification of changes in incipient carious lesions. Twelve subjects with 127 test teeth exhibiting white spot lesions on the buccal surfaces after completed orthodontic therapy were enrolled in the study. Visual examination was performed at baseline and after 12 months. The subjects were divided into two groups: one group received repeated professional tooth cleaning combined with oral hygiene instruction; and the control group received repeated oral hygiene instruction only. The white spot lesions were measured by DIAGNOdent at baseline, and at 3, 6, 9, and 12 months thereafter. There was a significant difference in the DIAGNOdent readings between the first and the final evaluations. However, there was no statistically significant difference between the two treatment groups regarding changes of DIAGNOdent values over time. In conclusion, it may be possible to use DIAGNOdent for longitudinal quantification of carious lesions on smooth surfaces over a period of 1 yr under in vivo conditions. The combination of professional tooth cleaning and oral hygiene instruction had a similar efficacy to professional tooth cleaning only for promoting the remineralization of white spot lesions. PMID:17026510

  4. 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

  5. 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

  6. Validating a local Arterial Input Function method for improved perfusion quantification in stroke

    PubMed Central

    Willats, Lisa; Christensen, Soren; K Ma, Henry; A Donnan, Geoffrey; Connelly, Alan; Calamante, Fernando

    2011-01-01

    In bolus-tracking perfusion magnetic resonance imaging (MRI), temporal dispersion of the contrast bolus due to stenosis or collateral supply presents a significant problem for accurate perfusion quantification in stroke. One means to reduce the associated perfusion errors is to deconvolve the bolus concentration time-course data with local Arterial Input Functions (AIFs) measured close to the capillary bed and downstream of the arterial abnormalities causing dispersion. Because the MRI voxel resolution precludes direct local AIF measurements, they must be extrapolated from the surrounding data. To date, there have been no published studies directly validating these local AIFs. We assess the effectiveness of local AIFs in reducing dispersion-induced perfusion error by measuring the residual dispersion remaining in the local AIF deconvolved perfusion maps. Two approaches to locating the local AIF voxels are assessed and compared with a global AIF deconvolution across 19 bolus-tracking data sets from patients with stroke. The local AIF methods reduced dispersion in the majority of data sets, suggesting more accurate perfusion quantification. Importantly, the validation inherently identifies potential areas for perfusion underestimation. This is valuable information for the identification of at-risk tissue and management of stroke patients. PMID:21629260

  7. 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.

  8. Methods and approaches for the comprehensive characterization and quantification of cellular proteomes using mass spectrometry

    PubMed Central

    Mirza, Shama P.; Olivier, Michael

    2009-01-01

    Proteomics has been proposed as one of the key technologies in the postgenomic era. So far, however, the comprehensive analysis of cellular proteomes has been a challenge because of the dynamic nature and complexity of the multitude of proteins in cells and tissues. Various approaches have been established for the analyses of proteins in a cell at a given state, and mass spectrometry (MS) has proven to be an efficient and versatile tool. MS-based proteomics approaches have significantly improved beyond the initial identification of proteins to comprehensive characterization and quantification of proteomes and their posttranslational modifications (PTMs). Despite these advances, there is still ongoing development of new technologies to profile and analyze cellular proteomes more completely and efficiently. In this review, we focus on MS-based techniques, describe basic approaches for MS-based profiling of cellular proteomes and analysis methods to identify proteins in complex mixtures, and discuss the different approaches for quantitative proteome analysis. Finally, we briefly discuss novel developments for the analysis of PTMs. Altered levels of PTM, sometimes in the absence of protein expression changes, are often linked to cellular responses and disease states, and the comprehensive analysis of cellular proteome would not be complete without the identification and quantification of the extent of PTMs of proteins. PMID:18162499

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

    PubMed

    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

  10. Radiation dose determines the method for quantification of DNA double strand breaks.

    PubMed

    Bulat, Tanja; Keta, Otilija; Korićanac, Lela; Žakula, Jelena; Petrović, Ivan; Ristić-Fira, Aleksandra; Todorović, Danijela

    2016-03-01

    Ionizing radiation induces DNA double strand breaks (DSBs) that trigger phosphorylation of the histone protein H2AX (γH2AX). Immunofluorescent staining visualizes formation of γH2AX foci, allowing their quantification. This method, as opposed to Western blot assay and Flow cytometry, provides more accurate analysis, by showing exact position and intensity of fluorescent signal in each single cell. In practice there are problems in quantification of γH2AX. This paper is based on two issues: the determination of which technique should be applied concerning the radiation dose, and how to analyze fluorescent microscopy images obtained by different microscopes. HTB140 melanoma cells were exposed to γ-rays, in the dose range from 1 to 16 Gy. Radiation effects on the DNA level were analyzed at different time intervals after irradiation by Western blot analysis and immunofluorescence microscopy. Immunochemically stained cells were visualized with two types of microscopes: AxioVision (Zeiss, Germany) microscope, comprising an ApoTome software, and AxioImagerA1 microscope (Zeiss, Germany). Obtained results show that the level of γH2AX is time and dose dependent. Immunofluorescence microscopy provided better detection of DSBs for lower irradiation doses, while Western blot analysis was more reliable for higher irradiation doses. AxioVision microscope containing ApoTome software was more suitable for the detection of γH2AX foci. PMID:26959322

  11. 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.

  12. 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. PMID:18303841

  13. 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. PMID:26698717

  14. Preanalytical Conditions and DNA Isolation Methods Affect Telomere Length Quantification in Whole Blood.

    PubMed

    Tolios, Alexander; Teupser, Daniel; Holdt, Lesca M

    2015-01-01

    Telomeres are located at chromosome ends and their length (TL) has been associated with aging and human diseases such as cancer. Whole blood DNA is frequently used for TL measurements but the influence of preanalytical conditions and DNA isolation methods on TL quantification has not been thoroughly investigated. To evaluate potential preanalytical as well as methodological bias on TL, anonymized leftover EDTA-whole blood samples were pooled according to leukocyte counts and were incubated with and without actinomycin D to induce apoptosis as a prototype of sample degradation. DNA was isolated from fresh blood pools and after freezing at -80°C. Commercially available kits using beads (Invitrogen), spin columns (Qiagen, Macherey-Nagel and 5prime) or precipitation (Stratec/Invisorb) and a published isopropanol precipitation protocol (IPP) were used for DNA isolation. TL was assessed by qPCR, and normalized to the single copy reference gene 36B4 using two established single-plex and a new multiplex protocol. We show that the method of DNA isolation significantly affected TL (e.g. 1.86-fold longer TL when comparing IPP vs. Invitrogen). Sample degradation led to an average TL decrease of 22% when using all except for one DNA isolation method (5prime). Preanalytical storage conditions did not affect TL with exception of samples that were isolated with the 5prime kit, where a 27% increase in TL was observed after freezing. Finally, performance of the multiplex qPCR protocol was comparable to the single-plex assays, but showed superior time- and cost-effectiveness and required > 80% less DNA. Findings of the current study highlight the need for standardization of whole blood processing and DNA isolation in clinical study settings to avoid preanalytical bias of TL quantification and show that multiplex assays may improve TL/SCG measurements. PMID:26636575

  15. Preanalytical Conditions and DNA Isolation Methods Affect Telomere Length Quantification in Whole Blood

    PubMed Central

    Tolios, Alexander; Teupser, Daniel; Holdt, Lesca M.

    2015-01-01

    Telomeres are located at chromosome ends and their length (TL) has been associated with aging and human diseases such as cancer. Whole blood DNA is frequently used for TL measurements but the influence of preanalytical conditions and DNA isolation methods on TL quantification has not been thoroughly investigated. To evaluate potential preanalytical as well as methodological bias on TL, anonymized leftover EDTA-whole blood samples were pooled according to leukocyte counts and were incubated with and without actinomycin D to induce apoptosis as a prototype of sample degradation. DNA was isolated from fresh blood pools and after freezing at -80°C. Commercially available kits using beads (Invitrogen), spin columns (Qiagen, Macherey-Nagel and 5prime) or precipitation (Stratec/Invisorb) and a published isopropanol precipitation protocol (IPP) were used for DNA isolation. TL was assessed by qPCR, and normalized to the single copy reference gene 36B4 using two established single-plex and a new multiplex protocol. We show that the method of DNA isolation significantly affected TL (e.g. 1.86-fold longer TL when comparing IPP vs. Invitrogen). Sample degradation led to an average TL decrease of 22% when using all except for one DNA isolation method (5prime). Preanalytical storage conditions did not affect TL with exception of samples that were isolated with the 5prime kit, where a 27% increase in TL was observed after freezing. Finally, performance of the multiplex qPCR protocol was comparable to the single-plex assays, but showed superior time- and cost-effectiveness and required > 80% less DNA. Findings of the current study highlight the need for standardization of whole blood processing and DNA isolation in clinical study settings to avoid preanalytical bias of TL quantification and show that multiplex assays may improve TL/SCG measurements. PMID:26636575

  16. A novel computer aided quantification method of focal arteriolar narrowing using colour retinal image.

    PubMed

    Roy, Pallab Kanti; Bhuiyan, Alauddin; Lee, Kim; Wong, Tien Yin; Ramamohanarao, Kotagiri

    2016-07-01

    We present a novel method for the quantification of focal arteriolar narrowing (FAN) in human retina, a precursor for hypertension, stroke and other cardiovascular diseases. A reliable and robust arteriolar boundary mapping method is proposed where intensity, gradient and spatial prior knowledge about the arteriolar shape is incorporated into a graph based optimization method to obtain the arteriolar boundary. Following the mapping of the arteriolar boundaries, arteriolar widths are analysed to quantify the severity of focal arteriolar narrowing (FAN). We evaluate our proposed method on a dataset of 116 retinal arteriolar segments which are manually graded by two expert graders. The experimental results indicate a strong correlation between the quantified FAN measurement scores provided by our method and two experts graded FAN severity levels. Our proposed FAN measurement score: percent narrowing (PN) shows high correlation (Spearman correlation coefficient of 0.82(p<0.0001) for grader-1 and 0.84(p<0.0001) for grade-2) with the manually graded FAN severity levels provided by two expert graders. In addition to that, the proposed method shows better reproducibility (Spearman correlation coefficient ρ=0.92(p<0.0001)) compared to two expert graders ( [Formula: see text] (p<0.0001) and [Formula: see text] ) in two successive sessions. The quantitative measurements provided by the proposed method can help us to establish a more reliable link between FAN and known systemic and eye diseases. PMID:27160638

  17. 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.

  18. 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. PMID:26275477

  19. 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....

  20. A new quantification method for assessing plasma concentrations of pemetrexed and its polyglutamate metabolites.

    PubMed

    Stoop, Marcel P; Visser, Sabine; van Dijk, Evert; Aerts, Joachim G J V; Stricker, Bruno H; Luider, Theo M

    2016-09-01

    Currently no quantification method exists for potentially therapeutically relevant polyglutamate metabolites of the drug pemetrexed which is used for the treatment of lung carcinoma patients. We developed and tested an LC-MS/MS-based analytical assay that uses isotope-labeled internal standards to quantify pemetrexed and its (poly)glutamate metabolites in clinical human plasma samples of lung carcinoma patients. UHPLC chromatography and triple quadrupole mass spectrometry showed an LLOQ of 0.2nmol/L for pemetrexed and an LLOQ of 0.5nmol/L for the two metabolites (one glutamate and two glutamate moieties covalently bound to the pemetrexed molecule, for which no other quantification methods have previously been published). The recoveries for PMTX and its metabolites ranged between 30% and 67%. Precision and accuracy at a concentration of 20nmol/L for all four analytes was well below 15% CV. The precision (RSD) in the biological replicates of the separate days (within-run precision) as well as the reproducibility over several days (between-run precision), tested in the range of 5-250nmol/L, were all below 15%. Autosampler, benchtop and freeze-thaw cycle stability of the analytes was also demonstrated. To illustrate the new assay in a relevant biological context, concentrations of pemetrexed and the two metabolites were quantified in plasma samples of lung carcinoma patients treated with pemetrexed. The assay is straightforward, relatively easy to perform, and has potential for use in therapeutic drug monitoring in non-small cell lung carcinoma patients. PMID:27209449

  1. A novel method for quantification of human hemoglobin from dried blood spots by use of tandem mass spectrometry.

    PubMed

    Yu, Chaowen; Zhang, Juan; Yuan, Zhaojian; Liu, Hao; Wang, Xingbin; Wang, Ming; Zou, Lin

    2015-10-01

    Quantification of human hemoglobin (Hb) is essential for diagnosis of anemia, especially for screening for thalassemia and sickle cell disease. The main methods currently used for quantification of Hb, including spectrophotometry, fluorimetry, and electrochemical assays, are all based on the structural integrity of Hb, which could be affected by hemolysis and degradation. When used for disease screening, whole blood specimens cannot meet requirements for sample collecting, transport, and storage. Here, we report a novel MS-MS method for quantification of Hb from dried blood spots (DBS) by use of a triple-quadrupole mass spectrometer. Proteospecific peptides from α-globin chains were selected after tryptic digestion. The precursor → product ion transitions of representative peptides were studied to identify the best choice with regard to sensitivity and chromatographic properties. For quantification, stable isotope-labeled peptides were used as internal standards. The concentration of Hb in each sample was obtained by calculation on the basis of established equations. The precision of the method was within 15 % and accuracy was in the range -7 to 13.0 %. Compared with routine clinical results obtained by use of the automated hematology analyzer (AHA) assay, the correlation, r (2), was >0.993. When used for determination of anemia levels the sensitivity of the assay was 95.7 % and specificity 96.5 %. Our new approach for quantification of the concentration of Hb from DBS is feasible, and precision is acceptable. The method could be used for determination of anemia levels when screening for hemoglobin disorders. Graphical Abstract Quantification of human hemoglobin from digested dried blood spot samples using tandem mass spectrometry. PMID:26345440

  2. Quantification of organ motion based on an adaptive image-based scale invariant feature method

    SciTech Connect

    Paganelli, Chiara; Peroni, Marta

    2013-11-15

    Purpose: The availability of corresponding landmarks in IGRT image series allows quantifying the inter and intrafractional motion of internal organs. In this study, an approach for the automatic localization of anatomical landmarks is presented, with the aim of describing the nonrigid motion of anatomo-pathological structures in radiotherapy treatments according to local image contrast.Methods: An adaptive scale invariant feature transform (SIFT) was developed from the integration of a standard 3D SIFT approach with a local image-based contrast definition. The robustness and invariance of the proposed method to shape-preserving and deformable transforms were analyzed in a CT phantom study. The application of contrast transforms to the phantom images was also tested, in order to verify the variation of the local adaptive measure in relation to the modification of image contrast. The method was also applied to a lung 4D CT dataset, relying on manual feature identification by an expert user as ground truth. The 3D residual distance between matches obtained in adaptive-SIFT was then computed to verify the internal motion quantification with respect to the expert user. Extracted corresponding features in the lungs were used as regularization landmarks in a multistage deformable image registration (DIR) mapping the inhale vs exhale phase. The residual distances between the warped manual landmarks and their reference position in the inhale phase were evaluated, in order to provide a quantitative indication of the registration performed with the three different point sets.Results: The phantom study confirmed the method invariance and robustness properties to shape-preserving and deformable transforms, showing residual matching errors below the voxel dimension. The adapted SIFT algorithm on the 4D CT dataset provided automated and accurate motion detection of peak to peak breathing motion. The proposed method resulted in reduced residual errors with respect to standard SIFT

  3. 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. PMID:26613377

  4. A simple and sensitive HPLC method for quantification of the metabolin of meclofenoxate in human plasma.

    PubMed

    Ni, Bin; Zhang, Junren; Zou, Jianjun; Zhao, Wei; Li, JianHua

    2010-01-01

    A simple and sensitive high-performance liquid chromatographic method was developed for quantification of the metabolin of meclofenoxate, chlorophenoxyacetic acid, in human plasma. Ibuprofen was used as an internal standard. The present method used protein precipitation for extraction of chlorophenoxyacetic acid from human plasma. Separation was carried out on a reversed-phase C(18) column. The column effluent was monitored by UV detection at 254 nm. The mobile phase was a mixture of methanol and water containing 1.0% glacial acetic acid (70:30 v/v) at a flow rate of 1.0 mL/min. The column temperature was 20 degrees C. This method was linear over the range of 0.047-28.20 microg/mL with a regression coefficient greater than 0.99. The mean recovery of chlorophenoxyacetic acid and IS were (79.54 +/- 6.33)% and (78.48 +/- 2.14)%, respectively, and the method was found to be precise, accurate, and specific during the study. The method was successfully applied for pharmacokinetic study of chlorophenoxyacetic acid in human. PMID:20515527

  5. 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

  6. 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...

  7. 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

  8. A simple and sensitive enzymatic method for cholesterol quantification in macrophages and foam cells

    PubMed Central

    Robinet, Peggy; Wang, Zeneng; Hazen, Stanley L.; Smith, Jonathan D.

    2010-01-01

    A precise and sensitive method for measuring cellular free and esterified cholesterol is required in order to perform studies of macrophage cholesterol loading, metabolism, storage, and efflux. Until now, the use of an enzymatic cholesterol assay, commonly used for aqueous phase plasma cholesterol assays, has not been optimized for use with solid phase samples such as cells, due to inefficient solubilization of total cholesterol in enzyme compatible solvents. We present an efficient solubilization protocol compatible with an enzymatic cholesterol assay that does not require chemical saponification or chromatographic separation. Another issue with enzyme compatible solvents is the presence of endogenous peroxides that interfere with the enzymatic cholesterol assay. We overcame this obstacle by pretreatment of the reaction solution with the enzyme catalase, which consumed endogenous peroxides resulting in reduced background and increased sensitivity in our method. Finally, we demonstrated that this method for cholesterol quantification in macrophages yields results that are comparable to those measured by stable isotope dilution gas chromatography with mass spectrometry detection. In conclusion, we describe a sensitive, simple, and high-throughput enzymatic method to quantify cholesterol in complex matrices such as cells. PMID:20688754

  9. Collaborative validation of the quantification method for suspected allergens and test of an automated data treatment.

    PubMed

    Chaintreau, Alain; Cicchetti, Esmeralda; David, Nathalie; Earls, Andy; Gimeno, Pascal; Grimaud, Béatrice; Joulain, Daniel; Kupfermann, Nikolai; Kuropka, Gryta; Saltron, Frédéric; Schippa, Christine

    2011-10-28

    Previous publications investigated different data treatment strategies for quantification of volatile suspected allergens by GC/MS. This publication presents the validation results obtained on "ready to inject" samples under reproducibility conditions following inter-laboratory ring-testing. The approach is based on the monitoring of three selected ions per analyte using two different GC capillary columns. To aid the analysts a decisional tree is used for guidance during the interpretation of the analytical results. The method is evaluated using a fragrance oil concentrate spiked with all suspected allergens to mimic the difficulty of a real sample extract or perfume oil. At the concentrations of 10 and 100mg/kg, imposed by Directive 76/768/EEC for labeling of leave-on and rinse-off cosmetics, the mean bias is +14% and -4%, respectively. The method is linear for all analytes, and the prediction intervals for each analyte have been determined. To speed up the analyst's task, an automated data treatment is also proposed. The method mean bias is slightly shifted towards negative values, but the method prediction intervals are close to that resulting from the decisional tree. PMID:21945622

  10. Kinetic detection method for the quantification of isoenzymes on electrophoretic media

    SciTech Connect

    Hampton, R.S.; Rutan, S.C. )

    1993-04-01

    A method has been developed for the quantitative determination of isozymes that combines electrophoresis, charge coupled device imaging, denaturation, and kinetic analysis. Detection of the Isozymes after electrophoresis is achieved by introducing a substrate that upon reaction with the isozymes forms a colored or fluorescent product. A tetrazolium dye and 4-methylumbelfferyl phosphate (MUP) are used to visualize lactose dehydrogenase (LDH) and alkaline phosphatase (ALP), respectively. The rate of product formation can be related to the activity of the isozymes. Poorly resolved bands can be distinguished by the addition of a denaturant, guanidine hydrochloride that differently deactivities the isozymes. The data are then analyzed using zero-and first-order kinetic models. Studies with LDH show that this method is as accurate and precise as traditional fixed time methods of analysis for the qualification of well-separated isozyme bands on planar separation media, with an average precision of 6.4% as compared to 7.4% with a fixed time analysis. The ALP studies provided information on the capability of the method for quantification of poorly resolved bands after electrophoresis. Linear calibration curves were obtained for the bovine intestinal and liver ALP in the range of 0-200 and 0-700 units/L, respectively. The deactivation constant is consistent for liver isozyme samples containing 180-640 units/L. 41 refs., 5 figs., 3 tabs.

  11. 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. PMID:26003718

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

    PubMed

    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

    2010-09-01

    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 PM(1), 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 mug/m(3) of PM(2.5) inside the bus, respectively, with significantly higher crankcase self pollution (SP) when windows were closed. Approximately two-thirds of in-cabin PM(2.5) 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

  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. PMID:24057775

  14. 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.

