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At least 1,063 records · Page 59Linked to original sources
PubMed2003

Simultaneous quantification of jasmonic acid and salicylic acid in plants by vapor-phase extraction and gas chromatography-chemical ionization-mass spectrometry.

Jasmonic acid and salicylic acid represent important signaling compounds in plant defensive responses against other organisms. Here, we present a new method for the easy, sensitive, and reproducible quantification of both compounds by vapor-phase extraction and gas chromatography-positive ion chemical ionization-mass spectrometry. The method is based on a one-step extraction, phase partitioning, methylation with HCl/methanol, and collection of methylated and, thus, volatilized compounds on Super Q filters, thereby omitting further purification steps. Eluted samples are analyzed and quantified by GC/MS with chemical ionization. Standard curves were linear over a range of 5-1000 ng for jasmonic acid and salicylic acid. The correlation coefficients were greater than 0.999 and the recovery rates estimated between 70 and 90% for salicylic acid and 90 and 100% for jasmonic acid. The limit of detection was about 500 fg by using single ion detection mode. Both, cis- and trans-isomers for jasmonic acid can be detected. A comparison with established methods indicates the new method to be highly efficient, allowing reliable quantification of both compounds from small amounts of plant material (5-400mg fresh weight).

Alamethicin↗
PubMed2003

Mass spectrometric quantification of acetylation at specific lysines within the amino-terminal tail of histone H4.

Electrospray ionization mass spectrometry, a leading method for the quantification of biomolecules, is useful for the analysis of posttranslational modifications of proteins. Here we describe a mass spectrometric approach for determining levels of acetylation at individual lysine residues within the amino-terminal tail of histone H4. Because of the high density of acetylatable lysine residues within this short span of amino acids, collision-induced dissociation tandem mass spectrometry was required. In addition, it was necessary to develop an algorithm to determine the fraction of acetylation at specific lysine residues from fragment ions containing more than one lysine residue. This is the first report of direct measurement of endogeneous levels of acetylation at individual lysine residues within the amino-terminal tail of yeast histone H4 and is the first use of tandem mass spectrometry for quantification of peptides containing multiple sites of modification.

Acetylation↗
PubMed2003

[Quantification of mitral regurgitation].

The echographic methods of quantification of mitral regurgitation are various. Semiquantitative methods using the color Doppler extension of the regurgitant jet are now replaced by more quantitative methods, including PISA, jet width, and regurgitant fraction. Although sometimes difficult, accurate quantification of mitral regurgitation is now possible in a majority of patients using transthoracic echocardiography.

Echocardiography, Doppler, Color↗
PubMed1996

Use of N-phenylmethazonium methosulphate oxidation of NADH in the quantification of oxygen uptake by oral bacteria under open-system conditions.

Technically, open systems are more suited than closed systems for the measurement of oxygen uptake by bacteria present at high cell densities as in dental plaque. Moreover, measurements are easier to perform and can be done faster and continuously. However, quantifications is more difficult and, except for steady-state conditions, has been semiquantitative. The stoichiometric oxidation of NADH by N-phenylmethazonium methosulphate (PMS) is a reaction used here to develop a formula that overcomes this problem and makes it possible to relate quantitatively the pO2 measured easily with an oxygen electrode to the amount of oxygen utilized by respiring bacteria provided with a given amount of substrate. The approach and formula consist of the summing of (i) the oxygen decrease observed during the period of bacterial oxygen consumption and (ii) the oxygen simultaneously entering from the atmosphere. To quantify the entry component, the rate of oxygen diffusion into the mixture needed to be determined: this was done by reducing or exhausting the oxygen content of the medium in various ways and determining the rate constant of atmospheric oxygen entry. The relation between the logarithm of the oxygen concentration (determined from pO2 values using the PMS-NADH reaction) and point of time in the oxygen resaturation process proved to be linear. This enabled calculation of a diffusion rate constant, k, for use in the oxygen utilization formula. The validity of this method for quantification of oxygen use was tested by comparing oxygen consumption determined from the pO2 tracing and the derived formula to the oxygen consumed when fixed amounts of NADH are oxidized by PMS. When oxygen uptake/substrate utilization molar ratios were calculated for each of several NADH concentrations, the resulting values were almost identical. The method proved reliable over most of the oxygen concentration range possible in this system.

