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PubMed2004

Field-enhanced sample injection for high-sensitivity analysis of peptides and proteins in capillary electrophoresis-mass spectrometry.

Field-enhanced sample injection (FESI) was used to improve the concentration sensitivity of a capillary electrophoresis (CE)-mass spectrometry (MS) system with sheath flow configuration. Using some bioactive peptides, more than 3000-fold improvement in signal was obtained, permitting analysis in the low nM (fmol/microl) levels. The system was further evaluated for analysis of complex peptide mixtures by using low concentration tryptic digests of standard proteins. Rapid identification of the original protein was obtained by database searching using the observed molecular masses of the peptides, and by comparison of actual MS-MS spectra of selected peptides with the predicted fragmentation patterns.

Amino Acid Sequence↗
PubMed1999

Assessment methods for alcohol consumption, prevalence of high risk drinking and harm: a sensitivity analysis.

BACKGROUND: There are no standardized ways to assess alcohol consumption in epidemiological studies. The main objective of the present study was to compare three widely used methods for assessing alcohol consumption with respect to resulting prevalence estimates for high risk drinking and harm as defined by morbidity and mortality indicators. METHODS: A within-subjects design was used to compare a quantity frequency, a graduated frequency, and a weekly drinking recall measure. Data consisted of a representative sample of 3961 adult residents of the province of Ontario, Canada, who participated in a multi-wave cross-sectional survey between 1990-1994. Cross-tabulation, Spearman correlation, and standard methodologies for prevalence-based cost-of-illness studies were used. RESULTS: The graduated frequency measure consistently yielded higher estimates of the prevalences of high risk drinking and harm. Differences were marked on all indicators, but were most pronounced for harmful drinking as defined by consuming an average of >60 g pure alcohol per day for males, and >40 g per day for females. Prevalence estimates of harmful drinking were almost five times higher for graduated frequency versus weekly drinking measures, and almost three times higher for graduated frequency versus quantity frequency measures. CONCLUSIONS: The characteristics of different measures of alcohol consumption should be considered in future research in epidemiology.

Adult↗
PubMed1980

An accurate and sensitive analysis by high-pressure liquid chromatography of conjugated and unconjugated bilirubin IX-alpha in various biological fluids.

An accurate and sensitive method was developed for the complete separation of the native tetrapyrroles, such as bilirubin and its mono- and di-conjugates of glucuronic acid, glucose and xylose, by ion-pair reversed-phase high-pressure liquid chromatography. The application of this method was demonstrated by the analysis of bile pigments in human bile and urine, and the method also makes it possible to estimate very low UDP-glucuronyltransferase activity, such as is found in the human foetal and neonatal liver.

Adult↗
PubMed1999

High-sensitivity analysis and sequencing of peptides and proteins by quadrupole ion trap mass spectrometry.

This paper describes experience with the commercially available LCQ quadrupole ion trap mass spectrometer applied to the off-line analysis of peptides and proteins. The standard front end of the electrospray probe was replaced with a micromanipulator which, with the aid of a magnifying device, allowed the use of a variety of miniaturized spraying interfaces. The low sample consumption and extended analysis times of these devices were ideally suitable to obtain improved results in terms of sensitivity and mass accuracy. This needed a careful optimization of the number of ions stored inside the trap (ion target parameter) and required spectrum averaging of many scans. A method is presented for the mathematical fitting of ZoomScan spectra to theoretical isotopic distributions, which allowed the mass determination of large peptides with more accuracy than that achieved by conventional deconvolution algorithms. A very simple on-line desalting configuration is also described which needed no external micro-high-performance liquid chromatographic pumps, and can be easily mounted using the built-in syringe delivery system of the LCQ. This set-up allowed extended analysis times of 'in-gel' protein digests in subpicomole amounts. Finally, the multiple fragmentation capabilities of the ion trap were found to be extremely useful for the analysis of peptide modifications such as phosphorylation and for sequencing individual peptides from highly complex MHC-bound peptide pools.

Animals↗
PubMed2001

Sensitivity analysis and application to trotting of a noninvasive method to calculate flexor tendon forces in the equine forelimb.