  15. 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

  16. 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

  17. Distribution assessment and quantification of counterfeit melamine in powdered milk by NIR imaging methods.

    PubMed

    Huang, Yue; Tian, Kuangda; Min, Shungeng; Xiong, Yanmei; Du, Guorong

    2015-06-15

    This paper presents a rapid calculation method for the imaging process in the identification and quantification of prohibited additives in milk. Data abstraction methods such as principal component analysis (PCA), classical least squares regression (CLS), and alternative least squares regression (ALS) were used. Different multivariate calculations provided possibilities of quantifying near-infrared (NIR) spectral data cube obtained from the surface of the complex mixture. The results of principal component decomposition confirmed that sample mixture identification is feasible using the PCA-CCI methods. Subsequently, CLSI was used for the direct quantitative analysis of the specific component. Behaving more conveniently than PLS without modeling, CLSI can obtain quantitative information as that melamine generally distribute at the low concentration range of 0-0.5 w/w. Moreover, ALSI can quantify the target component with higher accuracy than CLSI. Standard error of residue to predicted value is 0.0838. Lack of fit is 0.0841. Explanation of variables in the mixture is 99.30%, illustrating that the selective lack of rank is insignificant. Obviously, the most intuitive distribution images are constructed by ALSI among four imaging methods. PMID:25660874

  18. Development for 2D pattern quantification method on mask and wafer

    NASA Astrophysics Data System (ADS)

    Matsuoka, Ryoichi; Mito, Hiroaki; Toyoda, Yasutaka; Wang, Zhigang

    2010-03-01

    We have developed the effective method of mask and silicon 2-dimensional metrology. The aim of this method is evaluating the performance of the silicon corresponding to Hotspot on a mask. The method adopts a metrology management system based on DBM (Design Based Metrology). This is the high accurate contouring created by an edge detection algorithm used in mask CD-SEM and silicon CD-SEM. Currently, as semiconductor manufacture moves towards even smaller feature size, this necessitates more aggressive optical proximity correction (OPC) to drive the super-resolution technology (RET). In other words, there is a trade-off between highly precise RET and mask manufacture, and this has a big impact on the semiconductor market that centers on the mask business. 2-dimensional Shape quantification is important as optimal solution over these problems. Although 1-dimensional shape measurement has been performed by the conventional technique, 2-dimensional shape management is needed in the mass production line under the influence of RET. We developed the technique of analyzing distribution of shape edge performance as the shape management technique. On the other hand, there is roughness in the silicon shape made from a mass-production line. Moreover, there is variation in the silicon shape. For this reason, quantification of silicon shape is important, in order to estimate the performance of a pattern. In order to quantify, the same shape is equalized in two dimensions. And the method of evaluating based on the shape is popular. In this study, we conducted experiments for averaging method of the pattern (Measurement Based Contouring) as two-dimensional mask and silicon evaluation technique. That is, observation of the identical position of a mask and a silicon was considered. It is possible to analyze variability of the edge of the same position with high precision. The result proved its detection accuracy and reliability of variability on two-dimensional pattern (mask and

  19. 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. PMID:25379538

  20. 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

  1. An optimized and validated (1)H NMR method for the quantification of α-pinene in essentials oils.

    PubMed

    Cerceau, Cristiane I; Barbosa, Luiz C A; Filomeno, Claudinei A; Alvarenga, Elson S; Demuner, Antônio J; Fidencio, Paulo H

    2016-04-01

    The authenticity and composition of commercial essential oils requires strict quality control. Due to the importance of α-pinene containing essential oils, a rapid and efficient method for quantification of this terpene in oils of eucalyptus, pink pepper and turpentine using (1)H NMR was developed and validated. All evaluated parameters (selectivity, linearity, accuracy/precision, repeatability, robustness, stability of analyte and internal standard in solutions) showed satisfactory results. The limit of detection (LOD) and limit of quantification (LOQ) were 0.1 and 2.5mg respectively. These values indicated that α-pinene was detected in 35 mg samples containing at least 0.3% of this compound. In addition, a minimum of 8% of α-pinene in the sample was required for quantification. Furthermore, the standard deviations found in the (1)H NMR methodology were less than 1% and were lower than those obtained by gas chromatographic analysis. Statistical tests have shown that the results obtained by (1)H NMR methodology are similar to those obtained by GC-FID technique using external and internal standardization and normalization within 95% confidence. R&R values lower than 10% have shown that all the methods are appropriate and the (1)H NMR method is suitable for quantification of α-pinene in samples of essential oils since this method possessed the smallest R&R (1.81) value. PMID:26838386

  2. A novel method to prioritize RNAseq data for post-hoc analysis based on absolute changes in transcript abundance.

    PubMed

    McNutt, Patrick; Gut, Ian; Hubbard, Kyle; Beske, Phil

    2015-06-01

    The use of fold-change (FC) to prioritize differentially expressed genes (DEGs) for post-hoc characterization is a common technique in the analysis of RNA sequencing datasets. However, the use of FC can overlook certain population of DEGs, such as high copy number transcripts which undergo metabolically expensive changes in expression yet fail to exceed the ratiometric FC cut-off, thereby missing potential important biological information. Here we evaluate an alternative approach to prioritizing RNAseq data based on absolute changes in normalized transcript counts (ΔT) between control and treatment conditions. In five pairwise comparisons with a wide range of effect sizes, rank-ordering of DEGs based on the magnitude of ΔT produced a power curve-like distribution, in which 4.7-5.0% of transcripts were responsible for 36-50% of the cumulative change. Thus, differential gene expression is characterized by the high production-cost expression of a small number of genes (large ΔT genes), while the differential expression of the majority of genes involves a much smaller metabolic investment by the cell. To determine whether the large ΔT datasets are representative of coordinated changes in the transcriptional program, we evaluated large ΔT genes for enrichment of gene ontologies (GOs) and predicted protein interactions. In comparison to randomly selected DEGs, the large ΔT transcripts were significantly enriched for both GOs and predicted protein interactions. Furthermore, enrichments were were consistent with the biological context of each comparison yet distinct from those produced using equal-sized populations of large FC genes, indicating that the large ΔT genes represent an orthagonal transcriptional response. Finally, the composition of the large ΔT gene sets were unique to each pairwise comparison, indicating that they represent coherent and context-specific responses to biological conditions rather than the non-specific upregulation of a family of genes

  3. 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

  4. Drug detection and quantification directly from tissue using novel ionization methods for mass spectrometry.

    PubMed

    Wang, Beixi; Dearring, Chenelle L; Wager-Miller, James; Mackie, Ken; Trimpin, Sarah

    2015-01-01

    Solvent assisted ionization inlet (SAII) and matrix assisted ionization vacuum (MAIV) were used to quantify rapidly an antipsychotic drug, clozapine, directly from surfaces with minimal sample preparation. This simple surface analysis method based on SAII- and MAIV-mass spectrometry (MS) was developed to allow the detection of endogenous lipids, metabolites, and clozapine directly from sections of mouse brain tissue. A rapid surface assessment was achieved by SAII with the assistance of heat applied to the mass spectrometer inlet. MAIV provided an improved reproducibility without the need of a heated inlet. In addition, isotope dilution and standard addition were used without sample clean-up, and the results correlate well with liquid chromatography tandem MS using sample work-up. Using the simple surface methods, standard solutions containing clozapine and a deuterated internal standard (clozapine-d8) at different concentration ratios were used in the extraction and quantification of clozapine from brain tissue sections of a drug-treated mouse using different tissue thicknesses. The amount of clozapine extracted by these surface methods was independent of tissue thickness. PMID:26307700

  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. PMID:26270397

  6. An Innovative Method for Obtaining Consistent Images and Quantification of Histochemically Stained Specimens

    PubMed Central

    Sedgewick, Gerald J.; Ericson, Marna

    2015-01-01

    Obtaining digital images of color brightfield microscopy is an important aspect of biomedical research and the clinical practice of diagnostic pathology. Although the field of digital pathology has had tremendous advances in whole-slide imaging systems, little effort has been directed toward standardizing color brightfield digital imaging to maintain image-to-image consistency and tonal linearity. Using a single camera and microscope to obtain digital images of three stains, we show that microscope and camera systems inherently produce image-to-image variation. Moreover, we demonstrate that post-processing with a widely used raster graphics editor software program does not completely correct for session-to-session inconsistency. We introduce a reliable method for creating consistent images with a hardware/software solution (ChromaCal™; Datacolor Inc., NJ) along with its features for creating color standardization, preserving linear tonal levels, providing automated white balancing and setting automated brightness to consistent levels. The resulting image consistency using this method will also streamline mean density and morphometry measurements, as images are easily segmented and single thresholds can be used. We suggest that this is a superior method for color brightfield imaging, which can be used for quantification and can be readily incorporated into workflows. PMID:25575568

  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. A neutral pH thermal hydrolysis method for quantification of structured RNAs.

    PubMed

    Wilson, Stephen C; Cohen, Daniel T; Wang, Xin C; Hammond, Ming C

    2014-07-01

    Riboswitch aptamers adopt diverse and complex tertiary structural folds that contain both single-stranded and double-stranded regions. We observe that this high degree of secondary structure leads to an appreciable hypochromicity that is not accounted for in the standard method to calculate extinction coefficients using nearest-neighbor effects, which results in a systematic underestimation of RNA concentrations. Here we present a practical method for quantifying riboswitch RNAs using thermal hydrolysis to generate the corresponding pool of mononucleotides, for which precise extinction coefficients have been measured. Thermal hydrolysis can be performed at neutral pH without reaction quenching, avoids the use of nucleases or expensive fluorescent dyes, and does not require generation of calibration curves. The accuracy of this method for determining RNA concentrations has been validated using quantitative (31)P-NMR calibrated to an external standard. We expect that this simple procedure will be generally useful for the accurate quantification of any sequence-defined RNA sample, which is often a critical parameter for in vitro binding and kinetic assays. PMID:24860014

  9. 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

    2011-04-01

    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. PMID:21168299

  10. 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.

  11. An Optimized Method for Quantification of Pathogenic Leptospira in Environmental Water Samples.

    PubMed

    Riediger, Irina N; Hoffmaster, Alex R; Casanovas-Massana, Arnau; 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

  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. Comparison of colorimetric methods for the quantification of model proteins in aqueous two-phase systems.

    PubMed

    Glyk, Anna; Heinisch, Sandra L; Scheper, Thomas; Beutel, Sascha

    2015-05-15

    In the current study, the quantification of different model proteins in the presence of typical aqueous two-phase system components was investigated by using the Bradford and bicinchoninic acid (BCA) assays. Each phase-forming component above 1 and 5 wt% had considerable effects on the protein quantification in both assays, respectively, resulting in diminished protein recoveries/absorption values by increasing poly(ethylene glycol) (PEG)/salt concentration and PEG molecular weight. Therefore, a convenient dilution of both components (up to 1 and 5 wt%) before protein quantification is recommended in both assays, respectively, where the BCA assay is favored in comparison with the Bradford assay. PMID:25684109

  15. 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. PMID:26523666

  16. A comprehensive study of the delay vector variance method for quantification of nonlinearity in dynamical systems.

    PubMed

    Jaksic, V; 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

  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. 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. PMID:25053080

  19. 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

  20. Quantification of oxyresveratrol analog trans-2,4,3',5'-tetramethoxystilbene in rat plasma by a rapid HPLC method: application in a pre-clinical pharmacokinetic study.

    PubMed

    Lin, Hai-Shu; Choo, Qiu-Yi; Ho, Paul C

    2010-12-01

    A rapid HPLC method was developed and validated for the quantification of oxyresveratrol analog trans-2,4,3',5'-tetramethoxystilbene (oxyresveratrol tetramethyl ether, OTE) in rat plasma. Chromatographic separation was achieved on an RP-HPLC column, which was protected by a guard column through a 12 min gradient delivery of a mixture of acetonitrile-water at 50°C. The UV absorbance at 325 nm was recorded. The retention time of OTE and trans-stilbene (internal standard) was about 7.7 and 8.4 min, respectively. The calibration curves were linear (R(2) ≥ 0.9986) with a lower limit of quantification of 15 ng/mL. The intra- and inter-day variations, in terms of RSD, were all lower than 9.8% while the intra-day and inter-day bias ranged from -8.3 to +9.2%. The pharmacokinetics of OTE was assessed in rats using 2-hydroxypropyl-β-cyclodextrin as a dosing vehicle. After intravenous administration, OTE possessed a long terminal elimination half-life (t(1/2) (λz) = 481 ± 137 min) and slow clearance (Cl = 29.1 ± 3.7 mL/min/kg). Upon oral administration, OTE was rapidly absorbed. However, it only displayed minimal plasma exposure and its absolute oral bioavailability (F) was as low as 4.5 ± 3.2%. Fortunately, the levels of OTE after single oral administration were sufficient to inhibit human cytochrome P450 1B1. PMID:21077256

  1. ON A NEW NEAR-INFRARED METHOD TO ESTIMATE THE ABSOLUTE AGES OF STAR CLUSTERS: NGC 3201 AS A FIRST TEST CASE

    SciTech Connect

    Bono, G.; Di Cecco, A.; Sanna, N.; Buonanno, R.; Stetson, P. B.; VandenBerg, D. A.; Calamida, A.; Amico, P.; Marchetti, E.; D'Odorico, S.; Gilmozzi, R.; Dall'Ora, M.; Iannicola, G.; Caputo, F.; Corsi, C. E.; Ferraro, I.; Monelli, M.; Walker, A. R.; Zoccali, M.; Degl'Innocenti, S.

    2010-01-10

    We present a new method to estimate the absolute ages of stellar systems. This method is based on the difference in magnitude between the main-sequence turnoff (MSTO) and a well-defined knee located along the lower main sequence (MSK). This feature is caused by the collisionally induced absorption of molecular hydrogen, and it can easily be identified in near-infrared (NIR) and in optical-NIR color-magnitude diagrams of stellar systems. We took advantage of deep and accurate NIR images collected with the Multi-Conjugate Adaptive Optics Demonstrator temporarily available on the Very Large Telescope and of optical images collected with the Advanced Camera for Surveys Wide Field Camera on the Hubble Space Telescope and with ground-based telescopes to estimate the absolute age of the globular NGC 3201 using both the MSTO and the {delta}(MSTO-MSK). We have adopted a new set of cluster isochrones, and we found that the absolute ages based on the two methods agree to within 1{sigma}. However, the errors of the ages based on the {delta}(MSTO-MSK) method are potentially more than a factor of 2 smaller, since they are not affected by uncertainties in cluster distance or reddening. Current isochrones appear to predict slightly bluer ({approx}0.05 mag) NIR and optical-NIR colors than observed for magnitudes fainter than the MSK.

  2. 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).

  3. 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

  4. 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. PMID:23107159

  5. Quantification method analysis of the relationship between occupant injury and environmental factors in traffic accidents.

    PubMed

    Ju, Yong Han; Sohn, So Young

    2011-01-01

    Injury analysis following a vehicle crash is one of the most important research areas. However, most injury analyses have focused on one-dimensional injury variables, such as the AIS (Abbreviated Injury Scale) or the IIS (Injury Impairment Scale), at a time in relation to various traffic accident factors. However, these studies cannot reflect the various injury phenomena that appear simultaneously. In this paper, we apply quantification method II to the NASS (National Automotive Sampling System) CDS (Crashworthiness Data System) to find the relationship between the categorical injury phenomena, such as the injury scale, injury position, and injury type, and the various traffic accident condition factors, such as speed, collision direction, vehicle type, and seat position. Our empirical analysis indicated the importance of safety devices, such as restraint equipment and airbags. In addition, we found that narrow impact, ejection, air bag deployment, and higher speed are associated with more severe than minor injury to the thigh, ankle, and leg in terms of dislocation, abrasion, or laceration. PMID:21094332

  6. Uncertainty quantification for unsaturated flow in porous media: a stochastic collocation method

    NASA Astrophysics Data System (ADS)

    Barajas-Solano, D. A.; Tartakovsky, D. M.

    2011-12-01

    We present a stochastic collocation (SC) method to quantify epistemic uncertainty in predictions of unsaturated flow in porous media. SC provides a non-intrusive framework for uncertainty propagation in models based on the non-linear Richards' equation with arbitrary constitutive laws describing soil properties (relative conductivity and retention curve). To illustrate the approach, we use the Richards' equation with the van Genutchen-Mualem model for water retention and relative conductivity to describe infiltration into an initially dry soil whose uncertain parameters are treated as random fields. These parameters are represented using a truncated Karhunen-Loève expansion; Smolyak algorithm is used to construct a structured set of collocation points from univariate Gauss quadrature rules. A resulting deterministic problem is solved for each collocation point, and together with the collocation weights, the statistics of hydraulic head and infiltration rate are computed. The results are in agreement with Monte Carlo simulations. We demonstrate that highly heterogeneous soils (large variances of hydraulic parameters) require cubature formulas of high degree of exactness, while their short correlation lengths increase the dimensionality of the problem. Both effects increase the number of collocation points and thus of deterministic problems to solve, affecting the overall computational cost of uncertainty quantification.

  7. The Next-Generation PCR-Based Quantification Method for Ambient Waters: Digital PCR.

    PubMed

    Cao, Yiping; Griffith, John F; Weisberg, Stephen B

    2016-01-01

    Real-time quantitative PCR (qPCR) is increasingly being used for ambient water monitoring, but development of digital polymerase chain reaction (digital PCR) has the potential to further advance the use of molecular techniques in such applications. Digital PCR refines qPCR by partitioning the sample into thousands to millions of miniature reactions that are examined individually for binary endpoint results, with DNA density calculated from the fraction of positives using Poisson statistics. This direct quantification removes the need for standard curves, eliminating the labor and materials associated with creating and running standards with each batch, and removing biases associated with standard variability and mismatching amplification efficiency between standards and samples. Confining reactions and binary endpoint measurements to small partitions also leads to other performance advantages, including reduced susceptibility to inhibition, increased repeatability and reproducibility, and increased capacity to measure multiple targets in one analysis. As such, digital PCR is well suited for ambient water monitoring applications and is particularly advantageous as molecular methods move toward autonomous field application. PMID:27460373

  8. 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. PMID:21910963

  9. A method for quantification of gas plumes in thermal hyperspectral imagery

    NASA Astrophysics Data System (ADS)

    Messinger, David W.

    2005-06-01

    Several commercial and environmental applications require the detection and quantification of gaseous plumes from airborne platforms. Unlike active LIDAR imaging in a DIAL system, the signal received by a passive sensor depends not only on the gas concentration pathlength, but the temperature contrast between the gas column and the background as well. Further complicating the problem, the at-sensor radiance is a function of a non-linear combination of the gas concentration and temperature, both inherently unknown. A method is presented to estimate the gas concentration pathlength and temperature from LWIR Hyperspectral Imagery (HSI) without any assumptions about the gas properties or background radiance. A non-linear model is fit to the data using a Levenberg-Marquardt fitting procedure. This technique requires only a priori knowledge of the gas species present in the pixel of interest to reduce the complexity of the model. The resulting concentration pathlength and temperature are reported on a per-pixel basis. Results are shown for application to synthetic imagery created with the DIRSIG simulation. Concentration pathlength results are promising for a gas with strong, moderately broad features (Freon) but less so for a gas with weaker, narrow features (NH3). In neither case is the solution to the gas temperature satisfactory. This is further demonstrated through examination of the residual space in which the minimization is performed where it is shown that a unique minimum is not present in the space.

  10. In vivo reliability of an infrared fluorescence method for quantification of carious lesions in orthodontic patients.

    PubMed

    Aljehani, Abdulaziz; Bamzahim, Mohammad; Yousif, Mirgani Awad; Shi, Xie-Qi

    2006-01-01

    The aim of this study was to evaluate the reliability of a laser-induced infrared fluorescence method, DIAGNOdent, for measuring orthodontically induced white spot lesions. The subjects comprised 13 orthodontic patients, aged 13-17 years, who had recently completed fixed appliance therapy: 137 test teeth were selected, with white spot lesions on the facial or buccal smooth surfaces. An initial visual inspection was performed to localise and record the measuring region. The predetermined measuring regions were scanned to locate the sites of the highest reading. The readings and their corresponding sites were registered on the print out photographs. Following the measurement by the first examiner, the second and the third examiners took DIAGNOdent readings independently at the same lesion sites indicated on the photographs, under identical conditions. One week later, DIAGNOdent readings of the same lesions were retaken by the three observers working independently. Intra- and inter- examiner agreements on DIAGNOdent quantification of lesion severity were analysed by Intra-class correlation coefficient (ICC). The ICC values for intra-examiner agreement for the three examiners were 0.91, 0.97, and 0.98, respectively, with a mean value of 0.95, indicating excellent agreement. The ICC values for inter-examiner agreement were comparatively lower: 0.69 and 0.82 for the first and second measurements, respectively. It was concluded that the reliability of the DIAGNOdent readings on white spot lesions associated with orthodontic banding was good. PMID:16813144

  11. A novel RP-HPLC method for the detection and quantification of roxithromycin in topical delivery studies.

    PubMed

    Aucamp, M E; Csongradi, C; Gerber, M; Du Plessis, J

    2016-04-01

    A novel HPLC method with UV detection for the identification and quantification of roxithromycin (ROX) during in vitro skin penetration studies has been developed and validated. The method proved to be simple and rapid with isocratic elution (flow rate: 1.0 mL/min) of ROX, using a C18 column and UV detection at 205 nm. The mobile phase consisted of 0.06 M potassium di-hydrogen orthophosphate buffer (pH adjusted to 7.4 with sodium hydroxide) and acetonitrile in a 50:50 (v/v) ratio. This method showed linearity across the concentration range of 5 - 1000 μg/mL with a correlation coefficient of 0.9999. An average recovery of 101.78% was obtained. Limit of detection (LOD) and lower limit of quantification (LLOQ) values proved that ROX can still be detected at a concentration level of 0.3 μg/mL and accurately quantified at a concentration of 0.5 μg/mL. The specificity testing during method validation proved that this method is suitable for the accurate detection and quantification of ROX even when combined with different compounds typically used during the formulation of topical delivery systems. PMID:27209694

  12. Rapid and simple UPLC-MS/MS method for precise phytochelatin quantification in alga extracts.

    PubMed

    Bräutigam, Anja; Wesenberg, Dirk; Preud'homme, Hugues; Schaumlöffel, Dirk

    2010-09-01

    Quantitative phytochelatin (PC) analysis is, due to oxidation sensitivity of the PCs, matrix effects, and time consuming sample preparation, still a challenging analytical task. In this study, a rapid, simple, and sensitive method for precise determination of native PCs in crude extracts of the green alga Chlamydomonas reinhardtii was developed. Algae were exposed 48 h to 70 μM Cd. Coupling of ultra performance liquid chromatography and electrospray ionization tandem mass spectrometry with multi-reaction mode transitions for detection permitted the required short-time, high-resolution separation and detection specificity. Thus, under optimized chromatographic conditions, 10 thiol peptides were baseline-separated within 7 min. Relative detection limits in the nanomolar range in microliter sample volumes were achieved (corresponding to absolute detection limits at femtomole level). Next to glutathione (GSH), the most abundant cadmium-induced PCs in C. reinhardtii, namely CysGSH, PC(2), PC(3), CysPC(2), and CysPC(3), were quantified with high reproducibility at concentrations between 15 and 198 nmol g(-1) fresh weight. The biological variation of PC synthesis of nine independently grown alga cultures was determined to be on average 13.7%. PMID:20632163

  13. 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. PMID:23019168

  14. 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.