Bacteria↗
PubMed2003

Improved TRAP-silver staining versus conventional radioactive TRAP assays: quantification of telomerase activity during immortalization and in pathological human endometrium.

OBJECTIVES: To develop a sensitive telomeric repeat amplification protocol (TRAP)-silver staining assay for telomerase activity quantification. DESIGN AND METHODS: TRAP assays were performed by using a TRAPeze telomerase kit with or without [alpha-32P]-dCTP. Amplification products were electrophoresed in polyacrylamide gels and detected by autoradiography or a modified silver staining protocol. Telomerase activity was quantified from radioactive counts or optical density of telomerase products from test extracts and controls. RESULTS: TRAP-silver staining assay was at least as sensitive as radioactive TRAP assay and quantified telomerase activity within linearity from 10 to 3,000 cell equivalents. Both methods quantified a weak telomerase activity in normal endometrial glandular epithelial cells (GEC) and a strong increase in immortalized GEC. In human pathologic endometria (n=24), telomerase activity was correlated with lesion seriousness and distinguished simple hyperplasias from nonhyperplasic or cancerous lesions. CONCLUSIONS: TRAP-silver staining assay is suitable for cell and tissue telomerase activity routine quantification.

Adult↗
PubMed2001

Analysis of enantiomers giving partially overlapped peaks by using different treatments of the chromatographic ultraviolet signals: quantification of pseudoephedrine enantiomers.

Different strategies for the quantification of partially coeluting optical isomers have been investigated. The methods tested are based on the use of different features as the analytical UV signals: peak heights, perpendicular drop areas, first and second derivatives of the chromatograms, peak areas obtained by deconvolution of the overlapped peaks with data fitting optimization, and a multivariate model (principal component regression, PCR). The amphetamine-derivative drug pseudoephedrine was selected as a model compound. For chromatography, LiChrospher 100 RP18 and a mobile-phase consisting of methanol and a solution of carboxymethyl-beta-cyclodextrin (the chiral selector) were used. The UV detector was set at 215 nm. The accuracy obtained with the tested methods at different degrees of overlapping and at different concentration ratios between enantiomers was evaluated. The results of this study demonstrated that the best option for quantification of partially overlapped UV peaks of enantiomers and to obtain the enatiomeric excess is the use of a PCR model using peak heights, perpendicular drop peak areas and deconvoluted peak areas as the original variables. The predictive ability of the proposed calibration model is of about 2-8 times better (depending on the overlapping degree) than that achieved with the other models tested.

Ephedrine↗
PubMed2002

Identification and quantification of polar naphthalene derivatives in contaminated groundwater of a former gas plant site by liquid chromatography-electrospray ionization tandem mass spectrometry.

A liquid chromatography (LC) method followed by electrospray ionization (ESI) and tandem mass spectrometry (MS-MS) was developed for the quantification of acidic naphthalene derivatives in the concentration range 0.1 to 100 microg/l without excessive sample preparation. For optimal sensitivity the LC-MS-MS measurements were performed recording mass fragmentation by collision induced dissociation in the multiple reaction mode. The collision energy was optimized for every analyte. The matrix effects of the sample were investigated by spiking standards of 1-naphthoic acid with humic acid (HA) and with calcium chloride. While HA decreased the signal intensity an increase was observed in the presence of calcium chloride. For the investigated groundwater samples of a tar oil contaminated site a complete separation of the analytes from the sample matrix by reversed-phase separation could be obtained. The absence of matrix effects on quantification results was confirmed by comparison of results based on external calibration with those based on standard addition of the analytes to a groundwater sample. In four groundwater samples of the contaminated site naphthalene derivatives like 1-naphthoic acid, 2-naphthoic acid, 1-naphthylacetic acid, 2-naphthylacetic acid, 1-hydroxy-2-naphthoic acid, 2-hydroxy-3-naphthoic acid, and naphthyl-2-methylenesuccinic acid have been detected.