OBJECTIVE: To test the sensitivity to measurement and modeling errors of a method for noninvasive calculation of flexor tendon forces in the equine forelimb and to calculate tendon forces for Dutch Warmblood horses during trotting. SAMPLE POPULATION: A normative set of kinematic and ground-reaction force (GRF) data obtained from horses during trotting in another study. PROCEDURE: Forces in the flexor tendons were calculated from the data set before and after addition of fixed relative and absolute errors. Amount of error was based on normal accuracy of the variables. A similar analysis was performed for a measure of strain of the accessory ligament of the deep digital flexor tendon. RESULTS: The only errors that had a substantial influence on accuracy were modeling errors in the mechanical properties of the suspensory ligament and measurement errors in the point of application of the GRF and position of the marker on the distal interphalangeal joint. Influence of the measurement errors could be minimized by applying usual correction methods. CONCLUSIONS AND CLINICAL RELEVANCE: After correction of measurement errors, the method can be used to calculate mean tendon forces for a group of horses and to evaluate the influence of factors such as surface properties, type of shoe, speed, and fatigue on tendon forces. The method could become an important tool for use in research on the cause, prevention, and treatment of tendon injuries in horses.

Animals↗
PubMed1998

High sensitivity analysis of gene expression in single embryonic somites using coupled reverse transcription-polymerase chain reaction.

We have developed a highly sensitive and reproducible method to detect the expression of specific genes in small tissue samples, such as a single embryonic somite. The procedure, which utilizes coupled reverse transcription-polymerase chain reaction (RT-PCR), was developed for evaluating the sequence of gene expression occurring in single somites during chick embryonic development. Comparisons of results obtained from using combinations of various RNA isolation methods and reverse transcription methods demonstrate that a protocol using a commercially available RNA isolation reagent (Tri Reagent) followed by optimized PCR, successfully detects low levels of mRNAs.

Animals↗
PubMed1998

High sensitivity analysis of proteins and peptides by capillary electrophoresis-tandem mass spectrometry: recent developments in technology and applications.

Analytical biochemistry, in particular the analysis of regulatory proteins that control biological systems and pathways, is dependent on methods of ever-increasing sensitivity. Capillary electrophoresis (CE) has long been recognized as an ultrasensitive analytical technique. In spite of the high sensitivity, CE has not penetrated protein discovery research as a standard analytical method. In this review article we summarize recent technical developments which have significantly enhanced CE as a tool for the analysis of trace amounts of proteins. Specifically, we review recent advances in the development and application of capillary electrophoresis-mass spectrometry (CE-MS) and on-line analyte concentration techniques, and introduce the emerging field of microfluidics as a front end to mass spectrometry (MS).

Electrophoresis, Capillary↗
PubMed2006

Improved single-drop microextraction for high sensitive analysis.

This paper described a simple approach to prepare a small bell-mouthed extraction device for single-drop microextraction (SDME). Analytical sensitivity was improved by increasing the suspended acceptor volume. Because of the increased contact area and the rough inner surface of the extraction device, the stability of drop was markedly increased. The merits of the proposed method were demonstrated by using 1-octanol as extractant and with cyanazine, simazine and atrazine as model compounds. The related parameters and the effect of humic acid were systematically investigated. Under the optimized extraction conditions, the linear range, detection limit (S/N=3) and precision (RSD, n=6) were 0.2-50, 0.06microgL-1, 5.7% for cyanazine, 0.1-25, 0.03microgL-1, 6.7% for simazine, and 0.15-37.5, 0.04microgL-1, 5.0% for atrazine, respectively. The established method was applied to determine the target compounds in four real water samples, and the satisfactory spiked recoveries at two concentration levels were obtained. Moreover, the comparison of the proposed SDME with the traditional SDME was performed. These results indicated that the proposed improvement made SDME be a competitive analytical tool and an alternative of the traditional methods for the analysis of organic pollutants at trace level.

Analytic Sample Preparation Methods↗
PubMed1991

Sensitivity analysis of the effect of bioavailability or dosage form content on mean steady state phenytoin concentration.