  15. Evaluating open-path FTIR spectrometer data using different quantification methods, libraries, and background spectra obtained under varying environmental conditions

    SciTech Connect

    Tomasko, M.S.

    1995-12-31

    Studies were performed to evaluate the accuracy of open-path Fourier Transform Infrared (OP-FTIR) spectrometers using a 35 foot outdoor exposure chamber in Pittsboro, North Carolina. Results obtained with the OP-FTIR spectrometer were compared to results obtained with a reference method (a gas chromatograph equipped with a flame ionization detector, GC-FID). Concentration results were evaluated in terms of the mathematical methods and spectral libraries used for quantification. In addition, the research investigated the effect on quantification of using different backgrounds obtained at various times during the day. The chemicals used in this study were toluene, cyclohexane, and methanol; and these were evaluated over the concentration range of 5-30 ppm.

  16. 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.

  17. Spectrofluorimetric method for quantification of citalopram in pharmaceutical preparations and biological fluids through oxidation with Ce(IV)

    NASA Astrophysics Data System (ADS)

    Shah, Jasmin; Jan, M. Rasul; Khan, M. Naeem; Inayatullah

    2013-01-01

    A sensitive and simple spectrofluorimetric method for the quantification of citalopram in a pure form, in pharmaceutical preparations, and in human blood plasma and urine has been described. The proposed method was based on the oxidation of citalopram by Ce(IV) in acidic media to produce fluorescent Ce(III), and on the subsequent measurement of its fluorescence intensity at 353 nm after excitation at 252 nm. All variables affecting the oxidation of citalopram such as Ce(IV) concentration, the type and concentration of acid, temperature and heating time were studied and optimized. Under the optimized experimental conditions the linear range of concentration versus fluorescence intensity was found to lie between 0.02 and 1.4 g/ml. Limits of detection and quantification were determined to be 6.9·10-3 g/ml and 2.3·10-2 g/ml respectively. Effects of excipients commonly used in the quantification of citalopram have been studied and no interferences were found. The proposed method was successfully applied to determine citalopram in pure form, in pharmaceutical preparations, and in biological fluids. Good recoveries in the ranges of 95.31-101.67%, 88.50-96.67%, and 90.0-96.67% were obtained for the pharmaceutical preparations (tablets), blood plasma, and human urine respectively.

  18. Furan quantification in bread crust: development of a simple and sensitive method using headspace-trap GC-MS.

    PubMed

    Huault, Lucie; Descharles, Nicolas; Rega, Barbara; Bistac, Sophie; Bosc, Véronique; Giampaoli, Pierre

    2016-01-01

    To study reactivity in bread crust during the baking process in the pan, we followed furan mainly resulting from Maillard and caramelisation reactions in cereal products. Furan quantification is commonly performed with automatic HS-static GC-MS. However, we showed that the automatic HS-trap GC-MS method can improve the sensitivity of the furan quantification. Indeed, this method allowed the LOD to be decreased from 0.3 ng g(-1) with HS-static mode to 0.03 ng g(-1) with HS-trap mode under these conditions. After validation of this method for furan quantification in bread crust, a difference between the crust extracted from the bottom and from the sides of the bread was evident. The quantity of furan in the bottom crust was five times lower than in the side crust, revealing less reactivity on the bottom than on the sides of the bread during the baking process in the pan. Differences in water content may explain these variations in reactivity. PMID:26666729

  19. A validated SPME-GC-MS method for simultaneous quantification of club drugs in human urine.

    PubMed

    Brown, Stacy D; Rhodes, Daniel J; Pritchard, Boyd J

    2007-09-13

    A solid-phase microextraction-gas chromatographic-mass spectrometric (SPME-GC-MS) method has been developed and validated for measuring four club drugs in human urine. These drugs include gamma-hydroxybutyrate (GHB), ketamine (KET), methamphetamine (MAMP), and methylenedioxymethamphetamine (MDMA). These drugs are referred to as 'club drugs' because of their prevalence at parties and raves. Deuterium labeled internal standards for each of the four drugs was included in the assay to aid in quantitation. The drugs were spiked into human urine and derivatized using pyridine and hexylchloroformate to make them suitable for GC-MS analysis. The SPME conditions of extraction time/temperature and desorption time/temperature were optimized to yield the highest peak area for each of the four drugs. The final SPME parameters included a 90 degrees C extraction for 20min with a 1min desorption in the GC injector at 225 degrees C using a splitless injection. All SPME work was done using a 100microm PDMS fiber by Supelco. The ratio of pyridine to hexylchloroformate for derivatization was also optimized. The GC separation was carried out on a VF-5ht column by Varian (30m, 0.25mm i.d., 0.10microm film thickness) using a temperature program of 150-270 degrees C at 10 degrees C/min. The instrument used was a ThermoFinnigan Trace GC-Polaris Q interfaced with a LEAP CombiPal autosampler. The data was collected by using extracted ion chromatograms of marker m/z values for each drug from the total ion chromatograms (TIC) (full scan mode). Calibration curves with R(2)>0.99 were generated each day using the peak area ratios (peak area drug/peak area internal standard) versus concentration. The validated method resulted in intra-day and inter-day precision (% R.S.D.) of less than 15% and a % error of less than 15% for four concentrations in the range of 0.05-20microg/mL (MAMP) and 0.10-20microg/mL (GHB, KET, and MDMA). This method has the advantage of an easy sample preparation with

  20. 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. PMID:27279441

  1. Simultaneous Assessment of Cardiomyocyte DNA Synthesis and Ploidy: A Method to Assist Quantification of Cardiomyocyte Regeneration and Turnover.

    PubMed

    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

  2. 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

  3. Direct immunomagnetic quantification of lymphocyte subsets in blood.

    PubMed Central

    Brinchmann, J E; Vartdal, F; Gaudernack, G; Markussen, G; Funderud, S; Ugelstad, J; Thorsby, E

    1988-01-01

    A method is described where superparamagnetic polymer microspheres coated with monoclonal antibodies (MoAb) are used for the direct and fast quantification of the absolute number of cells of various lymphocyte subsets in blood. Blood samples were incubated with microspheres coated with a subset specific MoAb. Using a magnet the microsphere-rosetted cells were isolated and washed. Following lysis of the cell walls to detach the microspheres, the cell nuclei were stained with acridine orange and counted in a haemocytometer using an immunofluorescence microscope. With MoAb specific for CD2, CD4, CD8 and CD19, reproducible absolute counts of the corresponding lymphocyte subsets were obtained which correlated closely with those obtained by an indirect quantification method. PMID:3349645

  4. 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.

  5. 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

    2016-01-01

    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

  6. 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. PMID:26433002

  7. 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. PMID:25850374

  8. Eosinophil count - absolute

    MedlinePlus

    Eosinophils; Absolute eosinophil count ... the white blood cell count to give the absolute eosinophil count. ... than 500 cells per microliter (cells/mcL). Normal value ranges may vary slightly among different laboratories. Talk ...

  9. 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.

  10. Characterisation and optimisation of a method for the detection and quantification of atmospherically relevant carbonyl compounds in aqueous medium

    NASA Astrophysics Data System (ADS)

    Rodigast, M.; Mutzel, A.; Iinuma, Y.; Haferkorn, S.; Herrmann, H.

    2015-01-01

    Carbonyl compounds are ubiquitous in the atmosphere and either emitted primarily from anthropogenic and biogenic sources or they are produced secondarily from the oxidation of volatile organic compounds (VOC). Despite a number of studies about the quantification of carbonyl compounds a comprehensive description of optimised methods is scarce for the quantification of atmospherically relevant carbonyl compounds. Thus a method was systematically characterised and improved to quantify carbonyl compounds. Quantification with the present method can be carried out for each carbonyl compound sampled in the aqueous phase regardless of their source. The method optimisation was conducted for seven atmospherically relevant carbonyl compounds including acrolein, benzaldehyde, glyoxal, methyl glyoxal, methacrolein, methyl vinyl ketone and 2,3-butanedione. O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine hydrochloride (PFBHA) was used as derivatisation reagent and the formed oximes were detected by gas chromatography/mass spectrometry (GC/MS). The main advantage of the improved method presented in this study is the low detection limit in the range of 0.01 and 0.17 μmol L-1 depending on carbonyl compounds. Furthermore best results were found for extraction with dichloromethane for 30 min followed by derivatisation with PFBHA for 24 h with 0.43 mg mL-1 PFBHA at a pH value of 3. The optimised method was evaluated in the present study by the OH radical initiated oxidation of 3-methylbutanone in the aqueous phase. Methyl glyoxal and 2,3-butanedione were found to be oxidation products in the samples with a yield of 2% for methyl glyoxal and 14% for 2,3-butanedione.

  11. 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. PMID:26896631

  12. 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

  13. A method to quantify infectious airborne pathogens at concentrations below the threshold of quantification by culture

    PubMed Central

    Cutler, Timothy D.; Wang, Chong; Hoff, Steven J.; Zimmerman, Jeffrey J.

    2013-01-01

    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). PMID:24082399

  14. 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

  15. 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. PMID:26580828

  16. 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

  17. 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.

  18. A method for in situ absolute DD yield calibration of neutron time-of-flight detectors on OMEGA using CR-39-based proton detectors

    DOE PAGESBeta

    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,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

  19. 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.

  20. A method for in situ absolute DD yield calibration of neutron time-of-flight detectors on OMEGA using CR-39-based proton detectors.

    PubMed

    Waugh, C J; Rosenberg, M J; Zylstra, A B; Frenje, J A; Séguin, F H; Petrasso, R D; Glebov, V Yu; Sangster, T C; Stoeckl, C

    2015-05-01

    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. PMID:26026524

  1. A method for in situ absolute DD yield calibration of neutron time-of-flight detectors on OMEGA using CR-39-based proton detectors

    NASA Astrophysics Data System (ADS)

    Waugh, C. J.; Rosenberg, M. J.; Zylstra, A. B.; Frenje, J. A.; Séguin, F. H.; Petrasso, R. D.; Glebov, V. Yu.; Sangster, T. C.; Stoeckl, C.

    2015-05-01

    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.

  2. 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. PMID:25523465

  3. An Evaluation of Statistical Methods for Analyzing Follow-Up Gaussian Laboratory Data with a Lower Quantification Limit.

    PubMed

    Karon, John M; Wiegand, Ryan E; van de Wijgert, Janneke H; Kilmarx, Peter H

    2015-01-01

    Laboratory data with a lower quantification limit (censored data) are sometimes analyzed by replacing non-quantifiable values with a single value equal to or less than the quantification limit, yielding possibly biased point estimates and variance estimates that are too small. Motivated by a three-period, three-treatment crossover study of a candidate vaginal microbicide in human immunodeficiency virus (HIV)-infected women, we consider four analysis methods for censored Gaussian data with a single follow-up measurement: nonparametric methods, mixed models, mixture models, and dichotomous measures of a treatment effect. We apply these methods to the crossover study data and use simulation to evaluate the statistical properties of these methods in analyzing the treatment effect in a two-treatment parallel-arm or crossover study with censored Gaussian data. Our simulated data and our mixed and mixture models consider treated follow-up data with the same variance as the baseline data or with an inflated variance. Mixed models have the correct type I error, the best power, the least biased Gaussian parameter treatment-effect estimates, and appropriate confidence interval coverage for these estimates. A crossover study analysis with a period effect can greatly increase the required study sample size. For both designs and both variance assumptions, published sample-size estimation methods do not yield a good estimate of the sample size to obtain the stated power. PMID:24906060

  4. 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. PMID:16542570

  5. A simple and sensitive HPLC-UV method for the quantification of piceatannol analog trans-3,5,3',4'-tetramethoxystilbene in rat plasma and its application for a pre-clinical pharmacokinetic study.

    PubMed

    Lin, Hai-Shu; Tringali, Corrado; Spatafora, Carmela; Wu, Chun; Ho, Paul C

    2010-02-01

    A simple and sensitive HPLC-UV method was developed and validated for the quantification of piceatannol analog trans-3,5,3',4'-tetramethoxystilbene (M-PIC) in rat plasma. Following protein precipitation with three volumes of acetonitrile, the analytes were separated on a RP-HPLC column, which was protected by a guard column through gradient delivery of a mixture of acetonitrile-water at 40 degrees C. The UV absorbance at 325nm was recorded to quantify M-PIC. The retention time of M-PIC and trans-3,5-dimethoxystilbene (internal standard) was 7.4 and 8.4min, respectively. The calibration curves were linear (R(2)>0.9989) with a lower limit of quantification of 15ng/ml. The intra- and inter-day precisions, in terms of RSD, were all lower than 7.5%. The average analytical recovery ranged from 97.0 to 104.3% while the average absolute recovery ranged from 101.8 to 105.0%. This reliable HPLC method was subsequently applied to assess the pharmacokinetic profile of M-PIC in Sprague-Dawley rats using 2-hydroxypropyl-beta-cyclodextrin as a dosing vehicle. The terminal elimination half-life (t(1/2lambdaz)) and clearance (Cl) of M-PIC were 313+/-20min and 33.1+/-3.9ml/min/kg, respectively; and its absolute oral bioavailability was as high as 50.7+/-15.0%. M-PIC appeared to have a favorable pharmacokinetic profile and further pharmacological investigation on this phyto-stilbene was warranted. PMID:19836182

  6. 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

  7. Quantification of Volcanic CO2 Emissions Using the Eddy Covariance Method

    NASA Astrophysics Data System (ADS)

    Lewicki, J. L.; Hilley, G. E.; Dobeck, L.; Fischer, M. L.; Mcling, T. L.

    2012-12-01

    Eddy covariance (EC) is a micrometeorological technique proposed as a method to measure passive volcanic CO2 emissions from soil, vent, groundwater, and surface water sources. EC provides an automated, semi-continuous, and time and space-averaged CO2 flux measurement. Also, the measurement's "intermediate" spatial scale (m2-km2) has the potential to bridge the gap between relatively small-scale ground-based measurements (e.g., using the accumulation chamber, AC, method) and relatively large-scale satellite-based observations. We deployed and tested an EC system during two studies in an area of diffuse volcanic CO2 emissions on Mammoth Mountain, CA and near a bubbling spring in Soda Springs, ID. Half-hourly EC CO2 fluxes were measured on Mammoth Mountain during September-October 2006 and ranged from 218 to 3500 g m-2 d-1. Maps of surface CO2 flux were simulated based on AC measurements made repeatedly on a grid over a ten-day period. Large meteorologically driven variations in surface flux distributions and emission rates (16 to 52 t d-1) were observed. Using source weight function modeling, we compared EC to AC measurements of CO2 flux. Half-hour EC CO2 fluxes were moderately correlated (R2 = 0.42) with AC fluxes, whereas average-daily EC and AC fluxes were well correlated (R2 = 0.70). We then made EC measurements of CO2 flux on Mammoth Mountain during September-October 2010, which ranged from 85 to 1766 g m-2 d-1. Three AC soil CO2 flux surveys during this time were used to simulate maps of soil CO2 flux and estimate total emission rates. An inversion of measured EC CO2 fluxes and corresponding modeled source weight functions was carried out and recovered 58 to 77% of the CO2 emission rates estimated based on simulated AC soil CO2 fluxes within a 0.01 km2 area. Spatial distributions of modeled surface CO2 fluxes based on EC and AC observations showed moderate to good correspondence (R2 = 0.36 to 0.70). In September-October 2011, we deployed an EC system near a

  8. New method for caffeine quantification by planar chromatography coupled with electropray ionization mass spectrometry using stable isotope dilution analysis.

    PubMed

    Aranda, Mario; Morlock, Gertrud

    2007-01-01

    A new high-performance thin-layer chromatography/electrospray ionization mass spectrometry (HPTLC/ESI-MS) method for the quantification of caffeine in pharmaceutical and energy drink samples was developed using stable isotope dilution analysis (SIDA). After sample preparation, samples and caffeine standard were applied on silica gel 60 F254 HPTLC plates and over-spotted with caffeine-d3 used for correction of the plunger positioning. After chromatography, densitometric detection was performed by UV absorption at 274 nm. The bands were then eluted by means of a plunger-based extractor into the ESI interface of a single-quadrupole mass spectrometer. For quantification by MS the [M+H]+ ions of caffeine and caffeine-d3 were recorded in the positive ion single ion monitoring (SIM) mode at m/z 195 and 198, respectively. The calibration showed a linear regression with a determination coefficient (R2) of 0.9998. The repeatability (RSD, n=6) in matrix wasmethod accuracy was evaluated by comparing the results obtained by HPTLC/SIDA-ESI-MS with those from the validated HPTLC/UV method. The results for pharmaceutical and energy drink samples were (ng/band) 99.82+/-3.75 and 338.09+/-4.87 by HPTLC/SIDA-ESI-MS and 104.74+/-1.51 and 334.86+/-5.63 by HPTLC/UV. According to the F-test (homogeneity of variances) and the t-test (comparison of means) the two methods show no significant difference. The detection and quantification limits were 75 and 250 microg L-1 (0.75 and 2.5 ng/band), respectively, which were a factor of 13 lower than those established for HPTLC/UV. The positioning error (RSD+/-6%) was calculated by comparing HPTLC/SIDA-ESI-MS with HPTLC/ESI-MS. However, using SIDA the positioning error was nullified. HPTLC/SIDA-ESI-MS was demonstrated to be a

  9. HPLC MS/MS method for quantification of meprobamate in human plasma: application to 24/7 clinical toxicology.

    PubMed

    Delavenne, Xavier; Gay-Montchamp, Jean Pierre; Basset, Thierry

    2011-01-15

    We described the development and full validation of rapid and accurate liquid chromatography method, coupled with tandem mass spectrometry detection, for quantification of meprobamate in human plasma with [(13)C-(2)H(3)]-meprobamate as internal standard. Plasma pretreatment involved a one-step protein precipitation with acetonitrile. Separation was performed by reversed-phase chromatography on a Luna MercuryMS C18 (20 mm×4 mm×3 μm) column using a gradient elution mode. The mobile phase was a mix of distilled water containing 0.1% formic acid and acetonitrile containing 0.1% formic acid. The selected reaction monitoring transitions, in electrospray positive ionization, used for quantification were 219.2→158.2 m/z and 223.1→161.1m/z for meprobamate and internal standard, respectively. Qualification transitions were 219.2→97.0 and 223.1→101.1 m/z for meprobamate and internal standard, respectively. The method was linear over the concentration range of 1-300 mg/L. The intra- and inter-day precision values were below 6.4% and accuracy was within 95.3% and 103.6% for all QC levels (5, 75 and 200 mg/L). The lower limit of quantification was 1 mg/L. Total analysis time was reduced to 6 min including sample preparation. The present method is successfully applied to 24/7 clinical toxicology and demonstrated its usefulness to detect meprobamate poisoning. PMID:21185792

  10. 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. PMID:27283616

  11. 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

  12. Development of a gas chromatography-mass spectrometry method for the quantification of glucaric Acid derivatives in beverage substrates.

