Simultaneous determination of antidementia drugs in human plasma: Procedure transfer from HPLC-MS to UPLC-MS/MS.

A previously developed high performance liquid chromatography mass spectrometry (HPLC-MS) procedure for the simultaneous determination of antidementia drugs, including donepezil, galantamine, memantine, rivastigmine and its metabolite NAP 226-90, was transferred to an ultra performance liquid chromatography system coupled to a tandem mass spectrometer (UPLC-MS/MS). The drugs and their internal standards ([(2)H(7)]-donepezil, [(13)C,(2)H(3)]-galantamine, [(13)C(2),(2)H(6)]-memantine, [(2)H(6)]-rivastigmine) were extracted from 250μL human plasma by protein precipitation with acetonitrile. Chromatographic separation was achieved on a reverse phase column (BEH C18 2.1mm×50mm; 1.7μm) with a gradient elution of an ammonium acetate buffer at pH 9.3 and acetonitrile at a flow rate of 0.4mL/min and an overall run time of 4.5min. The analytes were detected on a tandem quadrupole mass spectrometer operated in positive electrospray ionization mode, and quantification was performed using multiple reaction monitoring. The method was validated according to the recommendations of international guidelines over a calibration range of 1-300ng/mL for donepezil, galantamine and memantine, and 0.2-50ng/mL for rivastimgine and NAP 226-90. The trueness (86-108%), repeatability (0.8-8.3%), intermediate precision (2.3-10.9%) and selectivity of the method were found to be satisfactory. Matrix effects variability was inferior to 15% for the analytes and inferior to 5% after correction by internal standards. A method comparison was performed with patients' samples showing similar results between the HPLC-MS and UPLC-MS/MS procedures. Thus, this validated UPLC-MS/MS method allows to reduce the required amount of plasma, to use a simplified sample preparation, and to obtain a higher sensitivity and specificity with a much shortened run-time.

Estimation of percentage body fat in 6- to 13-year-old children by skinfold thickness, body mass index and waist circumference.

We evaluated the accuracy of skinfold thicknesses, BMI and waist circumference for the prediction of percentage body fat (PBF) in a representative sample of 372 Swiss children aged 6-13 years. PBF was measured using dual-energy X-ray absorptiometry. On the basis of a preliminary bootstrap selection of predictors, seven regression models were evaluated. All models included sex, age and pubertal stage plus one of the following predictors: (1) log-transformed triceps skinfold (logTSF); (2) logTSF and waist circumference; (3) log-transformed sum of triceps and subscapular skinfolds (logSF2); (4) log-transformed sum of triceps, biceps, subscapular and supra-iliac skinfolds (logSF4); (5) BMI; (6) waist circumference; (7) BMI and waist circumference. The adjusted determination coefficient (R² adj) and the root mean squared error (RMSE; kg) were calculated for each model. LogSF4 (R² adj 0.85; RMSE 2.35) and logSF2 (R² adj 0.82; RMSE 2.54) were similarly accurate at predicting PBF and superior to logTSF (R² adj 0.75; RMSE 3.02), logTSF combined with waist circumference (R² adj 0.78; RMSE 2.85), BMI (R² adj 0.62; RMSE 3.73), waist circumference (R² adj 0.58; RMSE 3.89), and BMI combined with waist circumference (R² adj 0.63; RMSE 3.66) (P < 0.001 for all values of R² adj). The finding that logSF4 was only modestly superior to logSF2 and that logTSF was better than BMI and waist circumference at predicting PBF has important implications for paediatric epidemiological studies aimed at disentangling the effect of body fat on health outcomes.