Stochastic simulations were used to examine the sensitivity of mean phenytoin steady state concentrations (Css) to changes in the effective dosing rate produced by differences in average product content or bioavailability. Changes of +/- 4%, +/- 6%, +/- 8% and +/- 10% in the effective dosing rate (based on a starting dose yielding a Css of 15 mg/L) were examined. Monte Carlo simulations were performed for each change in dosing rate assuming a one-compartment open model with parallel Michaelis-Menten and first-order elimination. Parameter sets were comprised of a combination of values for maximal rate of saturable elimination (410 or 510 mg/day), the concentration at which the rate of saturable elimination is half maximum (Km, 4.4 or 5.7 mg/L), and linear clearance (CL, 0.15 or 1.5 L/day). These parameters were assumed to be log-normally distributed with coefficients of variation of 30%, 50%, and 15%. The percentages of "individuals" who would be predicted to have Css of less than 10 mg/L following a reduction in the effective dosing rate increased with decreasing Km and CL values. For a Km of 5.7 mg/L and CL of 1.5 L/day, 5% of the "individuals" had Css values of less than 10 mg/L with an 8% decrease in the dosing rate. If the dosing rate was reduced by 10%, then 14-16% of the "individuals" were predicted to have concentrations of less than 10 mg/L. All other combinations of Km and CL values yielded higher percentages of "individuals" with Css of less than 10 mg/L.(ABSTRACT TRUNCATED AT 250 WORDS)

Biological Availability↗
PubMed2003

Sensitivity analysis of a spatially-explicit stochastic simulation model of the evolution of resistance in Helicoverpa zea (Lepidoptera: Noctuidae) to Bt transgenic corn and cotton.

The sensitivities of a model simulating the evolution of resistance in Helicoverpa zea to Bt toxins in transgenic crops were investigated by examining effects of each of the model parameters on the frequency of resistance alleles after 8 yr. The functional dominance of resistance alleles and the initial frequency of those alleles had a major impact on resistance evolution. The survival of susceptible insects on the transgenic crops and the population dynamics of the insect, driven by winter survival and reproductive rates, were also important. In addition, agricultural practices including the proportion of the acreage planted to corn, and the larval threshold for spraying cotton fields affected the R-allele frequency. Many of these important parameters are inherently variable or cannot be measured with accuracy, so model output cannot be interpreted as being a forecast. However, this analysis is useful in focusing empirical research on those aspects of the insects' life system that have the largest effects on resistance development, and indicates ways in which to improve products and agricultural practices to increase the expected time to resistance. The model can thus be used as a scientific basis for devising a robust resistance management strategy for Bt crops.

Agriculture↗
PubMed2006

Adjoint sensitivity analysis of ozone nonattainment over the continental United States.

An application of the adjoint method in air quality management is demonstrated. We use a continental scale chemical transport model (STEM) to calculate the sensitivities of a nationwide U.S. ozone national ambient air quality standard (NAAQS) nonattainment metric to precursor emissions for the period July 1 to August 15, 2004. The model shows low bias and error (-4 and 24%, respectively), particularly for areas with high ozone concentrations. The nonattainment metric accounts for both 1-h and 8-h ozone standards, but is dominated by the 8-h exceedances (97% of the combined metric). Largest values of sensitivities are found to be with respect to emissions in the south and southeast U.S., Ohio River Valley, and California. When nonattainment sensitivities are integrated over the entire U.S., NOx emissions account for the largest contribution (62% of the total), followed by biogenic and anthropogenic VOCs (24% and 14%, respectively). For NOx emissions, point/area and mobile sources account for 54% and 46% of the total sensitivities, respectively. We also provide a state-by-state comparison for the nonattainment magnitude, nonattainment sensitivity, and emission magnitudes to explore the influence of interstate transport of ozone and its precursors, and policy implications of the results. Our analysis of the nationwide ozone nonattainment metric suggests that simple cap-and-trade programs may prove inadequate in achieving sought-after air quality objectives.

Air Pollutants↗
PubMed2006

Specific and sensitive analysis of nefopam and its main metabolite desmethyl-nefopam in human plasma by liquid chromatography-ion trap tandem mass spectrometry.