    PubMed

    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

  13. 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

  14. An UPLC-MS/MS method for separation and accurate quantification of tamoxifen and its metabolites isomers.

    PubMed

    Arellano, Cécile; Allal, Ben; Goubaa, Anwar; Roché, Henri; Chatelut, Etienne

    2014-11-01

    A selective and accurate analytical method is needed to quantify tamoxifen and its phase I metabolites in a prospective clinical protocol, for evaluation of pharmacokinetic parameters of tamoxifen and its metabolites in adjuvant treatment of breast cancer. The selectivity of the analytical method is a fundamental criteria to allow the quantification of the main active metabolites (Z)-isomers from (Z)'-isomers. An UPLC-MS/MS method was developed and validated for the quantification of (Z)-tamoxifen, (Z)-endoxifen, (E)-endoxifen, Z'-endoxifen, (Z)'-endoxifen, (Z)-4-hydroxytamoxifen, (Z)-4'-hydroxytamoxifen, N-desmethyl tamoxifen, and tamoxifen-N-oxide. The validation range was set between 0.5ng/mL and 125ng/mL for 4-hydroxytamoxifen and endoxifen isomers, and between 12.5ng/mL and 300ng/mL for tamoxifen, tamoxifen N-desmethyl and tamoxifen-N-oxide. The application to patient plasma samples was performed. PMID:25173109

  15. Bioanalytical LC-MS Method for the Quantification of Plasma Androgens and Androgen Glucuronides in Breast Cancer.

    PubMed

    Kalogera, Eleni; Pistos, Constantinos; Provatopoulou, Xeni; Christophi, Costas A; Zografos, George C; Stefanidou, Maria; Spiliopoulou, Chara; Athanaselis, Sotirios; Gounaris, Antonia

    2016-04-01

    The physiological and pathological development of the breast is strongly affected by the hormonal milieu consisting of steroid hormones. Mass spectrometry (MS) technologies of high sensitivity and specificity enable the quantification of androgens and consequently the characterization of the hormonal status. The aim of this study is the assessment of plasma androgens and androgen glucuronides, in the par excellence hormone-sensitive tissue of the breast, through the application of liquid chromatography-mass spectrometry (LC-MS). A simple and efficient fit-for-purpose method for the simultaneous identification and quantification of dehydroepiandrosterone sulfate (DHEAS), androstenedione (A4), androsterone glucuronide (ADTG) and androstane-3α, 17β-diol-17-glucuronide (3α-diol-17G) in human plasma was developed and validated. The presented method permits omission of derivatization, requires a single solid-phase extraction procedure and the chromatographic separation can be achieved on a single C18 analytical column, for all four analytes. The validated method was successfully applied for the analysis of 191 human plasma samples from postmenopausal women with benign breast disease (BBD), lobular neoplasia (LN), ductal carcinoma in situ and invasive ductal carcinoma (IDC). DHEAS plasma levels exhibited significant differences between LN, IDC and BBD patients (P < 0.05). Additionally, ADTG levels were significantly higher in patients with LN compared with those with BBD (P < 0.05). PMID:26762957

  16. A direct quantification method for measuring plasma MicroRNAs identified potential biomarkers for detecting metastatic breast cancer

    PubMed Central

    Liu, Cuicui; Meng, Lingyu; Qiao, Shanshan; Shen, Lei; Zhang, Yue; Lü, Jinhui; Li, Wenshu; Zhang, Yuzhen; Wang, Min; Pestell, Richard G.; Liang, Chunli; Yu, Zuoren

    2016-01-01

    Circulating miRNAs are protected from ribonuclease degradation by assembly into microvesicles and exosomes. Releasing miRNAs completely from these particles is the key step to quantify the circulating miRNAs. Currently purified RNA-based quantitative analysis is widely used while it is time and cost consuming with high risk for those circulating miRNAs with low abundance due to partial loss of RNA during the steps of total RNA extraction and small RNA enrichment. Herein, we optimized a simple, effective and time-saving method to directly measure plasma miRNAs without RNA isolation. It is based on complete miRNA release from the protein complexes, followed by miRNA-specific reverse transcription and quantitative real-time PCR amplification. By comparison to the RNA-based approach, the direct quantification method showed more efficiency for circulating miRNA analysis, higher accuracy and specificity. By application of the direct quantification method to clinical samples combined with the RNA-based miRNA screening analysis, upregulation of miR-106a in blood was validated in metastatic breast cancer patients, indicating miR-106a are a potential biomarker for metastatic breast cancer. PMID:26967564

  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. PMID:27283671

  18. Multi-level Monte Carlo finite volume methods for uncertainty quantification of acoustic wave propagation in random heterogeneous layered medium

    NASA Astrophysics Data System (ADS)

    Mishra, S.; Schwab, Ch.; Šukys, J.

    2016-05-01

    We consider the very challenging problem of efficient uncertainty quantification for acoustic wave propagation in a highly heterogeneous, possibly layered, random medium, characterized by possibly anisotropic, piecewise log-exponentially distributed Gaussian random fields. A multi-level Monte Carlo finite volume method is proposed, along with a novel, bias-free upscaling technique that allows to represent the input random fields, generated using spectral FFT methods, efficiently. Combined together with a recently developed dynamic load balancing algorithm that scales to massively parallel computing architectures, the proposed method is able to robustly compute uncertainty for highly realistic random subsurface formations that can contain a very high number (millions) of sources of uncertainty. Numerical experiments, in both two and three space dimensions, illustrating the efficiency of the method are presented.

  19. 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. PMID:17317068

  20. New capillary gel electrophoresis method for fast and accurate identification and quantification of multiple viral proteins in influenza vaccines.

    PubMed

    van Tricht, Ewoud; Geurink, Lars; Pajic, Bojana; Nijenhuis, Johan; Backus, Harold; Germano, Marta; Somsen, Govert W; Sänger-van de Griend, Cari E

    2015-11-01

    Current methods for the identification and/or quantification of viral proteins in influenza virus and virosome samples suffer from long analysis times, limited protein coverage and/or low accuracy and precision. We studied and optimized capillary gel electrophoresis (CGE) in order to achieve faster and enhanced characterization and quantification of viral proteins. Sample preparation as well the composition of the gel buffer was investigated in order to achieve adequate protein separation in relatively short times. The total sample preparation (reduction and deglycosylation) could be carried out efficiently within two hours. Hydrodynamic injection, separation voltage, and capillary temperature were optimized in full factorial design. The final method was validated and showed good performance for hemagglutinin fragment 1 (HA1), hemagglutinin fragment 2 (HA2), matrix protein (M) and nucleoprotein (NP). The CGE method allowed identification of different virus strains based on their specific protein profile. B/Brisbane inactivated virus and virosome samples could be analyzed within one day. The CGE results (titers) were comparable to single radial immune-diffusion (SRID), but the method has the advantage of a much faster time to results. CGE analysis of A/Christchurch from upstream process demonstrated the applicability of the method to samples of high complexity. The CGE method could be used in the same analyte concentration range as the RP-HPLC method, but showed better precision and accuracy. Overall, the total analysis time for the CGE method was much shorter, allowing analysis of 100 samples in 4 days instead of 10 days for SRID. PMID:26452923

  1. 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.

  2. Absolute Efficiency Calibration of a Beta-Gamma Detector

    SciTech Connect

    Cooper, Matthew W.; Ely, James H.; Haas, Derek A.; Hayes, James C.; McIntyre, Justin I.; Lidey, Lance S.; Schrom, Brian T.

    2013-04-10

    Abstract- Identification and quantification of nuclear events such as the Fukushima reactor failure and nuclear explosions rely heavily on the accurate measurement of radioxenon releases. One radioxenon detection method depends on detecting beta-gamma coincident events paired with a stable xenon measurement to determine the concentration of a plume. Like all measurements, the beta-gamma method relies on knowing the detection efficiency for each isotope measured. Several methods are commonly used to characterize the detection efficiency for a beta-gamma detector. The most common method is using a NIST certified sealed source to determine the efficiency. A second method determines the detection efficiencies relative to an already characterized detector. Finally, a potentially more accurate method is to use the expected sample to perform an absolute efficiency calibration; in the case of a beta-gamma detector, this relies on radioxenon gas samples. The complication of the first method is it focuses only on the gamma detectors and does not offer a solution for determining the beta efficiency. The second method listed is not similarly constrained, however it relies on another detector to have a well-known efficiency calibration. The final method using actual radioxenon samples to make an absolute efficiency determination is the most desirable, but until recently it was not possible to produce all four isotopically pure radioxenon. The production, by University of Texas (UT), of isotopically pure radioxenon has allowed the beta-gamma detectors to be calibrated using the absolute efficiency method. The first four radioxenon isotope calibration will be discussed is this paper.

  3. Use of the Relaxometry Technique for Quantification of Paramagnetic Ions in Aqueous Solutions and a Comparison with Other Analytical Methods.

    PubMed

    Gomes, Bruna Ferreira; 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, Cu(2+), Ni(2+), Cr(3+), and Mn(2+), 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. Methods for the quantification of GHG emissions at the landscape level for developing countries in smallholder contexts

    NASA Astrophysics Data System (ADS)

    Milne, Eleanor; Neufeldt, Henry; Rosenstock, Todd; Smalligan, Mike; Cerri, Carlos Eduardo; Malin, Daniella; Easter, Mark; Bernoux, Martial; Ogle, Stephen; Casarim, Felipe; Pearson, Timothy; Bird, David Neil; Steglich, Evelyn; Ostwald, Madelene; Denef, Karolien; Paustian, Keith

    2013-03-01

    Landscape scale quantification enables farmers to pool resources and expertise. However, the problem remains of how to quantify these gains. This article considers current greenhouse gas (GHG) quantification methods that can be used in a landscape scale analysis in terms of relevance to areas dominated by smallholders in developing countries. In landscape scale carbon accounting frameworks, measurements are an essential element. Sampling strategies need careful design to account for all pools/fluxes and to ensure judicious use of resources. Models can be used to scale-up measurements and fill data gaps. In recent years a number of accessible models and calculators have been developed which can be used at the landscape scale in developing country areas. Some are based on the Intergovernmental Panel on Climate Change (IPCC) method and others on dynamic ecosystem models. They have been developed for a range of different purposes and therefore vary in terms of accuracy and usability. Landscape scale assessments of GHGs require a combination of ground sampling, use of data from census, remote sensing (RS) or other sources and modelling. Fitting of all of these aspects together needs to be performed carefully to minimize uncertainties and maximize the use of scarce resources. This is especially true in heterogeneous landscapes dominated by smallholders in developing countries.

  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. 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

  7. 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.

  8. 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

  9. Absolute calibration of OH density in a nanosecond pulsed plasma filament in atmospheric pressure He-H2O: comparison of independent calibration methods

    NASA Astrophysics Data System (ADS)

    Verreycken, T.; van der Horst, R. M.; Sadeghi, N.; Bruggeman, P. J.

    2013-11-01

    The absolute density of OH radicals generated in a nanosecond pulsed filamentary discharge in atmospheric pressure He +0.84% H2O is measured independently by UV absorption and laser induced fluorescence (LIF) calibrated with Rayleigh scattering. For the calibration of LIF with Rayleigh scattering, two LIF models, with six levels and four levels, are studied to investigate the influence of the rotational and vibrational energy transfers. In addition, a chemical model is used to deduce the OH density in the afterglow from the relative LIF intensity as function of time. The different models show good correspondence and by comparing these different methods, the accuracy and the effect of assumptions on the obtained OH density are discussed in detail. This analysis includes an analysis of the sensitivity to parameters used in the LIF models.

  10. Evaluation of the Re-Os Geochronometer in Organic-rich Mudrocks as a Method for Constraining the Absolute Ages of Neoproterozoic Glaciogenic Deposits

    NASA Astrophysics Data System (ADS)

    Kendall, B. S.; Creaser, R. A.; Ross, G. M.

    2002-12-01

    Absolute-age constraints on the Neoproterozoic glaciations are generally poor due to a paucity of suitable plutonic and volcanic igneous rocks that are temporally and spatially related to Neoproterozoic glaciogenic deposits and are amenable to radiometric dating methods. In this study, the Re-Os isotope systematics of dark gray, sulfidic slates from the Old Fort Point Formation (OFP) of the Windermere Supergroup (near Jasper, Alberta) were examined to test the ability of the Re-Os geochronometer to provide an absolute age constraint for a Neoproterozoic glaciogenic deposit. The OFP has been interpreted as the deep water expression of post-glacial sea level rise and therefore is comparable stratigraphically to cap carbonates that immediately overlie glaciogenic deposits worldwide. Despite the relatively low Re (6-16 ppb) and Os (0.07-0.14 ppb) concentrations and total organic contents (~ 0.5% TOC) of the slates compared to other organic-rich mudrocks used in previous Re-Os isotope studies, precise well-fitted Re-Os isochrons have been obtained with two different dissolution methods. An age of 620.8 +/- 8.1 Ma (MSWD = 0.9; initial 187Os/188Os = 0.68 +/- 0.06) is obtained using conventional aqua regia dissolution. Using a method designed to selectively dissolve organic matter alone, an age of 609.0 +/- 8.3 Ma (MSWD = 1.5; initial 187Os/188Os = 0.62 +/- 0.05) is obtained. These absolute age results are in accord with existing age constraints (e.g., stratigraphically younger Hamill Group with a U-Pb zircon age of 569 Ma). The well-defined Re-Os systematics of the OFP slates demonstrates for the first time that the Re-Os system is not disturbed in organic-rich sediments during lower greenschist (-chlorite) grade metamorphic conditions. The whole-rock analysis of each individual sample yields consistently higher initial 187Os/188Os isotope ratios than the corresponding organic matter analysis and suggests that a significant radiogenic detrital Os component is present

  11. Re-evaluation of thin layer chromatography as an alternative method for the quantification of prostaglandins from rat Kupffer cells.

    PubMed

    Pestel, Sabine; Jungermann, Kurt; Schieferdecker, Henrike L

    2005-01-01

    In contrast to conventionally used immunoassays, thin layer chromatography (TLC)--by prelabeling of cells with radioactive arachidonic acid (AA)--allows to differentiate between cellularly built and added prostanoids and thus to investigate feedback effects of prostanoids on their own release. PGD2, TXB2 and PGE2 released from zymosan-stimulated Kupffer cells were separated with distinct RF-values, corresponding to those of the pure substances. Quantification of PGD2 and PGE2 gave comparable results with TLC and immunoassays, but measurement in the presence of added prostanoids was only possible with TLC. Moreover TLC was superior to immunoassays in having a longer linear range while being comparably sensitive. Cellularly built TXB2 in its radioactively labeled form was not detectable by TLC. Inhibition of TXB2 release by externally added AA or technical artifacts were excluded, suggesting that the cellular AA-pools used for prostaglandin and thromboxane synthesis differ in their accessibility for added AA. Thus, TLC is a simple, sensitive and precise method for the quantification of cellularly built prostaglandins but not of thromboxane even in the presence of added prostanoids. PMID:15789620

  12. 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. PMID:20437411

  13. New HPLC-MS method for the simultaneous quantification of the antileukemia drugs imatinib, dasatinib, and nilotinib in human plasma.

    PubMed

    De Francia, Silvia; D'Avolio, Antonio; De Martino, Francesca; Pirro, Elisa; Baietto, Lorena; Siccardi, Marco; Simiele, Marco; Racca, Silvia; Saglio, Giuseppe; Di Carlo, Francesco; Di Perri, Giovanni

    2009-06-15

    A new method using high performance liquid chromatography coupled with electrospray mass spectrometry is described for the quantification of plasma concentration of tyrosine kinase inhibitors imatinib, dasatinib and nilotinib. A simple protein precipitation extraction procedure was applied on 250 microl of plasma aliquots. Chromatographic separation of drugs and Internal Standard (quinoxaline) was achieved with a gradient (acetonitrile and water + formic acid 0.05%) on a C18 reverse phase analytical column with 20min of analytical run, at flow rate of 1 ml/min. Mean intra-day and inter-day precision for all compounds were 4.3 and 11.4%; mean accuracy was 1.5%; extraction recovery ranged within 95 and 114%. Calibration curves ranged from 10,000 to 62.5 ng/ml. The limit of quantification was set at 78.1 ng/ml for imatinib and at 62.5 ng/ml for dasatinib and nilotinib. This novel developed methodology allows a specific, sensitive and reliable simultaneous determination of the three tyrosine kinase inhibitors imatinib, dasatinib and nilotinib in a single chromatographic run, useful for drugs estimation in plasma of patients affected by chronic myeloid leukemia. PMID:19428316

  14. Simultaneous Detection and Quantification of Phytohormones by a Sensitive Method of Separation in Culture of Pseudomonas sp.

    PubMed

    Patel, Ravi R; Thakkar, Vasudev R; Subramanian, Ramalingam Bagavathi

    2016-06-01

    A high-performance thin-layer chromatography (HPTLC)-based sensitive, rapid and stringent protocol is designed for detection and quantification of five phytohormones simultaneously. Culture filtrate of Pseudomonas bacteria was acidified with 7 M HCl and extracted with an equal volume of ethyl acetate to separate abscisic acid (ABA), jasmonic acid (JA), gibberellic acid (GA3), and indole-3-acetic acid (IAA). Kinetin was extracted from the remaining water fraction of the same extract. Various extracts were loaded on silica gel 60 F254 foil using Linomat 5 spray on applicator. Standard phytohormones were also loaded adjacent to the sample, and the foils were developed with isopropanol-ammonia-water [10:1:1 (v/v)] as the mobile phase. A quantitative estimation of the separated ABA, kinetin, JA, GA3, and IAA was performed by measuring the absorbance at 260, 275, 295, 265, and 280 nm, respectively. HPTLC method was found to be cost effective, robust technique that can be routinely used for simultaneous phytohormone detection in plant or bacterial samples. The present work is not only useful for detection and quantification of phytohormones but also for screening of phytohormone producing microorganisms. PMID:26905268

  15. [Models for quantification of fluid saturation in two-phase flow system by light transmission method and its application].

    PubMed

    Zhang, Yan-Hong; Ye, Shu-Jun; Wu, Ji-Chun

    2014-06-01

    Based on light transmission method in quantification of liquid saturation and its application in two-phase flow system, two groups of sandbox experiments were set up to study the migration of gas or Dense Non-Aqueous Phase Liquids (DNAPLs) in water saturated porous media. The migration of gas or DNAPL was monitored in the study. Two modified Light Intensity-Saturation (LIS) models for water/gas two-phase system were applied and verified by the experiment data. Moreover two new LIS models for NAPL/water system were developed and applied to simulate the DNAPL infiltration experiment data. The gas injection experiment showed that gas moved upward to the top of the sandbox in the form of 'fingering' and finally formed continuous distribution. The results of DNAPL infiltration experiment showed that TCE mainly moved downward as the result of its gravity, eventually formed irregular plume and accumulated at the bottom of the sandbox. The outcomes of two LIS models for water/gas system (WG-A and WG-B) were consistent to the measured data. The results of two LIS models for NAPL/water system (NW-A and NW-B) fit well with the observations, and Model NW-A based on assumption of individual drainage gave better results. It could be a useful reference for quantification of NAPL/water saturation in porous media system. PMID:25158486

  16. An Improved Molecular Histology Method for Ion Suppression Monitoring and Quantification of Phosphatidyl Cholines During MALDI MSI Lipidomics Analyses.

    PubMed

    Jadoul, Laure; Smargiasso, Nicolas; Pamelard, Fabien; Alberts, Deborah; Noël, Agnès; De Pauw, Edwin; Longuespée, Rémi

    2016-02-01

    Tissue lipidomics is one of the latest omics approaches for biomarker discovery in pharmacology, pathology, and the life sciences at large. In this context, matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is the most versatile tool to map compounds within tissue sections. However, ion suppression events occurring during MALDI MSI analyses make it impossible to use this method for quantitative investigations without additional validation steps. This is especially true for lipidomics, since different lipid classes are responsible for important ion suppression events. We propose here an improved lipidomics method to assess local ion suppression of phospatidylcholines in tissues. Serial tissue sections were spiked with different amounts of PC(16:0 d31/18:1) using a nebulization device. Settings for standard nebulization were strictly controlled for a detection similar to when using spiked tissue homogenates. The sections were simultaneously analyzed by MALDI MSI using a Fourier transform ion cyclotron resonance analyzer. Such a spray-based approach allows taking into account the biochemical heterogeneity of the tissue for the detection of PC(16:0 d31/18:1). Thus, here we present the perspective to use this method for quantification purposes. The linear regression lines are considered as calibration curves and we calculate PC(16:0/18:1) quantification values for different ROIs. Although those values need to be validated by a using a different independent approach, the workflow offers an insight into new quantitative mass spectrometry imaging (q-MSI) methods. This approach of ion suppression monitoring of phosphocholines in tissues may be highly interesting for a large range of applications in MALDI MSI, particularly for pathology using translational science workflows. PMID:26871868

  17. An analytical method for the quantification of hERG1 channel gene expression in human colorectal cancer.

    PubMed

    Fortunato, Angelo; Gasparoli, Luca; Falsini, Sara; Boni, Luca; Luca, Boni; Arcangeli, Annarosa

    2013-12-01

    Cancer molecular investigation revealed a huge molecular heterogeneity between different types of cancers as well as among cancer patients affected by the same cancer type. This implies the necessity of a personalized approach for cancer diagnosis and therapy, on the basis of the development of standardized protocols to facilitate the application of molecular techniques in the clinical decision-making process. Ion channels encoding genes are acquiring increasing relevance in oncological translational studies, representing new candidates for molecular diagnostic and therapeutic purposes. Hence, the development of molecular protocols for the quantification of ion channels encoding genes in tumor specimens may have relevance for diagnostic and prognostic investigation. Two main hindrances must be overcome for these purposes: the use of formalin-fixed and paraffin-embedded samples for gene expression analysis and the physiological expression of ion channels in excitable cells, potentially present in the tumor sample. We here propose a method for hERG1 gene quantification in colorectal cancer samples in both cryopreserved and formalin-fixed and paraffin-embedded samples. An analytical method was developed to estimate hERG1 gene expression exclusively in epithelial cancer cells. Indeed, we found that the hERG1 gene was expressed at significant levels by myofibroblasts present in the tumor stroma. This method was based on the normalization on a smooth muscle-myofibroblast-specific gene, MYH11, with no need of microdissection. By applying this method, hERG1 expression turned out to correlate with VEGF-A expression, confirming previous immunohistochemical data. PMID:24193004

  18. 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

  19. Scoliosis quantification: an overview

    PubMed Central

    Kawchuk, Greg; McArthur, Ross

    1997-01-01

    Scoliotic curvatures have long been a focus of attention for clinicians and research scientists alike. The study, treatment and ultimately, the prevention of this prevalent health condition are impeded by the absence of an accurate, reliable, convenient and safe method of scoliosis quantification. The purpose of this paper is to provide an overview of the current methods of scoliosis quantification for clinicians who address this condition in their practices.

  20. 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.