A statistical methodology for the comparison of blue gel pen inks analyzed by laser desorption/ionization mass spectrometry

A statistical methodology for the objective comparison of LDI-MS mass spectra of blue gel pen inks was evaluated. Thirty-three blue gel pen inks previously studied by RAMAN were analyzed directly on the paper using both positive and negative mode. The obtained mass spectra were first compared using relative areas of selected peaks using the Pearson correlation coefficient and the Euclidean distance. Intra-variability among results from one ink and inter-variability between results from different inks were compared in order to choose a differentiation threshold minimizing the rate of false negative (i.e. avoiding false differentiation of the inks). This yielded a discriminating power of up to 77% for analysis made in the negative mode. The whole mass spectra were then compared using the same methodology, allowing for a better DP in the negative mode of 92% using the Pearson correlation on standardized data. The positive mode results generally yielded a lower differential power (DP) than the negative mode due to a higher intra-variability compared to the inter-variability in the mass spectra of the ink samples.

No use for waist-for-height ratio in addition to body mass index to identify children with elevated blood pressure.

Abstract Background. In children, waist-for-height ratio (WHtR) has been proposed to identify subjects at higher risk of cardiovascular diseases. The utility of WHtR to identify children with elevated blood pressure (BP) is unclear. Design. Cross-sectional population-based study of schoolchildren. Methods. Weight, height, waist circumference and BP were measured in all sixth-grade schoolchildren of the canton de Vaud (Switzerland) in 2005/06. WHtR was computed as waist [cm]/height [cm]. Elevated BP was defined according to sex-, age- and height-specific US reference data. The area under the receiver operating characteristic curve (AUC) statistic was computed to compare the ability of body mass index (BMI) z-score and WHtR, alone or in combination, to identify children with elevated BP. Results. 5207 children participated (76% response) [2621 boys, 2586 girls; mean (± SD) age, 12.3 ± 0.5 years; range: 10.1-14.9]. The prevalence of elevated BP was 11%. Mean WHtR was 0.44 ± 0.05 (range: 0.29- 0.77) and 11% had high WHtR (> 0.5). BMI z-score and WHtR were strongly correlated (Spearman correlation coefficient r = 0.76). Both indices were positively associated with elevated BP. AUCs for elevated BP was relatively low for BMI z-score (0.62) or for WHtR (0.62), and was not substantially improved when both indices were considered together (0.63). Conclusions. The ability of BMI z-score or WHtR to identify children aged 10-14 with elevated BP was weak. Adding WHtR did not confer additional discriminative power to BMI alone. These findings do not support the measurement of WHtR in addition to BMI to identify children with elevated BP.

Dielectric constant obtained from TDR and volumetric moisture of soils in southern Brazil

Soil moisture is the property which most greatly influences the soil dielectric constant, which is also influenced by soil mineralogy. The aim of this study was to determine mathematical models for soil moisture and the dielectric constant (Ka) for a Hapludalf, two clayey Hapludox and a very clayey Hapludox and test the reliability of universal models, such as those proposed by Topp and Ledieu and their co-workers in the 80's, and specific models to estimate soil moisture with a TDR. Soil samples were collected from the 0 to 0.30 m layer, sieved through a mesh of 0.002 m diameter and packed in PVC cylinders with a 0.1 m diameter and 0.3 m height. Seven samples of each soil class were saturated by capillarity and a probe composed of two rods was inserted in each one of them. Moisture readings began with the saturated soil and concluded when the soil was near permanent wilting point. In each step, the samples were weighed on a precision scale to calculate volumetric moisture. Linear and polynomial models were adjusted for each soil class and for all soils together between soil moisture and the dielectric constant. Accuracy of the models was evaluated by the coefficient of determination, the standard error of estimate and the 1:1 line. The models proposed by Topp and Ledieu and their co-workers were not adequate for estimating the moisture in the soil classes studied. The adjusted linear and polynomial models for the entire set of data of the four soil classes did not have sufficient accuracy for estimating soil moisture. The greater the soil clay and Fe oxide content, the greater the dielectric constant of the medium for a given volumetric moisture. The specific models, θ = 0.40283 - 0.04231 Ka + 0.00194 Ka² - 0.000022 Ka³ (Hapludox) θ = 0.01971 + 0.02902 Ka - 0.00086 Ka² + 0.000012 Ka³ (Hapludox -PF), θ = 0.01692 - 0.00507 Ka (Hapludalf) and θ = 0.08471 + 0.01145 Ka (Hapludox-CA), show greater accuracy and reliability for estimating soil moisture in the soil classes studied.