A specific and sensitive liquid chromatography-tandem mass spectrometric (LC-MS-MS) method using an ion trap spectrometer was developed for quantitation of nefopam and desmethyl-nefopam in human plasma. Nefopam, desmethyl-nefopam and the internal standard (ethyl loflazepate) were extracted in a single step with diethyl ether from 1 mL of alkalinized plasma. The mobile phase consisted of acetonitrile with 0.1% formic acid (50:50, v:v). It was delivered at a flow-rate of 0.3 mL/min. The effluent was monitored by MS-MS in positive-ion mode. Ionisation was performed using an electrospray ion source operating at 200 degrees C. Nefopam and desmethyl-nefopam were identified and quantified in full scan MS-MS mode using a homemade MS-MS library. Calibration curves were linear over the concentration range of 0.78-100 ng/mL with determination coefficients >0.996. This method was fast (total run time<6 min), accurate (bias<12.5%), and reproducible (intra- and inter-assay precision<17.5%) with a quantitation limit of 0.78 ng/mL. The high specificity and sensitivity achieved by this method allowed the determination of nefopam and desmethyl-nefopam plasma levels in patients following either intermittent or continuous intravenous administration of nefopam.

Calibration↗
PubMed2007

Functional network topology learning and sensitivity analysis based on ANOVA decomposition.

A new methodology for learning the topology of a functional network from data, based on the ANOVA decomposition technique, is presented. The method determines sensitivity (importance) indices that allow a decision to be made as to which set of interactions among variables is relevant and which is irrelevant to the problem under study. This immediately suggests the network topology to be used in a given problem. Moreover, local sensitivities to small changes in the data can be easily calculated. In this way, the dual optimization problem gives the local sensitivities. The methods are illustrated by their application to artificial and real examples.

Analysis of Variance↗
PubMed2001

Investigating regional differences in short-term effects of air pollution on daily mortality in the APHEA project: a sensitivity analysis for controlling long-term trends and seasonality.

Short-term effects of air pollution on daily mortality in eight western and five central-eastern European countries have been reported previously, as part of the APHEA project. One intriguing finding was that the effects were lower in central-eastern European cities. The analysis used sinusoidal terms for seasonal control and polynomial terms for meteorologic variables, but this is a more rigid approach than the currently accepted method, which uses generalized additive models (GAM). We therefore reanalyzed the original data to examine the sensitivity of the results to the statistical model. The data were identical to those used in the earlier analyses. The outcome was the daily total number of deaths, and the pollutants analyzed were black smoke (BS) and sulfur dioxide (SO(2)). The analyses were restricted to days with pollutant concentration < 200 microg/m(3) and < 150 microg/m(3) alternately. We used Poisson regression in a GAM model, and combined individual city regression coefficients using fixed and random-effect models. An increase in BS by 50 microg/m(3) was associated with a 2.2% and 3.1% increase in mortality when analysis was restricted to days < 200 microg/m(3) and < 150 microg/m(3), respectively. The corresponding figures were 5.0% and 5.6% for a similar increase in SO(2). These estimates are larger than the ones published previously: by 69% for BS and 55% for SO(2). The increase occurred only in central-eastern European cities. The ratio of western to central-eastern cities for estimates was reduced to 1.3 for BS (previously 4.8) and 2.6 for SO(2) (previously 4.4). We conclude that part of the heterogeneity in the estimates of air pollution effects between western and central-eastern cities reported in previous publications was caused by the statistical approach used and the inclusion of days with pollutant levels above 150 microg/m(3). However, these results must be investigated further.

Adolescent↗
PubMedDate not supplied

Incidence of leukaemia in young people around the La Hague nuclear waste reprocessing plant: a sensitivity analysis.

The objectives are to investigate the incidence of childhood leukaemia in young people around the French nuclear waste reprocessing plant (La Hague, Normandy) for the period 1978-1992, and to check the sensitivity of the results to the choice of disease clustering tests and reference rates. The performance of three statistical procedures are compared: a conventional approach; a Poisson maximum test, and an extraction mapping technique. On the whole, the results are in general agreement and demonstrate the apparent existence of a distinct cluster of childhood leukaemia, contiguous and south-east of the La Hague nuclear facility.

Adolescent↗