  1. Performance of automated platelet quantification using different analysers in comparison with an immunological reference method in thrombocytopenic patients

    PubMed Central

    Trabuio, Ernesto; Valverde, Sara; Antico, Francesco; Manoni, Fabio; Gessoni, Gianluca

    2009-01-01

    Background Rapidly available and accurate platelet counts play an important role in the evaluation of haemorrhagic status and in assessing the need for platelet transfusions. We, therefore, evaluated platelet counting performance of haematology analysers using optical, impedance and immunological methods in thrombocytopenic patients. Materials and Methods We considered 99 patients with a platelet (plt) count under 50x109 plt/L. We compared the platelet counts obtained using ADVIA 2120 (optical method), Cell-Dyn Sapphire (optical, impedance and immunological methods with CD61) and a reference, double staining (CD41+CD61) immunological method. Results The platelet counts of all the considered methods showed good correlation with those of the reference method, despite an overestimation in platelet quantification. The degree of inaccuracy was greater for platelet counts under 20 x109 plt/L. Conclusions Clinicians who use platelet thresholds below 20 x109 plt/L for making clinical decisions must be aware of the limitations in precision and accuracy of cell counters at this level of platelet count. Inaccurate counts of low platelet numbers could create problems if attempts are made to reduce the threshold below 20x109 plt/L. PMID:19290080

  2. Absolute quantitative analysis for sorbic acid in processed foods using proton nuclear magnetic resonance spectroscopy.

    PubMed

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

    2012-07-13

    An analytical method using solvent extraction and quantitative proton nuclear magnetic resonance (qHNMR) spectroscopy was applied and validated for the absolute quantification of sorbic acid (SA) in processed foods. The proposed method showed good linearity. The recoveries for samples spiked at the maximum usage level specified for food in Japan and at 0.13 g kg(-1) (beverage: 0.013 g kg(-1)) were larger than 80%, whereas those for samples spiked at 0.063 g kg(-1) (beverage: 0.0063 g kg(-1)) were between 56.9 and 83.5%. The limit of quantification was 0.063 g kg(-1) for foods (and 0.0063 g kg(-1) for beverages containing Lactobacillus species). Analysis of the SA content of commercial processed foods revealed quantities equal to or greater than those measured using conventional steam-distillation extraction and high-performance liquid chromatography quantification. The proposed method was rapid, simple, accurate, and precise, and provided International System of Units traceability without the need for authentic analyte standards. It could therefore be used as an alternative to the quantification of SA in processed foods using conventional method. PMID:22704472

  3. Limitations of amorphous content quantification by isothermal calorimetry using saturated salt solutions to control relative humidity: alternative methods.

    PubMed

    Khalef, Nawel; Pinal, Rodolfo; Bakri, Aziz

    2010-04-01

    Despite the high sensitivity of isothermal calorimetry (IC), reported measurements of amorphous content by this technique show significant variability even for the same compound. An investigation into the reasons behind such variability is presented using amorphous lactose and salbutamol sulfate as model compounds. An analysis was carried out on the heat evolved as a result of the exchange of water vapor between the solid sample during crystallization and the saline solution reservoir. The use of saturated salt solutions as means of control of the vapor pressure of water within sealed ampoules bears inherent limitations that lead in turn to the variability associated with the IC technique. We present an alternative IC method, based on an open cell configuration that effectively addresses the limitations encountered with the sealed ampoule system. The proposed approach yields an integral whose value is proportional to the amorphous content in the sample, thus enabling reliable and consistent quantifications. PMID:19774655

  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

    2016-01-01

    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. PMID:26421962

  5. Quantification of Melt Inclusion Chemistry by LA-ICP-MS, EMP and SIMS: Advantages and Possible Limitations of Either Method

    NASA Astrophysics Data System (ADS)

    Pettke, T.; Halter, W. E.; Aigner-Torres, M.; Heinrich, C. A.

    2002-05-01

    Laser-ablation inductively-coupled-plasma mass-spectrometry (LA-ICP-MS) has unique advantages for chemical analysis of melt inclusions (MI), because it allows bulk compositional reconstitution of heterogeneous (crystallized) MI without prior thermal homogenization. Quantification is possible even if chemically complex minerals such as plagioclase or amphibole host the MI. We have analyzed glassy MI in plagioclase in one sample using EMP, SIMS (exposed MI) and 193 nm ArF Excimer LA-ICP-MS (unexposed MI), and rigorously compared the results in order to test the analytical accuracy of the data obtained by the various methods. We used a basalt sample, ALV-3352-7 (MORB from the East Pacific Rise, 17 - 19° S, STOWA cruise) with plagioclase phenocrysts ( ~10 vol-%, An82) in a glassy matrix. Plagioclase contains abundant glassy MI (10 - 300 μ m) some of which show a shrinkage bubble. The plagioclase phenocrysts, the matrix glass and the MI are chemically uniform. For the matrix glass, major element analyses by EMP ( ~0.1 to 50 wt-%) and trace-element analyses by SIMS ( ~2 to 100 ppm) are in excellent agreement (+/- 3% RSD on average) with LA-ICP-MS results obtained with a single 40-element menu. This demonstrates analytical accuracy of the three methods. Using MgO or FeO analyses from EMP of exposed MI as an internal standard for the quantification of LA-ICP-MS data of unexposed MI provides identical composition, hence the numerical re-integration of inlcusion compositions (Halter et al., 2002, Chem. Geol. 183, 63-86) is also accurate. Careful evaluation of the EMP data of exposed MI and matrix glass demonstrates significant chemical differences between the two; the glass in the MI apparently fractionated more plagioclase than did the matrix glass. Using MgO of the matrix glass as an internal standard for the quantification of MI analyzed by LA-ICP-MS (i.e., assuming that the MI and the matrix glass originally contained the same MgO concentration) indicates that

  6. A general method for the calculation of absolute trace gas concentrations in air and breath from selected ion flow tube mass spectrometry data

    NASA Astrophysics Data System (ADS)

    Spanel, Patrik; Dryahina, Kseniya; Smith, David

    2006-03-01

    A complete description is presented of a numerical method that allows the calculation, in real time, of absolute concentrations of trace gases, including volatile organic compounds and water vapour, from selected ion flow tube mass spectrometry, SIFT-MS, data. No assumptions are made concerning the SIFT-MS instrument size or its configuration and thus the calculation can be applied to the currently available, relatively large instruments and the anticipated new generation of smaller SIFT-MS instruments. This numerical method clearly distinguishes those parameters that are obviously specific to a particular instrument, including flow tube geometry, degree of mass discrimination in the analytical mass spectrometer and flow tube reaction time, from general fundamental processes, in particular the differential diffusive loss of ions along the flow tube that is dependent on the properties of those ions involved in the determination of the concentrations of particular trace gases. The essential reaction and transport kinetics are outlined, which describe the formation and loss of the product ions formed in the chemical ionisation of the trace gases by the precursor ions. A generalised calculation of the required ionic diffusion coefficients is introduced with options either for their accurate determination from the molecular geometry of ions or for less accurate but simpler estimates obtained using just the ionic mass. Based on the above ideas, a straightforward calculation sequence is shown to determine trace gas concentrations by SIFT-MS, and its utility demonstrated by an example of the analysis of acetone in exhaled breath.

  7. 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. PMID:23790900

  8. 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

  9. The polymerase chain reaction: A stochastic model, methods of quantification, and applications to HIV. [HIV (human immunodeficiency virus)

    SciTech Connect

    Harris, O.A.

    1992-01-01

    This thesis is concerned with the development of the polymerase chain reaction (PCR) as an accurate and reliable measure of specific DNA copy number. This development is motivated by the need to quantify the number of copies of HIV in infected cells. In particular the extent of HIV infection, in terms of proviral load, can be determined by using PCR, leading to more accurate evaluation of drug treatments. The thesis is presented in four parts: (I) the assay, (II) the mathematics, (III) the models, and finally (IV) the applications. The first section includes a complete description of the assay. This section also includes descriptions of DNA structure and of cellular DNA replication. The second section contains the background material and presentation of new developments in the mathematics and statistics needed for the modeling of the assay. The assay is modeled as a branching process, and various aspects of the reaction dictate different types of branching processes. As a result, three types of processes are presented, classic Galton-Watson, generation-dependent, and population-size-dependent. These models lead to quantification procedures involving weighted linear regression and inverse prediction. In addition, new material is presented for the development of comparison methods and confidence intervals in this setting. The third section contains the actual modeling of the reaction through the three different types of branching processes mentioned. Complete characterizations of the distributions of the processes are derived for two of the models, from which new parametric statistical tests for the quantification of DNA, in particular of HIV, are developed. For the third model, simulations are used to explore the process and its moments. This model necessarily leads to a submodel reminiscent to that found in stochastic epidemics. The final section illustrates the application of methods developed to data from HIV-infected patients.

  10. 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%. PMID:26688345

  11. Absolute biological needs.

    PubMed

    McLeod, Stephen

    2014-07-01

    Absolute needs (as against instrumental needs) are independent of the ends, goals and purposes of personal agents. Against the view that the only needs are instrumental needs, David Wiggins and Garrett Thomson have defended absolute needs on the grounds that the verb 'need' has instrumental and absolute senses. While remaining neutral about it, this article does not adopt that approach. Instead, it suggests that there are absolute biological needs. The absolute nature of these needs is defended by appeal to: their objectivity (as against mind-dependence); the universality of the phenomenon of needing across the plant and animal kingdoms; the impossibility that biological needs depend wholly upon the exercise of the abilities characteristic of personal agency; the contention that the possession of biological needs is prior to the possession of the abilities characteristic of personal agency. Finally, three philosophical usages of 'normative' are distinguished. On two of these, to describe a phenomenon or claim as 'normative' is to describe it as value-dependent. A description of a phenomenon or claim as 'normative' in the third sense does not entail such value-dependency, though it leaves open the possibility that value depends upon the phenomenon or upon the truth of the claim. It is argued that while survival needs (or claims about them) may well be normative in this third sense, they are normative in neither of the first two. Thus, the idea of absolute need is not inherently normative in either of the first two senses. PMID:23586876

  12. Rapid method for quantification of seven synthetic pigments in colored Chinese steamed buns using UFLC-MS/MS without SPE.

    PubMed

    Gao, He-Gang; Gong, Wen-Jie; Zhao, Yong-Gang

    2015-01-01

    Synthetic pigments are still used instead of natural pigments in many foods and their residues in food could be an important risk to human health. A simple and rapid analytical method combining the low-cost extraction protocol with ultra-fast liquid chromatography-tandem quadrupole mass spectrometry (UFLC-MS/MS) was developed for the simultaneous determination of seven synthetic pigments used in colored Chinese steamed buns. For the first time, ethanol/ammonia solution/water (7:2:1, v/v/v) was used as extraction solution for the synthetic pigments in colored Chinese steamed buns. The results showed that the property of the extraction solution used in this method was more effective than critic acid solution, which is used in the polyamide adsorption method. The limits of quantification for the seven synthetic pigments ranged from 0.15 to 0.50 μg/kg. The present method was successfully applied to samples of colored Chinese steamed buns for food-safety risk monitoring in Zhejiang Province, China. The results found sunset yellow pigment in six out of 300 colored Chinese steamed buns (from 0.50 to 32.6 μg/kg). PMID:25765275

  13. 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. PMID:19789856

  14. 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...

  15. Optimized methods for total nucleic acid extraction and quantification of the bat white-nose syndrome fungus, Pseudogymnoascus destructans, from swab and environmental samples.

    PubMed

    Verant, Michelle L; Bohuski, Elizabeth A; Lorch, Jeffery M; Blehert, David S

    2016-03-01

    The continued spread of white-nose syndrome and its impacts on hibernating bat populations across North America has prompted nationwide surveillance efforts and the need for high-throughput, noninvasive diagnostic tools. Quantitative real-time polymerase chain reaction (qPCR) analysis has been increasingly used for detection of the causative fungus, Pseudogymnoascus destructans, in both bat- and environment-associated samples and provides a tool for quantification of fungal DNA useful for research and monitoring purposes. However, precise quantification of nucleic acid from P. destructans is dependent on effective and standardized methods for extracting nucleic acid from various relevant sample types. We describe optimized methodologies for extracting fungal nucleic acids from sediment, guano, and swab-based samples using commercial kits together with a combination of chemical, enzymatic, and mechanical modifications. Additionally, we define modifications to a previously published intergenic spacer-based qPCR test for P. destructans to refine quantification capabilities of this assay. PMID:26965231

  16. 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.

  17. Development and validation of an inductively coupled plasma mass spectrometry method for quantification of levothyroxine in dissolution studies.

    PubMed

    Pabla, Dimple; Akhlaghi, Fatemeh; Ahmed, Aftab; Zia, Hossein

    2008-04-01

    A simple, sensitive and reproducible inductively coupled plasma mass spectrometry (ICP-MS) method for the direct determination of levothyroxine (T4), based on the analysis of iodide content, in aqueous media was developed. The sample preparation consisted of addition of antimony, as the internal standard, and dilution with a 0.5% ammonia solution. The analytes were quantified at m/z 126.90 and 120.90 for iodide and antimony, respectively. The assay was linear in the concentration range of 0.1-50 ng/mL for iodide and 0.3-100 ng/mL for T4. The method was precise and accurate with lower limits of quantification (LLOQs) of 0.1 ng/mL for iodide and 0.3 ng/mL for T4. The inter-day accuracy was >94% for both analytes and the coefficient of variation (%CV) was less than 5%. The method has successfully been used for dissolution studies of T4 formulations and holds immense promise as a simple, precise and sensitive analytical technique for T4 concentration determination in in vitro studies. PMID:18320549

  18. 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. PMID:26821881

  19. 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. PMID:27154703

  20. Absolute optical instruments without spherical symmetry

    NASA Astrophysics Data System (ADS)

    Tyc, Tomáš; Dao, H. L.; Danner, Aaron J.

    2015-11-01

    Until now, the known set of absolute optical instruments has been limited to those containing high levels of symmetry. Here, we demonstrate a method of mathematically constructing refractive index profiles that result in asymmetric absolute optical instruments. The method is based on the analogy between geometrical optics and classical mechanics and employs Lagrangians that separate in Cartesian coordinates. In addition, our method can be used to construct the index profiles of most previously known absolute optical instruments, as well as infinitely many different ones.

  1. Under proper control, oxidation of proteins with known chemical structure provides an accurate and absolute method for the determination of their molar concentration.

    PubMed

    Guermant, C; Azarkan, M; Smolders, N; Baeyens-Volant, D; Nijs, M; Paul, C; Brygier, J; Vincentelli, J; Looze, Y

    2000-01-01

    Oxidation at 120 degrees C of inorganic and organic (including amino acids, di- and tripeptides) model compounds by K(2)Cr(2)O(7) in the presence of H(2)SO(4) (mass fraction: 0.572), Ag(2)SO(4) (catalyst), and HgSO(4) results in the quantitative conversion of their C-atoms into CO(2) within 24 h or less. Under these stressed, well-defined conditions, the S-atoms present in cysteine and cystine residues are oxidized into SO(3) while, interestingly, the oxidation states of all the other (including the N-) atoms normally present in a protein do remain quite unchanged. When the chemical structure of a given protein is available, the total number of electrons the protein is able to transfer to K(2)Cr(2)O(7) and thereof, the total number of moles of Cr(3+) ions which the protein is able to generate upon oxidation can be accurately calculated. In such cases, unknown protein molar concentrations can thus be determined through straightforward spectrophotometric measurements of Cr(3+) concentrations. The values of molar absorption coefficients for several well-characterized proteins have been redetermined on this basis and observed to be in excellent agreement with the most precise values reported in the literature, which fully assesses the validity of the method. When applied to highly purified proteins of known chemical structure (more generally of known atomic composition), this method is absolute and accurate (+/-1%). Furthermore, it is well adapted to series measurements since available commercial kits for chemical oxygen demand (COD) measurements can readily be adapted to work under the experimental conditions recommended here for the protein assay. PMID:10610688

  2. 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. PMID:26181824

  3. Preservation And Processing Methods For Molecular Genetic Detection And Quantification Of Nosema Ceranae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The prevalence of Nosema ceranae in managed honey bee colonies has increased dramatically in the past 10 – 20 years worldwide. A variety of genetic testing methods for species identification and prevalence are now available. However sample size and preservation method of samples prior to testing hav...

  4. Detection and quantification limits of the EPA Enterococcus qPCR method

    EPA Science Inventory

    The U.S. EPA will be recommending a quantitative polymerase chain reaction (qPCR) method targeting Enterococcus spp. as an option for monitoring recreational beach water quality in 2013 and has published preliminary proposed water quality criteria guidelines for the method. An im...

  5. The absolute path command

    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 canmore » provide the absolute path to a relative directory from the current working directory.« less

  6. 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.

  7. Two validated HPLC methods for the quantification of alizarin and other anthraquinones in Rubia tinctorum cultivars.

    PubMed

    Derksen, Goverdina C H; Lelyveld, Gerrit P; van Beek, Teris A; Capelle, Anthony; de Groot, A E

    2004-01-01

    Direct and indirect HPLC-UV methods for the quantitative determination of anthraquinones in dried madder root have been developed, validated and compared. In the direct method, madder root was extracted twice with refluxing ethanol-water. This method allowed the determination of the two major native anthraquinone glycosides lucidin primeveroside and ruberythric acid. In the indirect extraction method, the anthraquinone glycosides were first converted into aglycones by endogenous enzymes and the aglycones were subsequently extracted with tetrahydrofuran-water and then analysed. In this case the anthraquinones alizarin, purpurin and nordamnacanthal may be determined. The content of nordamnacanthal is proportional to the amount of lucidin primeveroside originally present. The indirect extraction method is easier to apply. Different madder cultivars were screened for their anthraquinone content. PMID:15599964

  8. A comprehensive quantification method for eicosanoids and related compounds by using liquid chromatography/mass spectrometry with high speed continuous ionization polarity switching.

    PubMed

    Yamada, Masaki; Kita, Yoshihiro; Kohira, Takahiro; Yoshida, Kenji; Hamano, Fumie; Tokuoka, Suzumi M; Shimizu, Takao

    2015-07-15

    Fatty acids and related metabolites, comprising several hundreds of molecular species, are an important target in disease metabolomics, as they are involved in various mammalian pathologies and physiologies. Selected reaction monitoring (SRM) analysis, which is capable of monitoring hundreds of compounds in a single run, has been widely used for comprehensive quantification. However, it is difficult to monitor a large number of compounds with different ionization polarity, as polarity switching requires a sub-second period per cycle in classical mass spectrometers. In the present study, we developed and evaluated a comprehensive quantification method for eicosanoids and related compounds by using LC/MS with high-speed continuous ionization polarity switching. The new method employs a fast (30ms/cycle) continuous ionization polarity switching, and differentiates 137 targets either by chromatography or by SRM transition. Polarity switching did not affect the lower limits of quantification, which ranged similarly from 0.5 to 200pg on column. Lipid extracts from mouse tissues were analyzed by this method, and 65 targets were quantitatively detected in the brain, including 6 compounds analyzed in the positive ion mode. We demonstrated that a fast continuous ionization polarity switching enables the quantification of a wide variety of lipid mediator species without compromising the sensitivity and reliability. PMID:26046978

  9. Quantification of Nitrous Oxide from Fugitive Emissions by Tracer Dilution Method using a Mobile Real-time Nitrous Oxide Analyzer

    NASA Astrophysics Data System (ADS)

    Mønster, J.; Rella, C.; Jacobson, G. A.; He, Y.; Hoffnagle, J.; Scheutz, C.

    2012-12-01

    Nitrous oxide is a powerful greenhouse gas considered 298 times stronger than carbon dioxide on a hundred years term (Solomon et al. 2007). The increasing global concentration is of great concern and is receiving increasing attention in various scientific and industrial fields. Nitrous oxide is emitted from both natural and anthropogenic sources. Inventories of source specific fugitive nitrous oxide emissions are often estimated on the basis of modeling and mass balance. While these methods are well-developed, actual measurements for quantification of the emissions can be a useful tool for verifying the existing estimation methods as well as providing validation for initiatives targeted at lowering unwanted nitrous oxide emissions. One approach to performing such measurements is the tracer dilution method (Galle et al. 2001), in which a tracer gas is released at the source location at a known flow. The ratio of downwind concentrations of both the tracer gas and nitrous oxide gives the ratios of the emissions rates. This tracer dilution method can be done with both stationary and mobile measurements; in either case, real-time measurements of both tracer and analyte gas is required, which places high demands on the analytical detection method. To perform the nitrous oxide measurements, a novel, robust instrument capable of real-time nitrous oxide measurements has been developed, based on cavity ring-down spectroscopy and operating in the near-infrared spectral region. We present the results of the laboratory and field tests of this instrument in both California and Denmark. Furthermore, results are presented from measurements using the mobile plume method with a tracer gas (acetylene) to quantify the nitrous oxide and methane emissions from known sources such as waste water treatment plants and composting facilities. Nitrous oxide (blue) and methane (yellow) plumes downwind from a waste water treatment facility.

  10. Evaluation of multistep derivatization methods for identification and quantification of oxygenated species in organic aerosol.

    PubMed

    Flores, Rosa M; Doskey, Paul V

    2015-10-30

    Two, 3-step methods for derivatizing mono- and multi-functional species with carbonyl (CO), carboxylic acid (-COOH), and alcohol (-OH) moieties were compared and optimized. In Method 1, the CO, -COOH, and -OH moieties were converted (1) to methyloximes (R-CN-OCH3) with O-methylhydroxylamine hydrochloride (MHA), (2) to methyl esters (OC-R-OCH3) with (trimethylsilyl)diazomethane in methanol (TMSD/MeOH), and (3) to trimethylsilyl ethers [R-OSi(CH3)3] with N,O-bis(trimethylsilyl)-trifluoroacetamide (BSTFA) containing 1% trimethylchlorosilane (TMCS), respectively. Steps 1 and 3 of both methods were identical; however, in Step 2 of Method 2, -COOH moieties were derivatized with 10% (v/v) boron trifluoride (BF3) in MeOH or n-butanol (n-BuOH). The BF3/MeOH and BF3/n-BuOH were ineffective at converting species with more than 2-OH moieties. Average standard deviations for derivatization of 36 model compounds by the 3-step methods using TMSD/MeOH and BF3/(MeOH) were 7.4 and 14.8%, respectively. Average derivatization efficiencies for Methods 1 and 2 were 88.0 and 114%, respectively. Despite the lower average derivatization efficiency of Method 1, distinct advantages included a greater certainty of derivatization yield for the entire suite of mono- and multi-functional species and fewer processing steps for sequential derivatization. Detection limits for Method 1 using GC×GC-ToF-MS were 0.3-54pgm(-3). Approximately 100 oxygenated organic species were identified and quantified in aerosol filtered from 39m(3) of air in an urban location. Levels of species were 0.013-17ngm(-3) and were nearly all above the Method 1 limit of detection. PMID:26427323

  11. A sensitive LC-MS/MS method for the quantification of febuxostat in human plasma and its pharmacokinetic application.