PEDO-TRANSFER FUNCTIONS FOR ESTIMATING SOIL BULK DENSITY IN CENTRAL AMAZONIA

Under field conditions in the Amazon forest, soil bulk density is difficult to measure. Rigorous methodological criteria must be applied to obtain reliable inventories of C stocks and soil nutrients, making this process expensive and sometimes unfeasible. This study aimed to generate models to estimate soil bulk density based on parameters that can be easily and reliably measured in the field and that are available in many soil-related inventories. Stepwise regression models to predict bulk density were developed using data on soil C content, clay content and pH in water from 140 permanent plots in terra firme (upland) forests near Manaus, Amazonas State, Brazil. The model results were interpreted according to the coefficient of determination (R2) and Akaike information criterion (AIC) and were validated with a dataset consisting of 125 plots different from those used to generate the models. The model with best performance in estimating soil bulk density under the conditions of this study included clay content and pH in water as independent variables and had R2 = 0.73 and AIC = -250.29. The performance of this model for predicting soil density was compared with that of models from the literature. The results showed that the locally calibrated equation was the most accurate for estimating soil bulk density for upland forests in the Manaus region.

Development and validation of a gas chromatography-negative chemical ionization tandem mass spectrometry method for the determination of ethyl glucuronide in hair and its application to forensic toxicology.

Ethyl glucuronide (EtG) is a minor and direct metabolite of ethanol. EtG is incorporated into the growing hair allowing retrospective investigation of chronic alcohol abuse. In this study, we report the development and the validation of a method using gas chromatography-negative chemical ionization tandem mass spectrometry (GC-NCI-MS/MS) for the quantification of EtG in hair. EtG was extracted from about 30 mg of hair by aqueous incubation and purified by solid-phase extraction (SPE) using mixed mode extraction cartridges followed by derivation with perfluoropentanoic anhydride (PFPA). The analysis was performed in the selected reaction monitoring (SRM) mode using the transitions m/z 347-->163 (for the quantification) and m/z 347-->119 (for the identification) for EtG, and m/z 352-->163 for EtG-d(5) used as internal standard. For validation, we prepared quality controls (QC) using hair samples taken post mortem from 2 subjects with a known history of alcoholism. These samples were confirmed by a proficiency test with 7 participating laboratories. The assay linearity of EtG was confirmed over the range from 8.4 to 259.4 pg/mg hair, with a coefficient of determination (r(2)) above 0.999. The limit of detection (LOD) was estimated with 3.0 pg/mg. The lower limit of quantification (LLOQ) of the method was fixed at 8.4 pg/mg. Repeatability and intermediate precision (relative standard deviation, RSD%), tested at 4 QC levels, were less than 13.2%. The analytical method was applied to several hair samples obtained from autopsy cases with a history of alcoholism and/or lesions caused by alcohol. EtG concentrations in hair ranged from 60 to 820 pg/mg hair.

Practical considerations for improving the productivity of mass spectrometry-based proteomics.

Mass spectrometry (MS) is currently the most sensitive and selective analytical technique for routine peptide and protein structure analysis. Top-down proteomics is based on tandem mass spectrometry (MS/ MS) of intact proteins, where multiply charged precursor ions are fragmented in the gas phase, typically by electron transfer or electron capture dissociation, to yield sequence-specific fragment ions. This approach is primarily used for the study of protein isoforms, including localization of post-translational modifications and identification of splice variants. Bottom-up proteomics is utilized for routine high-throughput protein identification and quantitation from complex biological samples. The proteins are first enzymatically digested into small (usually less than ca. 3 kDa) peptides, these are identified by MS or MS/MS, usually employing collisional activation techniques. To overcome the limitations of these approaches while combining their benefits, middle-down proteomics has recently emerged. Here, the proteins are digested into long (3-15 kDa) peptides via restricted proteolysis followed by the MS/MS analysis of the obtained digest. With advancements of high-resolution MS and allied techniques, routine implementation of the middle-down approach has been made possible. Herein, we present the liquid chromatography (LC)-MS/MS-based experimental design of our middle-down proteomic workflow coupled with post-LC supercharging.