Chromatography, Liquid↗
PubMed2002

Validation of a liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of 11 (fluoro)quinolone antibiotics in swine kidney.

A LC-MS-MS method has been validated for the simultaneous quantification of 11 (fluoro)quinolone antibiotics at the maximum residue level (MRL) in swine kidney. The studied compounds were danofloxacine, cinoxacine, ciprofloxacine, noxacine, enrofloxacine, flumequine, marbofloxacine, nalidixic acid, norfloxacine, ofloxacine and oxolinic acid. The method involves solid-phase extraction of these compounds followed by LC-MS-MS analysis using an electrospray ionisation interface. Limits of quantification < or = 50 microg/kg could be obtained in swine kidney, much lower than every MRL. The validation is discussed. This work was carried out in order to support the European Union policy on consumer health

Animals↗
PubMed2002

Gas chromatographic-mass spectrometric quantification of 4-(5-)methylimidazole in roasted coffee after ion-pair extraction.

A GC-MS method is described for quantification of 4-(5-)methylimidazole (4MI) in coffee. Although tested, GC-flame ionization detection proved inadequate for this purpose due to the complexity of the coffee matrix. The developed method was based on ion-pair extraction with bis-2-ethylhexylphosphate and derivatization with isobutylchloroformate. Quantification was carried out by the standard addition method using 2-ethylimidazole as internal standard. Reproducibility data from the complete procedure are presented. Mean recoveries were higher than 98%. The method was applied to green and roasted coffee samples from the two most important varieties, arabica and robusta, and to commercial "torrefacto" coffee blends. 4MI was not detected in the green coffee samples analysed and ranged from 0.307 to 1.241 mg/kg in roasted samples.

Coffee↗
PubMed2003

Quantification of the anticancer agent STI-571 in erythrocytes and plasma by measurement of sediment technology and liquid chromatography-tandem mass spectrometry.

An isocratic high-performance liquid chromatographic method coupled to tandem mass spectrometry for the quantification of the revolutionary and promising anticancer agent STI-571 (tradenames Gleevec, Glivec, Imatinib) in blood plasma and red blood cells (RBCs) is described. The method involves measurement of sediment technology for RBCs and a subsequent single protein precipitation step by the addition of acetonitrile to both the RBC isolate and plasma. The sample mixture was centrifuged (10 min, 3600 g), and the supernatant filtered through a HPLC filter (0.45 microm). The analytes of interest, STI-571 and the internal standard [2H8]STI-571 were eluted on a Waters Symmetry C18 column (50x2.1 mm I.D., 3.5 microm particle size) using a methanol-0.05% ammonium acetate (72:28, v/v) mixture. STI-571 and [2H8]STI-571 were detected by electrospray tandem mass spectrometry in the positive mode, and monitored in the multiple reaction monitoring transitions 494>394 and 502<394, respectively. The lower limit of quantitation of STI-571 was 2.1 ng/ml in RBCs and 1.8 ng/ml in plasma. The recovery from both plasma and RBCs was between 65 and 70%. The method proved to be robust, allowing simultaneous quantification of STI-571 in RBCs and plasma with sufficient precision, accuracy and sensitivity and is useful in monitoring the fate of this signal transduction inhibitor in whole blood of cancer patients.

Antineoplastic Agents↗
PubMed2003

Identification of 2-bromo-3,4,5,6-tetrachloroaniline and its quantification in the color additives D&C Red Nos. 27 and 28 (phloxine B) using solid-phase microextraction and gas chromatography-mass spectrometry.