    PubMed

    Vaka, Venkata Rami Reddy; Inamadugu, Jaswanth Kumar; Pilli, Nageswara Rao; Ramesh, Mullangi; Katreddi, Hussain Reddy

    2013-11-01

    An improved, simple and highly sensitive LC-MS/MS method has been developed and validated for quantification of febuxostat with 100 μL human plasma using febuxostat-d7 as an internal standard (IS) according to regulatory guidelines. The analyte and IS were extracted from human plasma via liquid-liquid extraction using diethyl ether. The chromatographic separation was achieved on a Zorbax C18 column using a mixture of acetonitrile and 5 mm ammonium formate (60:40, v/v) as the mobile phase at a flow rate of 0.5 mL/min. The total run time was 5.0 min and the elution of febuxostat and IS occurred at 1.0 and 1.5 min, respectively. A linear response function was established for the range of concentrations 1-6000 ng/mL (r > 0.99). The precursor to product ion transitions monitored for febuxostat and IS were m/z 317.1 → 261.1 and 324.2 → 262.1, respectively. The intra- and inter-day precisions (%RSD) were within 1.29-9.19 and 2.85-7.69%, respectively. The proposed method was successfully applied to pharmacokinetic studies in humans. PMID:23733262

  12. Analytical methods for the quantification of bisphenol A, alkylphenols, phthalate esters, and perfluoronated chemicals in biological samples.

    PubMed

    Nakazawa, Hiroyuki; Iwasaki, Yusuke; Ito, Rie

    2014-01-01

    Our modern society has created a large number of chemicals that are used for the production of everyday commodities including toys, food packaging, cosmetic products, and building materials. We enjoy a comfortable and convenient lifestyle with access to these items. In addition, in specialized areas, such as experimental science and various medical fields, laboratory equipment and devices that are manufactured using a wide range of chemical substances are also extensively employed. The association between human exposure to trace hazardous chemicals and an increased incidence of endocrine disease has been recognized. However, the evaluation of human exposure to such endocrine disrupting chemicals is therefore imperative, and the determination of exposure levels requires the analysis of human biological materials, such as blood and urine. To obtain as much information as possible from limited sample sizes, highly sensitive and reliable analytical methods are also required for exposure assessments. The present review focuses on effective analytical methods for the quantification of bisphenol A (BPA), alkylphenols (APs), phthalate esters (PEs), and perfluoronated chemicals (PFCs), which are chemicals used in the production of everyday commodities. Using data obtained from liquid chromatography/mass spectrometry (LC/MS) and LC/MS/MS analyses, assessments of the risks to humans were also presented based on the estimated levels of exposure to PFCs. PMID:24420241

  13. Optimized ultra performance liquid chromatography tandem high resolution mass spectrometry method for the quantification of paraquat in plasma and urine.

    PubMed

    Lu, Haihua; Yu, Jing; Wu, Linlin; Xing, Jingjing; Wang, Jun; Huang, Peipei; Zhang, Jinsong; Xiao, Hang; Gao, Rong

    2016-08-01

    A simple, sensitive and specific ultra performance liquid chromatography coupled to electrospray tandem high resolution mass spectrometry (UPLC-ESI-HRMS/MS) method has been developed and validated for quantification of paraquat in plasma and urine. The sample preparation was carried out by one-step protein precipitation with acetonitrile. The paraquat was separated with a HILIC column in 10min. Detection was performed using Q Exactive Orbitrap mass spectrometer by Targeted-MS/MS scan mode. Methodological parameters, such as ammonium formate concentration, formic acid concentration, spray voltage, capillary temperature, heater temperature and normalized collision energy were optimized to achieve the highest sensitivity. The calibration curve was linear over the concentration range of LOQ-1000ng/mL. LOD was 0.1 and 0.3ng/mL, LOQ was 0.3 and 0.8ng/mL for urine and plasma, respectively. The intra- and inter-day precisions were <7.97% and 4.78% for plasma and urine. The accuracies were within the range 93.51-100.90%. The plasma and urine matrices had negligible relative matrix effect in this study. This method was successfully applied to determine paraquat concentration in plasma samples with hemoperfusion from 5 suspected paraquat poisoning patients. PMID:27270261

  14. Quantification of halobetasol propionate and its impurities present in topical dosage forms by stability-indicating LC method.

    PubMed

    Nalwade, Santaji; Reddy, Vangala Ranga; Kulkarni, Dipak; Todamal, Sandip

    2015-01-01

    A novel, sensitive, stability-indicating, gradient, reverse-phase high-performance liquid chromatographic method has been developed for quantitative determination of halobetasol propionate and its impurities in topical dosage forms. The chromatographic separation was achieved on a Phenomenex Synergi polar reverse phase, 250 × 4.6 mm, 4 µm column. Mobile phase A comprises a mixture of 0.01 M KH2PO4 buffer containing 0.2% 1-octane sulfonic acid sodium salt (pH 3.0), acetonitrile and methanol in the ratio 80:15:05 (v/v/v), respectively, and mobile phase B contains a mixture of 0.01 M KH2PO4 buffer containing 0.2% 1-octane sulfonic acid sodium salt (pH 3.0), acetonitrile and methanol in the ratio 20:70:10 (v/v/v), respectively. The flow rate is 0.8 mL min(-1). The column compartment temperature is set at 40°C and the detection wavelength is set at 240 nm. The resolutions between Halobetasol propionate and all the impurities are >2.0 for all pairs of compounds. The drug product was subjected to International Conference on Harmonization (ICH)-prescribed hydrolytic, oxidative, photolytic and thermal stress conditions. The method is validated as per the ICH guidelines with respect to specificity, linearity, limit of detection, limit of quantification, accuracy, precision, robustness and ruggedness. PMID:24784115

  15. A GC-MS Method for Detection and Quantification of Cathine, Cathinone, Methcathinone and Ephedrine in Oral Fluid.

    PubMed

    Mohamed, Khaled M; Al-Hazmi, Abtehaj H; Alasiri, Alanoud M; Ali, Mahmoud El-Said

    2016-09-01

    The stimulating herbal drug khat (cathine, cathinone) and its analog methcathinone are common substances of abuse in most countries. A GC-MS method was developed and validated for the detection and quantification of cathine, cathinone, methcathinone and ephedrine in oral fluid specimens. The analytes and internal standard (amphetamine-d5) were extracted from 0.5 mL oral fluids by ethyl acetate, and then the dried extracts were derivatized with heptafluorobutyric anhydride at 70°C for 30 min. The MS was used in selected ion monitoring mode. Ions monitored were m/z 117, 240 and 330 for cathine, m/z 77, 105 and 240 for cathinone, m/z 105, 210 and 254 for methcathinone, m/z 210, 254 and 344 for ephedrine and m/z 244 and 336 for amphetamine-d5 The calibration curves were linear (r(2)> 0.98) in the concentration range 20-2,000 ng/mL for all analytes. The intra- and inter-assay imprecisions were within (1.6-12.5%) and (1.5-9.5%), respectively, for all analytes. Intra-assay accuracies were between -5.9 and 6.7% for all analytes. The method was successfully applied to detect and quantify the target analytes from oral fluid specimens collected from Khat and methcathinone users. PMID:27165573

  16. A new and reliable method for live imaging and quantification of reactive oxygen species in Botrytis cinerea: technological advancement.

    PubMed

    Marschall, Robert; Tudzynski, Paul

    2014-10-01

    Reactive oxygen species (ROS) are produced in conserved cellular processes either as by-products of the cellular respiration in mitochondria, or purposefully for defense mechanisms, signaling cascades or cell homeostasis. ROS have two diametrically opposed attributes due to their highly damaging potential for DNA, lipids and other molecules and due to their indispensability for signaling and developmental processes. In filamentous fungi, the role of ROS in growth and development has been studied in detail, but these analyses were often hampered by the lack of reliable and specific techniques to monitor different activities of ROS in living cells. Here, we present a new method for live cell imaging of ROS in filamentous fungi. We demonstrate that by use of a mixture of two fluorescent dyes it is possible to monitor H2O2 and superoxide specifically and simultaneously in distinct cellular structures during various hyphal differentiation processes. In addition, the method allows for reliable fluorometric quantification of ROS. We demonstrate that this can be used to characterize different mutants with respect to their ROS production/scavenging potential. PMID:25220147

  17. Improving the reliability of POD curves in NDI methods using a Bayesian inversion approach for uncertainty quantification

    NASA Astrophysics Data System (ADS)

    Ben Abdessalem, A.; Jenson, F.; Calmon, P.

    2016-02-01

    This contribution provides an example of the possible advantages of adopting a Bayesian inversion approach to uncertainty quantification in nondestructive inspection methods. In such problem, the uncertainty associated to the random parameters is not always known and needs to be characterised from scattering signal measurements. The uncertainties may then correctly propagated in order to determine a reliable probability of detection curve. To this end, we establish a general Bayesian framework based on a non-parametric maximum likelihood function formulation and some priors from expert knowledge. However, the presented inverse problem is time-consuming and computationally intensive. To cope with this difficulty, we replace the real model by a surrogate one in order to speed-up the model evaluation and to make the problem to be computationally feasible for implementation. The least squares support vector regression is adopted as metamodelling technique due to its robustness to deal with non-linear problems. We illustrate the usefulness of this methodology through the control of tube with enclosed defect using ultrasonic inspection method.

  18. LC-MS/MS method development for quantification of busulfan in human plasma and its application in pharmacokinetic study.

    PubMed

    Nadella, Taraka Ramarao; Suryadevara, Vidyadhara; Lankapalli, Sasidhar Reddyvallam; Mandava, Venkata Basaveswara Rao; Bandarupalli, Deepti

    2016-02-20

    A simple, rapid, specific and precise liquid chromatography-tandem mass spectrophotometric (LC-MS/MS) method was developed and validated for quantification of busulfan, in human plasma. busulfan d8 was used as internal standard, added to plasma sample prior to extraction using acetonitrile as a precipitating agent. Chromatographic separation was achieved on phenomenex kinetex C18 column (50mm×2.1mm, 2.6μm) with acteonitrile: 10mM ammonium formate buffer (80:20v/v) as an isocratic mobile phase with a flow rate of 0.5mLmin(-1). Quantitation was performed by transition of 264.1→151.1 (m/z) for busulfan and 272.1→159.1 (m/z) for busulfan d8. The lower limit of quantitation was 0.2ngmL(-1) with a 100μL plasma sample. The concentrations of nine working standards showed linearity between 0.2 and 100ngmL(-1) (r(2)≥0.9986). Chromatographic separation was achieved within 2.0min. The average extraction recoveries of 3quality control concentrations were 92.52% for busulfan and 90.75% for busulfan d8. The coefficient of variation was ≤15% for intra- and inter-batch assays. The developed method was successfully applied for the determination of Busulfan pharmacokinetics after oral administration. PMID:26736033

  19. Validation of RP-HPLC Method for Simultaneous Quantification of Bicalutamide and Hesperetin in Polycaprolactone-Bicalutamide-Hesperetin-Chitosan Nanoparticles.

    PubMed

    Arya, Abhishek; Khandelwal, Kiran; Singh, Aanchal; Ahmad, Hafsa; Agrawal, Satish; Khatik, Renuka; Mittapelly, Naresh; Dwivedi, Anil Kumar

    2015-10-01

    Bicalutamide is a non-steroidal anti-androgen drug used for the treatment of androgen-dependent prostate cancer. Hesperetin is a natural bioflavonoid that can be used in combination with bicalutamide to improve efficacy and decrease tolerance. The aim of the present work was to develop and validate a simple, sensitive, rapid reverse phase-high performance liquid chromatographic method for simultaneous estimation of bicalutamide and hesperetin. The validation parameters such as specificity, linearity, precision and accuracy, limit of detection (LOD) and limit of quantification (LOQ) were determined according to International Conference on Harmonization ICH Q2 (R1) guidelines. Chromatographic separation was achieved on Lichrocart(®) CN column (250 × 4 mm, 5 µm, MERCK) with isocratic elution. The retention times and detection wavelength, for hesperetin and bicalutamide were 4.28 min, 288 nm and 5.90 min, 270 nm respectively. The intra-day and inter-day assay precision and accuracy were found to be <2% over linearity of 50-2000 ng/mL with R(2) 0.999. LOD and LOQ, of bicalutamide and hesperetin was 14.70, 44.57 ng/mL and 16.11, 48.84 ng/mL, respectively. The method was successfully applied for encapsulation efficiency and drug release studies from bicalutamide and hesperetin loaded nanoparticles. PMID:26045585

  20. Simultaneous quantification of related substances of perindopril tert-butylamine using a novel stability indicating liquid chromatographic method.

    PubMed

    Szabó, Zoltán-István; Réti, Zenkő-Zsuzsánna; Gagyi, László; Kis, Erika Lilla; Sipos, Emese

    2015-03-01

    A novel stability indicating gradient reverse-phased high-performance liquid chromatographic method has been developed for the quantification of impurities of perindopril tert-butylamine (PER) in pharmaceutical dosage form. Separation of the active substance and its known (Impurities B, C, D, E, F) and unknown impurities was achieved on a BDS Hypersil C18 column (250 mm × 4.6 mm, 5 µm), thermostated at 70°C, using a mobile phase comprised of aqueous solution of sodium 1-heptanesulfonate adjusted to pH 2 with perchloric acid and acetonitrile. The flow rate was maintained at 1.5 mL min(-1), injection volume of 20 µL was utilized and detection of analytes was performed at 215 nm. The developed method was validated in accordance with current ICH Guidelines for all suggested parameters, including forced degradation studies and proved to be linear, accurate, precise and suitable for the impurity testing of PER, being subsequently applied during on-going stability studies of a newly developed generic formulation. PMID:25616989

  1. An improved method for susceptibility and radius quantification of cylindrical objects from MRI.

    PubMed

    Hsieh, Ching-Yi; Cheng, Yu-Chung N; Neelavalli, Jaladhar; Haacke, E Mark; Stafford, R Jason

    2015-05-01

    A new method is developed to measure the magnetic susceptibilities and radii of small cylinder-like objects at arbitrary orientations accurately. This method for most biological substances only requires a standard gradient echo sequence with one or two echo times, depending on the orientation of an object relative to the main magnetic field. For objects oriented at the magic angle, however, this method is not applicable. As a byproduct of this method, the cross-sectional area as well as signals inside and outside the object can be determined. The uncertainty of each measurement is estimated from the error propagation method. Partial volume, dephasing, and phase aliasing effects are naturally included in the equations of this method. A number of simulations, phantom, and pilot in-vivo human studies are carried out to validate the theory. When the maximal phase value at the boundary of a given cylindrical object is larger than 3 radians, and the phase inside the object is more than 1 radian, the susceptibility can be accurately quantified within 15%. The radius of the object can be determined to subpixel accuracy. This is the case when the signal-to-noise ratio inside the object is about 6:1 or higher and the radius of the object is about one pixel or larger. These conditions are realistic when considering medullary and pial veins for example. PMID:25633922

  2. An improved method for susceptibility and radius quantification of cylindrical objects from MRI

    PubMed Central

    Hsieh, Ching-Yi; Cheng, Yu-Chung N.; Neelavalli, Jaladhar; Haacke, E. Mark; Stafford, R. Jason

    2015-01-01

    A new method is developed to measure the magnetic susceptibilities and radii of small cylinder-like objects at arbitrary orientations accurately. This method for most biological substances only requires a standard gradient echo sequence with one or two echo times, depending on the orientation of an object relative to the main magnetic field. For objects oriented at the magic angle, however, this method is not applicable. As a byproduct of this method, the cross-sectional area as well as signals inside and outside the object can be determined. The uncertainty of each measurement is estimated from the error propagation method. Partial volume, dephasing, and phase aliasing effects are naturally included in the equations of this method. A number of simulations, phantom, and pilot in-vivo human studies are carried out to validate the theory. When the maximal phase value at the boundary of a given cylindrical object is larger than 3 radians, and the phase inside the object is more than 1 radian, the susceptibility can be accurately quantified within 15%. The radius of the object can be determined to subpixel accuracy. This is the case when the signal-to-noise ratio inside the object is about 6:1 or higher and the radius of the object is about one pixel or larger. These conditions are realistic when considering medullary and pial veins for example. PMID:25633922

  3. In situ DNA hybridized chain reaction (FISH-HCR) as a better method for quantification of bacteria and archaea within marine sediment

    NASA Astrophysics Data System (ADS)

    Buongiorno, J.; Lloyd, K. G.; Shumaker, A.; Schippers, A.; Webster, G.; Weightman, A.; Turner, S.

    2015-12-01

    Nearly 75% of the Earth's surface is covered by marine sediment that is home to an estimated 2.9 x 1029 microbial cells. A substantial impediment to understanding the abundance and distribution of cells within marine sediment is the lack of a consistent and reliable method for their taxon-specific quantification. Catalyzed reporter fluorescent in situ hybridization (CARD-FISH) provides taxon-specific enumeration, but this process requires passing a large enzyme through cell membranes, decreasing its precision relative to general cell counts using a small DNA stain. In 2015, Yamaguchi et al. developed FISH hybridization chain reaction (FISH-HCR) as an in situ whole cell detection method for environmental microorganisms. FISH-HCR amplifies the fluorescent signal, as does CARD-FISH, but it allows for milder cell permeation methods that might prevent yield loss. To compare FISH-HCR to CARD-FISH, we examined bacteria and archaea cell counts within two sediment cores, Lille Belt (~78 meters deep) and Landsort Deep (90 meters deep), which were retrieved from the Baltic Sea Basin during IODP Expedition 347. Preliminary analysis shows that CARD-FISH counts are below the quantification limit for most depths across both cores. By contrast, quantification of cells was possible with FISH-HCR in all examined depths. When quantification with CARD-FISH was above the limit of detection, counts with FISH-HCR were up to 11 fold higher for Bacteria and 3 fold higher for Archaea from the same sediment sample. Further, FISH-HCR counts follow the trends of on board counts nicely, indicating that FISH-HCR may better reflect the cellular abundance within marine sediment than other quantification methods, including qPCR. Using FISH-HCR, we found that archaeal cell counts were on average greater than bacterial cell counts, but within the same order of magnitude.

  4. Establishment of quantitative PCR methods for the quantification of geosmin-producing potential and Anabaena sp. in freshwater systems.

    PubMed

    Su, Ming; Gaget, Virginie; Giglio, Steven; Burch, Michael; An, Wei; Yang, Min

    2013-06-15

    Geosmin has often been associated with off-flavor problems in drinking water with Anabaena sp. as the major producer. Rapid on-site detection of geosmin-producers as well as geosmin is important for a timely management response to potential off-flavor events. In this study, quantitative polymerase chain reaction (qPCR) methods were developed to detect the levels of Anabaena sp. and geosmin, respectively, by designing two PCR primer sets to quantify the rpoC1 gene (ARG) and geosmin synthase one (GSG) in Anabaena sp. in freshwater systems. The ARG density determined by qPCR assay is highly related to microscopic cell count (r(2) = 0.726, p < 0.001), and the limit of detection (LOD) and limit of quantification (LOQ) of the qPCR method were 0.02 pg and 0.2 pg of DNA, respectively. At the same time, the relationship between geosmin concentrations measured by gas chromatography-mass spectrometry (GC-MS) and GSG copies was also established (r(2) = 0.742, p < 0.001) with similar LOD and LOQ values. Using the two qPCR protocols, we succeeded in measuring different levels of ARG and GSG copies in different freshwater systems with high incidence environmental substrata and diverse ecological conditions, showing that the methods developed could be applied for environmental monitoring. Moreover, comparing to the microscopic count and GC-MS analytical methods, the qPCR methods can reduce the time-to-results from several days to a few hours and require considerably less traditional algal identification and taxonomic expertise. PMID:23622984

  5. Comparison of dynamic susceptibility contrast-MRI perfusion quantification methods in the presence of delay and dispersion

    NASA Astrophysics Data System (ADS)

    Maan, Bianca; Simões, Rita Lopes; Meijer, Frederick J. A.; Klaas Jan Renema, W.; Slump, Cornelis H.

    2011-03-01

    The perfusion of the brain is essential to maintain brain function. Stroke is an example of a decrease in blood flow and reduced perfusion. During ischemic stroke the blood flow to tissue is hampered due to a clot inside a vessel. To investigate the recovery of stroke patients, follow up studies are necessary. MRI is the preferred imaging modality for follow up because of the absence of radiation dose concerns, contrary to CT. Dynamic Susceptibility Contrast (DSC) MRI is an imaging technique used for measuring perfusion of the brain, however, is not standard applied in the clinical routine due to lack of immediate patient benefit. Several post processing algorithms are described in the literature to obtain cerebral blood flow (CBF). The quantification of CBF relies on the deconvolution of a tracer concentration-time curve in an arterial and a tissue voxel. There are several methods to obtain this deconvolution based on singular-value decomposition (SVD). This contribution describes a comparison between the different approaches as currently there is no best practice for (all) clinical relevant situations. We investigate the influence of tracer delay, dispersion and recirculation on the performance of the methods. In the presence of negative delays, the truncated SVD approach overestimates the CBF. Block-circulant and reformulated SVD are delay-independent. Due to its delay dependent behavior, the truncated SVD approach performs worse in the presence of dispersion as well. However all SVD approaches are dependent on the amount of dispersion. Moreover, we observe that the optimal truncation parameter varies when recirculation is added to noisy data, suggesting that, in practice, these methods are not immune to tracer recirculation. Finally, applying the methods to clinical data resulted in a large variability of the CBF estimates. Block-circulant SVD will work in all situations and is the method with the highest potential.