Fate of the herbicide glyphosate and its metabolite AMPA in soils and their transfer to surface waters: A multi-scale approach in the Lavaux vineyards, western Switzerland

The use of herbicides in agriculture may lead to environmental problems, such as surface water pollution, with a potential risk for aquatic organisms. The herbicide glyphosate is the most used active ingredient in the world and in Switzerland. In the Lavaux vineyards it is nearly the only molecule applied. This work aimed at studying its fate in soils and its transfer to surface waters, using a multi-scale approach: from molecular (10-9 m) and microscopic scales (10-6 m), to macroscopic (m) and landscape ones (103 m). First of all, an analytical method was developed for the trace level quantification of this widely used herbicide and its main by-product, aminomethylphosphonic acid (AMPA). Due to their polar nature, their derivatization with 9-fluorenylmethyl chloroformate (FMOC-Cl) was done prior to their concentration and purification by solid phase extraction. They were then analyzed by ultra performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS). The method was tested in different aqueous matrices with spiking tests and validated for the matrix effect correction in relevant environmental samples. Calibration curves established between 10 and 1000ng/l showed r2 values above 0.989, mean recoveries varied between 86 and 133% and limits of detection and quantification of the method were as low as 5 and 10ng/l respectively. At the parcel scale, two parcels of the Lavaux vineyard area, located near the Lutrive River at 6km to the east of Lausanne, were monitored to assess to which extent glyphosate and AMPA were retained in the soil or exported to surface waters. They were equipped at their bottom with porous ceramic cups and runoff collectors, which allowed retrieving water samples for the growing seasons 2010 and 2011. Results revealed that the mobility of glyphosate and AMPA in the unsaturated zone was likely driven by the precipitation regime and the soil characteristics, such as slope, porosity structure and layer permeability discrepancy. Elevated glyphosate and AMPA concentrations were measured at 60 and 80 cm depth at parcel bottoms, suggesting their infiltration in the upper parts of the parcels and the presence of preferential flow in the studied parcels. Indeed, the succession of rainy days induced the gradual saturation of the soil porosity, leading to rapid infiltration through macropores, as well as surface runoff formation. Furthermore, the presence of more impervious weathered marls at 100 cm depth induced throughflows, the importance of which for the lateral transport of the herbicide molecules was determined by the slope steepness. Important rainfall events (>10 mm/day) were clearly exporting molecules from the soil top layer, as indicated by important concentrations in runoff samples. A mass balance showed that total loss (10-20%) mainly occurred through surface runoff (96%) and, to a minor extent, by throughflows in soils (4%), with subsequent exfiltration to surface waters. Observations made in the Lutrive River revealed interesting details of glyphosate and AMPA dynamics in urbanized landscapes, such as the Lavaux vineyards. Indeed, besides their physical and chemical properties, herbicide dynamics at the catchment level strongly depend on application rates, precipitation regime, land use and also on the presence of drains or constructed channels. Elevated concentrations, up to 4970 ng/l, observed just after the application, confirmed the diffuse export of these compounds from the vineyard area by surface runoff during main rain events. From April to September 2011, a total load of 7.1 kg was calculated, with 85% coming from vineyards and minor urban sources and 15% from arable crops. Small vineyard surfaces could generate high concentrations of herbicides and contribute considerably to the total load calculated at the outlet, due to their steep slopes (~10%). The extrapolated total amount transferred yearly from the Lavaux vineyards to the Lake of Geneva was of 190kg. At the molecular scale, the possible involvement of dissolved organic matter (DOM) in glyphosate and copper transport was studied using UV/Vis fluorescence spectroscopy. Combined with parallel factor (PARAFAC) analysis, this technique allowed characterizing DOM of soil and surface water samples from the studied vineyard area. Glyphosate concentrations were linked to the fulvic-like spectroscopic signature of DOM in soil water samples, as well as to copper, suggesting the formation of ternary complexes. In surface water samples, its concentrations were also correlated to copper ones, but not in a significant way to the fulvic-like signature. Quenching experiments with standards confirmed field tendencies in the laboratory, with a stronger decrease in fluorescence intensity for fulvic-like fluorophore than for more aromatic ones. Lastly, based on maximum concentrations measured in the river, an environmental risk for these compounds was assessed, using laboratory tests and ecotoxicity data from the literature. In our case and with the methodology applied, the risk towards aquatic species was found negligible (RF<1).