  6. In-vivo quantification of natural incipient caries lesions using the quantitative light-induced fluoroscence method: a reproducibility study

    NASA Astrophysics Data System (ADS)

    Tranaeus, Sofia; Shi, Xie-Qi; Trollsas, Karin; Lindgren, Lars-Erik; Angmar-Mansson, Birgit

    2000-03-01

    A new method for detection and quantification of natural incipient caries lesions, the Quantitative Light-induced Fluorescence method (QLF), has recently been developed. The aim of this study was to test the repeatability and reproducibility of the analytical part of the method. In vivo captured images (CCD-video camera, Panasonic WV-KS 152, with an argon ion laser as light source) of 15 different incipient caries lesions on smooth surfaces were analyzed by three analysts. The images were analyzed three times in a randomized order, twice for the first reconstructed area (P1A1 and P1A2), and then once for a second one (P2A1). Three parameters were measured, lesion area (mm2), average change in fluorescence (%), and maximum change in fluorescence (%) in the lesion. Repeated measures ANOVA were used to calculate the intra-, and inter-examiner reliability. Intra-examiner reliability for all three analysts showed an intra-class correlation coefficient, R, between 0.93 and 0.99 (for the analyses with the first patch, P1A1 and P1A2, as well as between the first and the second patch, P1A1 and P2A1). Inter-examiner reliability showed an inter-class correlation coefficient, R, between 0.95 and 0.99 (for analyses P1A1, P1A2 and P2A1). It was concluded that the Quantitative Light- induced fluorescence method showed excellent repeatability and reproducibility concerning the analytical part of the method.

  7. A comparative study for PSP toxins quantification by using MBA and HPLC official methods in shellfish.

    PubMed

    Ben-Gigirey, B; Rodríguez-Velasco, M L; Otero, A; Vieites, J M; Cabado, A G

    2012-10-01

    Commission Regulation (EC) N° 2074/2005 recognises the biological method as the reference method for Paralytic Shellfish Poisoning (PSP) toxins detection in molluscs. It was amended by Commission Regulation (EC) N° 1664/2006 that accepted the so-called Lawrence method as an alternative to the reference method. The goal of this study was to compare AOAC Official Methods of Analysis 959.08 (Biological method) and 2005.06 (Prechromatographic Oxidation and Liquid Chromatography with fluorescence detection) in samples with different toxin profiles. The influence of extraction solvent in the total samples toxicity was also evaluated. A total of 40 samples including mussels, clams, scallops, razor-clams, cockles, oysters and barnacles were analysed by both official methods. Samples were selected with Alexandrium and Gymnodinium toxic profiles, from different origin and including several presentations: fresh, frozen, canned and boiled. Acetic and hydrochloric acid extractions were performed in all samples and the extracts were simultaneously analysed by both methods. Most samples were naturally contaminated and two samples were spiked. Comparison of both official methods, mouse bioassay (MBA) with HCl extraction and Liquid Chromatography with fluorescence detection (HPLC-FLD) with acetic acid extraction, led to an 85% of consistent results regarding compliance with legal limit, including samples below and above it. The linear correlation coefficient was r² = 0.69 and the paired t test (two tails, α = 0.05) indicated that there were not significant differences among both sets of data. Nevertheless, toxicity differences were found in several samples. In 15 out of 18 shellfish with a Gymnodinium toxic profile, higher toxicity levels were obtained by MBA. This fact was more evident in 7 samples, partially related to the lack of standards and the impossibility of analysing dc-NEO, C1, 2 and GTX6 at the beginning of the study. However, other factors concerning the extraction

  8. [Rapid FTIR-ATR Method for the Quantification of Bitumen Property].

    PubMed

    Tang, Jie-qiong; Ma, Qing-feng; Shi, Jing-tao; Yuan, Hong-fu; Song, Chun-feng; Xie, Jin-chun; Li, Xiao-yu

    2016-03-01

    The quality of bitumen directly affects road performance and road life. Traditional analytical methods-for wax content, softening point and penetration of bitumen are tedious and time-consuming. A new fast method, with which the three properties can be determined at same time, is proposed in this paper. The spectra of 220 bitumen were collected and their wax content, softening point and penetration data were determined according to the standard JTJ052-2000. The quantitative calibration models for wax content, softening point and penetration were established using partial least squares (PLS), with SECV 0.13, 0.88, 3.18 and SEP 0.14, 1.06, 3.90, less than the reproducibility error stipulated in the standard method. Three samples were in random selected to test the repeatability, the results met the precision requirement of the standard method. With its advantages of better repeatability, fast, easy operation, the new method can be used as an alternative for the determination of wax content, softening point and penetration of bitumen. PMID:27400503

  9. A Novel Method for Identification and Quantification of Consistently Differentially Methylated Regions

    PubMed Central

    Hsiao, Ching-Lin; Hsieh, Ai-Ru; Lian, Ie-Bin; Lin, Ying-Chao; Wang, Hui-Min; Fann, Cathy S. J.

    2014-01-01

    Advances in biotechnology have resulted in large-scale studies of DNA methylation. A differentially methylated region (DMR) is a genomic region with multiple adjacent CpG sites that exhibit different methylation statuses among multiple samples. Many so-called “supervised” methods have been established to identify DMRs between two or more comparison groups. Methods for the identification of DMRs without reference to phenotypic information are, however, less well studied. An alternative “unsupervised” approach was proposed, in which DMRs in studied samples were identified with consideration of nature dependence structure of methylation measurements between neighboring probes from tiling arrays. Through simulation study, we investigated effects of dependencies between neighboring probes on determining DMRs where a lot of spurious signals would be produced if the methylation data were analyzed independently of the probe. In contrast, our newly proposed method could successfully correct for this effect with a well-controlled false positive rate and a comparable sensitivity. By applying to two real datasets, we demonstrated that our method could provide a global picture of methylation variation in studied samples. R source codes to implement the proposed method were freely available at http://www.csjfann.ibms.sinica.edu.tw/eag/programlist/ICDMR/ICDMR.html. PMID:24818602

  10. More accurate matrix-matched quantification using standard superposition method for herbal medicines.

    PubMed

    Liu, Ying; Shi, Xiao-Wei; Liu, E-Hu; Sheng, Long-Sheng; Qi, Lian-Wen; Li, Ping

    2012-09-01

    Various analytical technologies have been developed for quantitative determination of marker compounds in herbal medicines (HMs). One important issue is matrix effects that must be addressed in method validation for different detections. Unlike biological fluids, blank matrix samples for calibration are usually unavailable for HMs. In this work, practical approaches for minimizing matrix effects in HMs analysis were proposed. The matrix effects in quantitative analysis of five saponins from Panax notoginseng were assessed using high-performance liquid chromatography (HPLC). Matrix components were found to interfere with the ionization of target analytes when mass spectrometry (MS) detection were employed. To compensate the matrix signal suppression/enhancement, two matrix-matched methods, standard addition method with the target-knockout extract and standard superposition method with a HM extract were developed and tested in this work. The results showed that the standard superposition method is simple and practical for overcoming matrix effects for quantitative analysis of HMs. Moreover, the interference components were observed to interfere with light scattering of target analytes when evaporative light scattering detection (ELSD) was utilized for quantitative analysis of HMs but was not indicated when Ultraviolet detection (UV) were employed. Thus, the issue of interference effects should be addressed and minimized for quantitative HPLC-ELSD and HPLC-MS methodologies for quality control of HMs. PMID:22835696

  11. Improved Method for the Quantification of Methane Concentrations in Unconsolidated Lake Sediments.

    PubMed

    Tyroller, Lina; Tomonaga, Yama; Brennwald, Matthias S; Ndayisaba, Cyprien; Naeher, Sebastian; Schubert, Carsten; North, Ryan P; Kipfer, Rolf

    2016-07-01

    There is conclusive evidence that the methods most commonly used to sample methane (CH4) dissolved in the pore water of lake sediments produce results that are likely to be affected by gas loss or gas exchange with the atmosphere. To determine the in situ amount of CH4 per unit mass of pore water in sediments, we developed and validated a new method that combines techniques developed for noble-gas analysis in pore waters with a standard headspace technique to quantify the CH4 present in the pore space in dissolved and gaseous form. The method was tested at two sites: Lake Lungern, where CH4 concentrations were close to saturation; and Lake Rotsee, where CH4 concentrations are known to exceed saturation and where CH4 bubble formation and gas ebullition are commonly observed. We demonstrate that the new method, in contrast to the available methods, more reliably captures the total amount of CH4 per unit mass of pore water consisting of both dissolved and free CH4 (i.e., gas bubbles) in the pore space of the sediment. PMID:27244276

  12. Immunologic, spectrophotometric and nucleic acid based methods for the detection and quantification of airborne pollen

    PubMed Central

    Rittenour, William R.; Hamilton, Robert G.; Beezhold, Donald H.; Green, Brett J.

    2015-01-01

    Microscopic identification of pollen morphological phenotypes has been the traditional method used to identify and quantify pollen collected by air monitoring stations worldwide. Although this method has enabled a semi-standardized approach for the assessment of pollen exposure, limitations including labor intensiveness, required expertise, examiner bias, and the inability to differentiate species, genera, and in some cases families have limited data derived from the these stations. Recent advances in chemical, biochemical and molecular detection methods have provided standardized alternatives to this microscopic approach. In this review, we examine the applicability of alternative methodologies, in particular nucleic acid based assays involving the quantitative polymerase chain reaction, for the standardized detection of airborne pollen. PMID:22342607

  13. Quantification of rat brain SPECT with 123I-ioflupane: evaluation of different reconstruction methods and image degradation compensations using Monte Carlo simulation

    NASA Astrophysics Data System (ADS)

    Roé-Vellvé, N.; Pino, F.; Falcon, C.; Cot, A.; Gispert, J. D.; Marin, C.; Pavía, J.; Ros, D.

    2014-08-01

    SPECT studies with 123I-ioflupane facilitate the diagnosis of Parkinson’s disease (PD). The effect on quantification of image degradations has been extensively evaluated in human studies but their impact on studies of experimental PD models is still unclear. The aim of this work was to assess the effect of compensating for the degrading phenomena on the quantification of small animal SPECT studies using 123I-ioflupane. This assessment enabled us to evaluate the feasibility of quantitatively detecting small pathological changes using different reconstruction methods and levels of compensation for the image degrading phenomena. Monte Carlo simulated studies of a rat phantom were reconstructed and quantified. Compensations for point spread function (PSF), scattering, attenuation and partial volume effect were progressively included in the quantification protocol. A linear relationship was found between calculated and simulated specific uptake ratio (SUR) in all cases. In order to significantly distinguish disease stages, noise-reduction during the reconstruction process was the most relevant factor, followed by PSF compensation. The smallest detectable SUR interval was determined by biological variability rather than by image degradations or coregistration errors. The quantification methods that gave the best results allowed us to distinguish PD stages with SUR values that are as close as 0.5 using groups of six rats to represent each stage.

  14. Quantification of rat brain SPECT with (123)I-ioflupane: evaluation of different reconstruction methods and image degradation compensations using Monte Carlo simulation.

    PubMed

    Roé-Vellvé, N; Pino, F; Falcon, C; Cot, A; Gispert, J D; Marin, C; Pavía, J; Ros, D

    2014-08-21

    SPECT studies with (123)I-ioflupane facilitate the diagnosis of Parkinson's disease (PD). The effect on quantification of image degradations has been extensively evaluated in human studies but their impact on studies of experimental PD models is still unclear. The aim of this work was to assess the effect of compensating for the degrading phenomena on the quantification of small animal SPECT studies using (123)I-ioflupane. This assessment enabled us to evaluate the feasibility of quantitatively detecting small pathological changes using different reconstruction methods and levels of compensation for the image degrading phenomena. Monte Carlo simulated studies of a rat phantom were reconstructed and quantified. Compensations for point spread function (PSF), scattering, attenuation and partial volume effect were progressively included in the quantification protocol. A linear relationship was found between calculated and simulated specific uptake ratio (SUR) in all cases. In order to significantly distinguish disease stages, noise-reduction during the reconstruction process was the most relevant factor, followed by PSF compensation. The smallest detectable SUR interval was determined by biological variability rather than by image degradations or coregistration errors. The quantification methods that gave the best results allowed us to distinguish PD stages with SUR values that are as close as 0.5 using groups of six rats to represent each stage. PMID:25069105

  15. Development and validation of a liquid chromatography isotope dilution mass spectrometry method for the reliable quantification of alkylphenols in environmental water samples by isotope pattern deconvolution.

    PubMed

    Fabregat-Cabello, Neus; Sancho, Juan V; Vidal, Andreu; González, Florenci V; Roig-Navarro, Antoni Francesc

    2014-02-01

    We present here a new measurement method for the rapid extraction and accurate quantification of technical nonylphenol (NP) and 4-t-octylphenol (OP) in complex matrix water samples by UHPLC-ESI-MS/MS. The extraction of both compounds is achieved in 30min by means of hollow fiber liquid phase microextraction (HF-LPME) using 1-octanol as acceptor phase, which provides an enrichment (preconcentration) factor of 800. On the other hand we have developed a quantification method based on isotope dilution mass spectrometry (IDMS) and singly (13)C1-labeled compounds. To this end the minimal labeled (13)C1-4-(3,6-dimethyl-3-heptyl)-phenol and (13)C1-t-octylphenol isomers were synthesized, which coelute with the natural compounds and allows the compensation of the matrix effect. The quantification was carried out by using isotope pattern deconvolution (IPD), which permits to obtain the concentration of both compounds without the need to build any calibration graph, reducing the total analysis time. The combination of both extraction and determination techniques have allowed to validate for the first time a HF-LPME methodology at the required levels by legislation achieving limits of quantification of 0.1ngmL(-1) and recoveries within 97-109%. Due to the low cost of HF-LPME and total time consumption, this methodology is ready for implementation in routine analytical laboratories. PMID:24423386

  16. Comparison of methods for acid quantification: impact of resist components on acid-generating efficiency

    NASA Astrophysics Data System (ADS)

    Cameron, James F.; Fradkin, Leslie; Moore, Kathryn; Pohlers, Gerd

    2000-06-01

    Chemically amplified deep UV (CA-DUV) positive resists are the enabling materials for manufacture of devices at and below 0.18 micrometer design rules in the semiconductor industry. CA-DUV resists are typically based on a combination of an acid labile polymer and a photoacid generator (PAG). Upon UV exposure, a catalytic amount of a strong Bronsted acid is released and is subsequently used in a post-exposure bake step to deprotect the acid labile polymer. Deprotection transforms the acid labile polymer into a base soluble polymer and ultimately enables positive tone image development in dilute aqueous base. As CA-DUV resist systems continue to mature and are used in increasingly demanding situations, it is critical to develop a fundamental understanding of how robust these materials are. One of the most important factors to quantify is how much acid is photogenerated in these systems at key exposure doses. For the purpose of quantifying photoacid generation several methods have been devised. These include spectrophotometric methods, ion conductivity methods and most recently an acid-base type titration similar to the standard addition method. This paper compares many of these techniques. First, comparisons between the most commonly used acid sensitive dye, tetrabromophenol blue sodium salt (TBPB) and a less common acid sensitive dye, Rhodamine B base (RB) are made in several resist systems. Second, the novel acid-base type titration based on the standard addition method is compared to the spectrophotometric titration method. During these studies, the make up of the resist system is probed as follows: the photoacid generator and resist additives are varied to understand the impact of each of these resist components on the acid generation process.

  17. Comparative evaluation of synaptophysin-based methods for quantification of synapses.

    PubMed

    Calhoun, M E; Jucker, M; Martin, L J; Thinakaran, G; Price, D L; Mouton, P R

    1996-12-01

    Development, ageing, and a variety of neurological disorders are characterized by selective alterations in specific populations of nerve cells which are, in turn, associated with changes in the numbers of synapses in the target fields of these neurons. To begin to delineate the significance of changes in synapses in development, ageing, and disease, it is first essential to quantify the number of synapses in defined regions of the CNS. In the past, investigators have used EM methods to assess synapse numbers or density, but these approaches are costly, labour intensive, and technically difficult, particularly in autopsy material. To begin to define reliable strategies useful for studies of both animals and humans, we used three techniques to measure synaptophysin-immunoreactivity in rat brain. The levels of synaptophysin protein were determined by Western blots of five hippocampal subregions; the intensity of synaptophysin-immunoreactivity in dentate gyrus and stratum oriens was determined by optical densitometry of immunocytochemically stained sections; and the total number of synaptophysin-immunoreactivity presynaptic boutons in dentate gyrus and stratum oriens was assessed by unbiased stereology. Each approach has advantages and disadvantages. Western blotting is the least time-consuming of the three methods and allows simultaneous processing of multiple samples. In systematically sampled histological sections, both densitometry and stereology allow precise definition of the region of interest, and the stereological optical dissector method allows quantitation of the numbers of synaptophysin-immunoreactive boutons. Stereology was the only method that clearly demonstrated greater synaptophysin-immunoreactivity in the dentate gyrus as compared to stratum oriens. The use of systematic sampling and the dissector technique offer a high degree of anatomical resolution (lacking in Western blot methods) and has quantitative advantage over the greyscale-based density

  18. Comparative evaluation of different extraction and quantification methods for forensic RNA analysis.

    PubMed

    Grabmüller, Melanie; Madea, Burkhard; Courts, Cornelius

    2015-05-01

    Since about 2005, there is increasing interest in forensic RNA analysis whose versatility may very favorably complement traditional DNA profiling in forensic casework. There is, however, no method available specifically dedicated for extraction of RNA from forensically relevant sample material. In this study we compared five commercially available and commonly used RNA extraction kits and methods (mirVana™ miRNA Isolation Kit Ambion; Trizol® Reagent, Invitrogen; NucleoSpin® miRNA Kit Macherey-Nagel; AllPrep DNA/RNA Mini Kit and RNeasy® Mini Kit both Qiagen) to assess their relative effectiveness of yielding RNA of good quality and their compatibility with co-extraction of DNA amenable to STR profiling. We set up samples of small amounts of dried blood, liquid saliva, semen and buccal mucosa that were aged for different time intervals for co-extraction of RNA and DNA. RNA quality was assessed by determination of 'RNA integrity number' (RIN) and quantitative PCR based expression analysis. DNA quality was assessed via monitoring STR typing success rates. By comparison, the different methods exhibited considerable differences between RNA and DNA yields, RNA quality values and expression levels, and STR profiling success, with the AllPrep DNA/RNA Mini Kit and the NucleoSpin® miRNA Kit excelling at DNA co-extraction and RNA results, respectively. Overall, there was no 'best' method to satisfy all demands of comprehensible co-analysis of RNA and DNA and it appears that each method has specific merits and flaws. We recommend to cautiously choose from available methods and align its characteristics with the needs of the experimental setting at hand. PMID:25625965

  19. Coumarins in horse chestnut flowers: isolation and quantification by UPLC method.

    PubMed

    Dudek-Makuch, Marlena; Matławska, Irena

    2013-01-01

    The coumarins: scopoletin, esculetin and fraxetin were isolated from the flowers of horse chestnut (Aesculus hippocastanum L., Hippocastanaceae) and identified by spectrophotometric methods (UV, 1H, 13C NMR, ESI-MS). Their content, determined using the Ultra Performance Liquid Chromatography (UPLC), was 0.41, 0.13 and 0.05%, respectively. PMID:23757942

  20. Comparison of different mass transport calculation methods for wind erosion quantification purposes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Quantitative estimation of the material transported by the wind is essential in the study and control of wind erosion, although methods for its calculation are still controversial. Sampling the dust cloud at discrete heights, fitting an equation to the data, and integrating this equation from the so...

  1. Detection and Quantification of Silicon in Floricultural Crops Utilizing Three Distinct Analytical Methods

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Silicon (Si) is increasingly used in agriculture due to its apparent beneficial effects in stimulating stress resistance in some crops. Several methods for detecting and quantifying silicon in plants have been reported including electron beam analysis (EBA), which is the use of scanning electron mi...

  2. Quantification of Greenhouse Gas Emissions from Soil Applied Swine Effluent by Different Methods

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Greenhouse gas (CO2, CH4, and N2O) emissions were measured from a field experiment in which pre-plant swine effluent application methods where evaluated for no-till corn grain production. The treatments included a control, an inorganic fertilizer treatment that received 179 kg N ha-1 as urea ammoni...

  3. Quantification of histone modifications by parallel-reaction monitoring: a method validation.

    PubMed

    Sowers, James L; Mirfattah, Barsam; Xu, Pei; Tang, Hui; Park, In Young; Walker, Cheryl; Wu, Ping; Laezza, Fernanda; Sowers, Lawrence C; Zhang, Kangling

    2015-10-01

    Abnormal epigenetic reprogramming is one of the major causes leading to irregular gene expression and regulatory pathway perturbations, in the cells, resulting in unhealthy cell development or diseases. Accurate measurements of these changes of epigenetic modifications, especially the complex histone modifications, are very important, and the methods for these measurements are not trivial. By following our previous introduction of PRM to targeting histone modifications (Tang, H.; Fang, H.; Yin, E.; Brasier, A. R.; Sowers, L. C.; Zhang, K. Multiplexed parallel reaction monitoring targeting histone modifications on the QExactive mass spectrometer. Anal. Chem. 2014, 86 (11), 5526-34), herein we validated this method by varying the protein/trypsin ratios via serial dilutions. Our data demonstrated that PRM with SILAC histones as the internal standards allowed reproducible measurements of histone H3/H4 acetylation and methylation in the samples whose histone contents differ at least one-order of magnitude. The method was further validated by histones isolated from histone H3 K36 trimethyltransferase SETD2 knockout mouse embryonic fibroblasts (MEF) cells. Furthermore, histone acetylation and methylation in human neural stem cells (hNSC) treated with ascorbic acid phosphate (AAP) were measured by this method, revealing that H3 K36 trimethylation was significantly down-regulated by 6 days of treatment with vitamin C. PMID:26356480

  4. A non-invasive method for in situ quantification of subpopulation behaviour in mixed cell culture.

    PubMed

    MacArthur, Ben D; Tare, Rahul S; Please, Colin P; Prescott, Philip; Oreffo, Richard O C

    2006-02-22

    Ongoing advances in quantitative molecular- and cellular-biology highlight the need for correspondingly quantitative methods in tissue-biology, in which the presence and activity of specific cell-subpopulations can be assessed in situ. However, many experimental techniques disturb the natural tissue balance, making it difficult to draw realistic conclusions concerning in situ cell behaviour. In this study, we present a widely applicable and minimally invasive method which combines fluorescence cell labelling, retrospective image analysis and mathematical data processing to detect the presence and activity of cell subpopulations, using adhesion patterns in STRO-1 immunoselected human mesenchymal populations and the homogeneous osteoblast-like MG63 continuous cell line as an illustration. Adhesion is considered on tissue culture plastic and fibronectin surfaces, using cell area as a readily obtainable and individual cell specific measure of spreading. The underlying statistical distributions of cell areas are investigated and mappings between distributions are examined using a combination of graphical and non-parametric statistical methods. We show that activity can be quantified in subpopulations as small as 1% by cell number, and outline behaviour of significant subpopulations in both STRO-1+/- fractions. This method has considerable potential to understand in situ cell behaviour and thus has wide applicability, for example in developmental biology and tissue engineering. PMID:16849218

  5. Uncertainty Quantification of Prompt Fission Neutron Spectra Using the Unified Monte Carlo Method

    NASA Astrophysics Data System (ADS)

    Rising, M. E.; Talou, P.; Prinja, A. K.

    2014-04-01

    In the ENDF/B-VII.1 nuclear data library, the existing covariance evaluations of the prompt fission neutron spectra (PFNS) were computed by combining the available experimental differential data with theoretical model calculations, relying on the use of a first-order linear Bayesan approach, the Kalman filter. This approach assumes that the theoretical model response to changes in input model parameters be linear about the a priori central values. While the Unified Monte Carlo (UMC) method remains a Bayesian approach, like the Kalman filter, this method does not make any assumption about the linearity of the model response or shape of the a posteriori distribution of the parameters. By sampling from a distribution centered about the a priori model parameters, the UMC method computes the moments of the a posteriori parameter distribution. As the number of samples increases, the statistical noise in the computed a posteriori moments decrease and an appropriately converged solution corresponding to the true mean of the a posteriori PDF results. The UMC method has been successfully implemented using both a uniform and Gaussian sampling distribution and has been used for the evaluation of the PFNS and its associated uncertainties. While many of the UMC results are similar to the first-order Kalman filter results, significant differences are shown when experimental data are excluded from the evaluation process. When experimental data are included a few small nonlinearities are present in the high outgoing energy tail of the PFNS.

  6. Quantification of myocardial perfusion using CMR with a radial data acquisition: comparison with a dual-bolus method

    PubMed Central

    2010-01-01

    Background Quantitative estimates of myocardial perfusion generally require accurate measurement of the arterial input function (AIF). The saturation of signal intensity in the blood that occurs with most doses of contrast agent makes obtaining an accurate AIF challenging. This work seeks to evaluate the performance of a method that uses a radial k-space perfusion sequence and multiple saturation recovery times (SRT) to quantify myocardial perfusion with cardiovascular magnetic resonance (CMR). Methods Perfusion CMR was performed at 3 Tesla with a saturation recovery radial turboFLASH sequence with 72 rays. Fourteen subjects were given a low dose (0.004 mmol/kg) of dilute (1/5 concentration) contrast agent (Gd-BOPTA) and then a higher non-dilute dose of the same volume (0.02 mmol/kg). AIFs were calculated from the blood signal in three sub-images with differing effective saturation recovery times. The full and sub-images were reconstructed iteratively with a total variation constraint. The images from the full 72 ray data were processed to obtain six tissue enhancement curves in two slices of the left ventricle in each subject. A 2-compartment model was used to determine absolute flows Results The proposed multi-SRT method resulted in AIFs that were similar to those obtained with the dual-bolus method. Myocardial blood flow (MBF) estimates from the dual-bolus and the multi-SRT methods were related by MBFmulti-SRT = 0.85MBFdual-bolus + 0.18 (r = 0.91). Conclusions The multi-SRT method, which uses a radial k-space perfusion sequence, can be used to obtain an accurate AIF and thus quantify myocardial perfusion for doses of contrast agent that result in a relatively saturated AIF. PMID:20653961

  7. Development of an electrospray LC-MS/MS method for quantification of Δ(9) -tetrahydrocannabinol and its main metabolite in oral fluid.

    PubMed

    Bylda, Caroline; Leinenbach, Andreas; Thiele, Roland; Kobold, Uwe; Volmer, Dietrich A

    2012-01-01

    A fast and sensitive reference method for quantification of Δ(9) -tetrahydrocannabinol (THC) and its main metabolite 11-nor-9-carboxy-Δ(9) -tetrahydrocannabinol (THCCOOH) in oral fluid is described in this study. Samples were collected using an oral specimen collection device, followed by solid-phase extraction and liquid chromatography-tandem mass spectrometry analysis. Chromatographic separation of the analytes was achieved by gradient elution on a reversed-phase column with subsequent detection by electrospray triple quadrupole mass spectrometry in positive ionization multiple reaction monitoring mode. Quantification was performed by means of deuterated analogues of the analytes as internal standards. Total run time of the assay was 12 min. The method allowed sensitive quantification of both analytes at a limit of quantification of 0.2 ng/ml. This sensitivity is essential for analysis of samples collected with the Intercept Oral Fluid Collection device (OraSure) and an assay for simultaneous quantification of THC and THCCOOH in saliva has not yet been described. The calibration curves for THC and THCCOOH were linear in the range between 0.25 and 8 ng/ml (r(2)  > 0.99). Ion suppression effects from endogenous or exogenous interferences were investigated using selected model substances (albumin, ascorbic acid, bilirubin, hemoglobin, breath spray, cigarette, chewing gum, chewing tobacco, candy, tooth whitening, and Tums antacid). These substances were chosen because of the high probability of their presence in the collected samples. None of the 11 endogenous model interferences altered the accuracy of analysis, demonstrating good robustness of the method with respect to interferences in common hygiene products, medicine, tobacco and naturally occurring endogenous substances. PMID:22374692

  8. Determination of absolute threshold and just noticeable difference in the sensory perception of pungency.

    PubMed

    Orellana-Escobedo, L; Ornelas-Paz, J J; Olivas, G I; Guerrero-Beltran, J A; Jimenez-Castro, J; Sepulveda, D R

    2012-03-01

    Absolute threshold and just noticeable difference (JND) were determined for the perception of pungency using chili pepper in aqueous solutions. Absolute threshold and JND were determined using 2 alternative forced-choice sensory tests tests. High-performance liquid chromatography technique was used to determine capsaicinoids concentration in samples used for sensory analysis. Sensory absolute threshold was 0.050 mg capsaicinoids/kg sample. Five JND values were determined using 5 reference solutions with different capsaicinoids concentration. JND values changed proportionally as capsaicinoids concentration of the reference sample solutions changed. Weber fraction remained stable for the first 4 reference capsaicinoid solutions (0.05, 0.11, 0.13, and 0.17 mg/kg) but changed when the most concentrated reference capsaicinoids solution was used (0.23 mg/kg). Quantification limit for instrumental analysis was 1.512 mg/kg capsaicinoids. Sensory methods employed in this study proved to be more sensitive than instrumental methods. Practical Application: A better understanding of the process involved in the sensory perception of pungency is currently required because "hot" foods are becoming more popular in western cuisine. Absolute thresholds and differential thresholds are useful tools in the formulation and development of new food products. These parameters may help in defining how much chili pepper is required in a formulated product to ensure a perceptible level of pungency, as well as in deciding how much more chili pepper is required in a product to produce a perceptible increase in its pungency. PMID:22384966

  9. An efficient and scalable extraction and quantification method for algal derived biofuel.

    PubMed

    Lohman, Egan J; Gardner, Robert D; Halverson, Luke; Macur, Richard E; Peyton, Brent M; Gerlach, Robin

    2013-09-01

    Microalgae are capable of synthesizing a multitude of compounds including biofuel precursors and other high value products such as omega-3-fatty acids. However, accurate analysis of the specific compounds produced by microalgae is important since slight variations in saturation and carbon chain length can affect the quality, and thus the value, of the end product. We present a method that allows for fast and reliable extraction of lipids and similar compounds from a range of algae, followed by their characterization using gas chromatographic analysis with a focus on biodiesel-relevant compounds. This method determines which range of biologically synthesized compounds is likely responsible for each fatty acid methyl ester (FAME) produced; information that is fundamental for identifying preferred microalgae candidates as a biodiesel source. Traditional methods of analyzing these precursor molecules are time intensive and prone to high degrees of variation between species and experimental conditions. Here we detail a new method which uses microwave energy as a reliable, single-step cell disruption technique to extract lipids from live cultures of microalgae. After extractable lipid characterization (including lipid type (free fatty acids, mono-, di- or tri-acylglycerides) and carbon chain length determination) by GC-FID, the same lipid extracts are transesterified into FAMEs and directly compared to total biodiesel potential by GC-MS. This approach provides insight into the fraction of total FAMEs derived from extractable lipids compared to FAMEs derived from the residual fraction (i.e. membrane bound phospholipids, sterols, etc.). This approach can also indicate which extractable lipid compound, based on chain length and relative abundance, is responsible for each FAME. This method was tested on three species of microalgae; the marine diatom Phaeodactylum tricornutum, the model Chlorophyte Chlamydomonas reinhardtii, and the freshwater green alga Chlorella vulgaris

  10. Quantification of serotonin O-sulphate by LC-MS method in plasma of healthy volunteers

    PubMed Central

    Lozda, Raimonds; Purviņš, Indulis

    2014-01-01

    The objective of this study was to test the hypothesis that serotonin O-sulphate (5-HT-SO4) could be quantified in human plasma using modern liquid chromatography–mass spectrometry (LC-MS) method as well as develop and validate that method. First, a suitable LC-MS method for detection of 5-HT-SO4 in human plasma samples was developed and validated. Second, a Pilot phase involving four healthy volunteers was executed, where a basal plasma level of 5-HT-SO4 was measured for all subjects and for one after the intake of 100 mg of a 5-hydroxytryptophan (5-HTP) -containing food supplement used to promote serotonergic stimulation of the central nervous system. The basal level of 0.9–2.8 ng/mL of 5-HT-SO4 was observed. The changes of plasma 5HT-O-SO4 showed 1.2 ng/mL before and 22.6 ng/mL 1 h after stimulation. Finally, nine healthy volunteers were selected for the Study phase, where a basal plasma level of 5-HT-SO4 was measured before and after the intake of 5-HTP. One hour after stimulation, six study subjects showed a decrease in 5-HT-SO4 levels while three subjects showed an increase. The changes of plasma 5HT-O-SO4 from the Study phase showed an average 5-HT-SO4 level of 19.2 ng/mL before and 15.7 ng/mL 1 h after stimulation indicating ability of method to emphasize quantitative changes. This was the first study in which naturally occurring 5-HT-SO4 was detected in the samples of human plasma obtained from healthy volunteers. The method developed herein is specific to the measurement of 5-HT-SO4, sensitive enough to quantify intra-individual changes in the samples of plasma and opens up new possibilities to evaluate pathways of serotonin metabolism by minimally invasive methods. The discovery of novel biomarkers using such approaches is increasingly required to expedite development of mechanism-based therapeutics and patient stratification. PMID:24782770

  11. Rapid method of quantification of tight-junction organization using image analysis.

    PubMed

    Terryn, Christine; Sellami, Mehdi; Fichel, Caroline; Diebold, Marie-Danielle; Gangloff, Sophie; Le Naour, Richard; Polette, Myriam; Zahm, Jean-Marie

    2013-02-01

    The spatial organization of proteins in a cell population or in tissues is an important parameter to study the functionality of biological specimens. In this article, we have focused on tight junctions which form network-like features in immunofluorescence microscopy images. Usually, the organization or disorganization of tight junctions is noticed qualitatively. The aim of this article is to present a simple method to quantify the organization level of tight junction network using image analysis with a dedicated macro developed with Image J software. The method has been validated with simulated images displaying regular decrease of network organization. Then, the macro has been applied to immunofluorescence microscopy images of cells in culture and of tissue sections. PMID:23212973

  12. Quantification of refractory organic substances in freshwaters: further insight into the response of the voltammetric method.

    PubMed

    Quentel, François; Filella, Montserrat

    2008-11-01

    A recently published method for quantifying refractory organic matter (often referred to as humic substances) in freshwaters was applied to a wide range of International Humic Substance Society (IHSS) humic compounds in order to (i) gain a better understanding of the mechanism of the voltammetric response which is the basis of the analytical method and (ii) provide guidance on choosing the optimal standard to be used. At the same time, the sensitivity of the technique has been increased by switching from the pulse mode initially proposed to the square-wave mode. The results obtained show that (i) differences in adsorption onto the electrode rather than differences in complexation strength are responsible for the differences in the intensity of the signal obtained for the different humic compounds, (ii) carboxylate, N- and S-containing groups do not play a role in the voltammetric signal. PMID:18791870

  13. The MESERAN Method: Rapid Quantification of Non-Volatile Organic Residue (NVOR)

    SciTech Connect

    Benkovich, M.G.

    2002-06-13

    The precision analytical technique known as MESERAN Analysis permits quantitative measurement of the level of preexisting nonvolatile organic residue (NVOR) on a substrate from <1 nanogram (ng)/cm{sup 2} to > 100 micrograms ({micro}g)/cm{sup 2} in 2 minutes. MESERAN Analysis is also applicable to determining NVOR in solvents and solvent extracts. The MESERAN method is able to quantify organic contamination levels down to and below 1 ng by depositing as little as 10 microliters ({micro}L) of solvent containing a known amount of contamination on a clean substrate, allowing it to evaporate, and measuring the evaporated residue. The method will be described in detail and NVOR measurements determined from MESERAN data will be presented.

  14. LC-MS quantification of protein drugs: validating protein LC-MS methods with predigestion immunocapture.

    PubMed

    Duggan, Jeffrey; Ren, Bailuo; Mao, Yan; Chen, Lin-Zhi; Philip, Elsy

    2016-09-01

    A refinement of protein LC-MS bioanalysis is to use predigestion immunoaffinity capture to extract the drug from matrix prior to digestion. Because of their increased sensitivity, such hybrid assays have been successfully validated and applied to a number of clinical studies; however, they can also be subject to potential interferences from antidrug antibodies, circulating ligands or other matrix components specific to patient populations and/or dosed subjects. The purpose of this paper is to describe validation experiments that measure immunocapture efficiency, digestion efficiency, matrix effect and selectivity/specificity that can be used during method optimization and validation to test the resistance of the method to these potential interferences. The designs and benefits of these experiments are discussed in this report using an actual assay case study. PMID:27532431

  15. New miRNA labeling method for bead-based quantification

    PubMed Central

    2010-01-01

    Background microRNAs (miRNAs) are small single-stranded non-coding RNAs that act as crucial regulators of gene expression. Different methods have been developed for miRNA expression profiling in order to better understand gene regulation in normal and pathological conditions. miRNAs expression values obtained from large scale methodologies such as microarrays still need a validation step with alternative technologies. Results Here we have applied with an innovative approach, the Luminex® xMAP™ technology validate expression data of differentially expressed miRNAs obtained from high throughput arrays. We have developed a novel labeling system of small RNA molecules (below 200 nt), optimizing the sensitive cloning method for miRNAs, termed miRNA amplification profiling (mRAP). The Luminex expression patterns of three miRNAs (miR-23a, miR-27a and miR-199a) in seven different cell lines have been validated by TaqMan miRNA assay. In all cases, bead-based meas were confirmed by the data obtained by TaqMan and microarray technologies. Conclusions We demonstrate that the measure of individual miRNA by the bead-based method is feasible, high speed, sensitive and low cost. The Luminex® xMAP™ technology also provides flexibility, since the central reaction can be scaled up with additional miRNA capturing beads, allowing validation of many differentially expressed miRNAs obtained from microarrays in a single experiment. We propose this technology as an alternative method to qRT-PCR for validating miRNAs expression data obtained with high-throughput technologies. PMID:20553585

  16. Comparison of Sample and Detection Quantification Methods for Salmonella Enterica from Produce

    NASA Technical Reports Server (NTRS)

    Hummerick, M. P.; Khodadad, C.; Richards, J. T.; Dixit, A.; Spencer, L. M.; Larson, B.; Parrish, C., II; Birmele, M.; Wheeler, Raymond

    2014-01-01

    The purpose of this study was to identify and optimize fast and reliable sampling and detection methods for the identification of pathogens that may be present on produce grown in small vegetable production units on the International Space Station (ISS), thus a field setting. Microbiological testing is necessary before astronauts are allowed to consume produce grown on ISS where currently there are two vegetable production units deployed, Lada and Veggie.

  17. Simple colorimetric method for quantification of surface carboxy groups on polymer particles.

    PubMed

    Hennig, Andreas; Hoffmann, Angelika; Borcherding, Heike; Thiele, Thomas; Schedler, Uwe; Resch-Genger, Ute

    2011-06-15

    We present a novel, simple, and fast colorimetric method to quantify the total number of carboxy groups on polymer microparticle and nanoparticle surfaces. This method exploits that small divalent transition metal cations (M(2+) = Ni(2+), Co(2+), Cd(2+)) are efficiently bound to these surface functional groups, which allows their extraction by a single centrifugation step. Remaining M(2+) in the supernatant is subsequently quantified spectrophotometrically after addition of the metal ion indicator pyrocatechol violet, for which Ni(2+) was identified to be the most suitable transition metal cation. We demonstrate that the difference between added and detected M(2+) is nicely correlated to the number of surface carboxy groups as determined by conductometry, thereby affording a validated measure for the trueness of this procedure. The variation coefficient of ~5% found in reproducibility studies underlines the potential of this novel method that can find conceivable applications for the characterization of different types of poly(carboxylic acid)-functionalized materials, e.g., for quality control by manufacturers of such materials. PMID:21561064

  18. Simultaneous quantification of paracetamol, acetylsalicylic acid and papaverine with a validated HPLC method.

    PubMed

    Kalmár, Eva; Gyuricza, Anett; Kunos-Tóth, Erika; Szakonyi, Gerda; Dombi, György

    2014-01-01

    Combined drug products have the advantages of better patient compliance and possible synergic effects. The simultaneous application of several active ingredients at a time is therefore frequently chosen. However, the quantitative analysis of such medicines can be challenging. The aim of this study is to provide a validated method for the investigation of a multidose packed oral powder that contained acetylsalicylic acid, paracetamol and papaverine-HCl. Reversed-phase high-pressure liquid chromatography was used. The Agilent Zorbax SB-C18 column was found to be the most suitable of the three different stationary phases tested for the separation of the components of this sample. The key parameters in the method development (apart from the nature of the column) were the pH of the aqueous phase (set to 3.4) and the ratio of the organic (acetonitrile) and the aqueous (25 mM phosphate buffer) phases, which was varied from 7:93 (v/v) to 25:75 (v/v) in a linear gradient, preceded by an initial hold. The method was validated: linearity, precision (repeatability and intermediate precision), accuracy, specificity and robustness were all tested, and the results met the ICH guidelines. PMID:24344050

  19. Development and validation of RP-HPLC method for quantification of glipizide in biological macromolecules.

    PubMed

    Pani, Nihar Ranjan; Acharya, Sujata; Patra, Sradhanjali

    2014-04-01

    Glipizide (GPZ) has been widely used in the treatment of type-2 diabetics as insulin secretogague. Multiunit chitosan based GPZ floating microspheres was prepared by ionotropic gelation method for gastroretentive delivery using sodiumtripolyphosphate as cross-linking agent. Pharmacokinetic study of microspheres was done in rabbit and plasma samples were analyzed by a newly developed and validated high-performance liquid chromatographic method. Method was developed on Hypersil ODS-18 column using a mobile phase of 10mM phosphate buffer (pH, 3.5) and methanol (25:75, v/v). Elute was monitored at 230 nm with a flow rate of 1 mL/min. Calibration curve was linear over the concentration range of 25.38-2046.45 ng/mL. Retention times of GPZ and internal standard (gliclazide) were 7.32 and 9.02 min respectively. Maximum plasma drug concentration, area under the plasma drug concentration-time curve and elimination half life for GPZ floating microspheres were 2.88±0.29 μg mL(-1), 38.46±2.26 μg h mL(-1) and 13.55±1.36 h respectively. When the fraction of drug dissolved from microspheres in pH 7.4 was plotted against the fraction of drug absorbed, a linear correlation (R(2)=0.991) was obtained in in vitro and in vivo correlation study. PMID:24418334

  20. Validated HPTLC Method for Quantification of Luteolin and Apigenin in Premna mucronata Roxb., Verbenaceae

    PubMed Central

    Patel, Nayan G.; Patel, Kalpana G.; Patel, Kirti V.; Gandhi, Tejal R.

    2015-01-01

    A simple, rapid, and precise high-performance thin-layer chromatographic method was developed for quantitative estimation of luteolin and apigenin in Premna mucronata Roxb., fami