Efficiency of informational transfer in regular and complex networks

We analyze the process of informational exchange through complex networks by measuring network efficiencies. Aiming to study nonclustered systems, we propose a modification of this measure on the local level. We apply this method to an extension of the class of small worlds that includes declustered networks and show that they are locally quite efficient, although their clustering coefficient is practically zero. Unweighted systems with small-world and scale-free topologies are shown to be both globally and locally efficient. Our method is also applied to characterize weighted networks. In particular we examine the properties of underground transportation systems of Madrid and Barcelona and reinterpret the results obtained for the Boston subway network.

Coolant Flow and Heat Transfer in Pebble-Bed Reactor Core

This thesis gathers knowledge about ongoing high-temperature reactor projects around the world. Methods for calculating coolant flow and heat transfer inside a pebble-bed reactor core are also developed. The thesis begins with the introduction of high-temperature reactors including the current state of the technology. Process heat applications that could use the heat from a high-temperature reactor are also introduced. A suitable reactor design with data available in literature is selected for the calculation part of the thesis. Commercial computational fluid dynamics software Fluent is used for the calculations. The pebble-bed is approximated as a packed-bed, which causes sink terms to the momentum equations of the gas flowing through it. A position dependent value is used for the packing fraction. Two different models are used to calculate heat transfer. First a local thermal equilibrium is assumed between the gas and solid phases and a single energy equation is used. In the second approach, separate energy equations are used for the phases. Information about steady state flow behavior, pressure loss, and temperature distribution in the core is obtained as results of the calculations. The effect of inlet mass flow rate to pressure loss is also investigated. Data found in literature and the results correspond each other quite well, considered the amount of simplifications in the calculations. The models developed in this thesis can be used to solve coolant flow and heat transfer in a pebble-bed reactor, although additional development and model validation is needed for better accuracy and reliability.

Mathematical models to describe the volumetric shrinkage rate of red beans during drying

The present study aimed to determine the volumetric shrinkage rate of bean (Phaseolus vulgaris L.) seeds during air-drying under different conditions of air, temperature and relative humidity, and to adjust several mathematical models to the empiric values observed, and select the one that best represents the phenomenon. Six mathematical models were adjusted to the experimental values to represent the phenomenon. It was determined the degree of adjustment of each model from the value of the coefficient of determination, the behavior of the distribution of the residuals, and the magnitude of the average relative and estimated errors. The rate of volumetric shrinkage that occurred in bean seeds during drying is between 25 and 37%. It basically depends on the final moisture content, regardless of the air conditions during drying. The Modified Bala & Woods' model best represented the process.

Construction and calibration of TDR probes for volumetric water content estimation in a Distroferric Red Latosol

The determination of volumetric water content of soils is an important factor in irrigation management. Among the indirect methods for estimating, the time-domain reflectometry (TDR) technique has received a significant attention. Like any other technique, it has advantages and disadvantages, but its greatest disadvantage is the need of calibration and high cost of acquisition. The main goal of this study was to establish a calibration model for the TDR equipment, Trase System Model 6050X1, to estimate the volumetric water content in a Distroferric Red Latosol. The calibration was carried out in a laboratory with disturbed soil samples under study, packed in PVC columns of a volume of 0.0078m³. The TDR probes were handcrafted with three rods and 0.20m long. They were vertically installed in soil columns, with a total of five probes per column and sixteen columns. The weightings were carried out in a digital scale, while daily readings of dielectric constant were obtained in TDR equipment. The linear model θν = 0.0103 Ka + 0.1900 to estimate the studied volumetric water content showed an excellent coefficient of determination (0.93), enabling the use of probes in indirect estimation of soil moisture.

Homogeneous assay technologies in drug screening: Quenching Resonance Energy Transfer (QRET) technique

Information gained from the human genome project and improvements in compound synthesizing have increased the number of both therapeutic targets and potential lead compounds. This has evolved a need for better screening techniques to have a capacity to screen number of compound libraries against increasing amount of targets. Radioactivity based assays have been traditionally used in drug screening but the fluorescence based assays have become more popular in high throughput screening (HTS) as they avoid safety and waste problems confronted with radioactivity. In comparison to conventional fluorescence more sensitive detection is obtained with time-resolved luminescence which has increased the popularity of time-resolved fluorescence resonance energy transfer (TR-FRET) based assays. To simplify the current TR-FRET based assay concept the luminometric homogeneous single-label utilizing assay technique, Quenching Resonance Energy Transfer (QRET), was developed. The technique utilizes soluble quencher to quench non-specifically the signal of unbound fraction of lanthanide labeled ligand. One labeling procedure and fewer manipulation steps in the assay concept are saving resources. The QRET technique is suitable for both biochemical and cell-based assays as indicated in four studies:1) ligand screening study of β2 -adrenergic receptor (cell-based), 2) activation study of Gs-/Gi-protein coupled receptors by measuring intracellular concentration of cyclic adenosine monophosphate (cell-based), 3) activation study of G-protein coupled receptors by observing the binding of guanosine-5’-triphosphate (cell membranes), and 4) activation study of small GTP binding protein Ras (biochemical). Signal-to-background ratios were between 2.4 to 10 and coefficient of variation varied from 0.5 to 17% indicating their suitability to HTS use.

Wind Turbine Direct-Drive Permanent-Magnet Generator with Direct Liquid Cooling for Mass Reduction

Today’s electrical machine technology allows increasing the wind turbine output power by an order of magnitude from the technology that existed only ten years ago. However, it is sometimes argued that high-power direct-drive wind turbine generators will prove to be of limited practical importance because of their relatively large size and weight. The limited space for the generator in a wind turbine application together with the growing use of wind energy pose a challenge for the design engineers who are trying to increase torque without making the generator larger. When it comes to high torque density, the limiting factor in every electrical machine is heat, and if the electrical machine parts exceed their maximum allowable continuous operating temperature, even for a short time, they can suffer permanent damage. Therefore, highly efficient thermal design or cooling methods is needed. One of the promising solutions to enhance heat transfer performances of high-power, low-speed electrical machines is the direct cooling of the windings. This doctoral dissertation proposes a rotor-surface-magnet synchronous generator with a fractional slot nonoverlapping stator winding made of hollow conductors, through which liquid coolant can be passed directly during the application of current in order to increase the convective heat transfer capabilities and reduce the generator mass. This doctoral dissertation focuses on the electromagnetic design of a liquid-cooled direct-drive permanent-magnet synchronous generator (LC DD-PMSG) for a directdrive wind turbine application. The analytical calculation of the magnetic field distribution is carried out with the ambition of fast and accurate predicting of the main dimensions of the machine and especially the thickness of the permanent magnets; the generator electromagnetic parameters as well as the design optimization. The focus is on the generator design with a fractional slot non-overlapping winding placed into open stator slots. This is an a priori selection to guarantee easy manufacturing of the LC winding. A thermal analysis of the LC DD-PMSG based on a lumped parameter thermal model takes place with the ambition of evaluating the generator thermal performance. The thermal model was adapted to take into account the uneven copper loss distribution resulting from the skin effect as well as the effect of temperature on the copper winding resistance and the thermophysical properties of the coolant. The developed lumpedparameter thermal model and the analytical calculation of the magnetic field distribution can both be integrated with the presented algorithm to optimize an LC DD-PMSG design. Based on an instrumented small prototype with liquid-cooled tooth-coils, the following targets have been achieved: experimental determination of the performance of the direct liquid cooling of the stator winding and validating the temperatures predicted by an analytical thermal model; proving the feasibility of manufacturing the liquid-cooled tooth-coil winding; moreover, demonstration of the objectives of the project to potential customers.