The present work describes (a) the identification and characterization of a contaminant, 2-bromo-3,4,5,6-tetrachloroaniline (2BTCA), in the color additives D&C Red Nos. 27 and 28 (phloxine B) and (b) the determination of the extent and level of 2BTCA contamination in certified lots of these colors. For these purposes, 2BTCA (a compound not previously reported in the literature) and its positional isomer 4-bromo-2,3,5,6-tetrachloroaniline (4BTCA) were synthetically prepared. 4BTCA was used as the internal standard for the quantification of 2BTCA in the colors. Test portions from 35 certified lots of D&C Red Nos. 27 and 28 were analyzed for 2BTCA using a solid-phase microextraction-GC-MS method. Those lots were submitted for certification by both domestic (seven) and foreign (four) manufacturers during the past 4 years. Of the test portions analyzed, 22 (62.9%) contained 2BTCA in amounts ranging from 0.15 to 435.7 ppm with an average value of approximately 131.7 ppm. The remaining 13 (37.1%) test portions contained no detectable 2BTCA or less than 0.01 ppm, which is the limit of quantification of the present method. The analyses revealed substantial differences in the level of 2BTCA across lots from the same manufacturer as well as among different manufacturers. The wide range of 2BTCA levels found in the analyzed lots suggests that the presence of 2BTCA in D&C Red Nos. 27 and 28 may be avoided or significantly reduced during the manufacturing process. A direct correlation was observed between the presence of 2BTCA and that of 3,4,5,6-tetrachlorophthalic acid in analyzed batches of D&C Red Nos. 27 and 28. A chemical pathway that could explain the presence of 2BTCA in these color additives, and ways to avoid its formation, are also proposed.

Aniline Compounds↗
PubMed2003

Detection and quantification of bovine, ovine and caprine milk percentages in protected denomination of origin cheeses by reversed-phase high-performance liquid chromatography of beta-lactoglobulins.

A method for detecting and quantifying bovine, ovine and caprine milk mixtures in milk and cheeses by means of reversed-phase high-performance liquid chromatography (RP-HPLC) of beta-lactoglobulins is described. Gradient elution was carried out with a flow rate of 0.5 ml/min and a temperature of 45 degrees C, using a mixture of two solvents: solvent A (0.1% TFA in water) and solvent B (0.09% TFA in 80% aqueous acetonitrile, v/v). The effluent was monitored at 215 nm. Under the conditions used different chromatographic patterns were obtained for bovine, ovine and caprine whey proteins. Each milk type presented different retention times for beta-lactoglobulin peaks. Binary mixtures of bovine and ovine or bovine and caprine raw milks containing 1, 2, 5, 10, 20, 30, 50, 75 and 95% (v/v) of bovine milk, as well as ovine and caprine milk mixtures containing 1, 2, 5, 10, 20, 30, 50, 75 and 95% (v/v) of ovine milk were used for cheese making. Cheeses were prepared and ripened, according to traditional methods. Milk mixtures, fresh and ripened cheeses were analyzed. A linear relationship was established between log 10 of beta-lactoglobulin peaks ratio (calculated as peak area values ratio) and log 10 of the relative percentage of bovine or ovine milk. The ratio between beta-lactoglobulin peaks was not affected by the degree of ripening. Thus, enabling the quantification of milk type percentage, with a detection limit of 2%. This technique allowed quantification of milk species within the concentration range of 5-95%. The method was successfully applied for authenticity evaluation and quantitative determination of ovine and caprine milk percentages of commercial protected denomination of origin (PDO) cheeses.

Animals↗
PubMed2003

Chemometric approach for the resolution and quantification of unresolved peaks in gas chromatography--selected-ion mass spectrometry data.

A semiautomated and integrated chemometric approach is presented for the resolution and quantification of unresolved target-analyte signals in gas chromatography-selected-ion monitoring (GC-SIM) data collected using scanning mass spectrometers. The chemometric approach utilizes an unskewing algorithm and two multivariate chemometric methods known as rank alignment and the generalized rank annihilation method (GRAM). The unskewing algorithm corrects the retention-time differences within a single GC-SIM data matrix caused by using a scanning mass spectrometer. Rank alignment objectively corrects the run-to-run retention-time difference between a sample GC-SIM data matrix and a standard addition GC-SIM data matrix. GRAM analysis uses the sample and standard addition data matrices to mathematically resolve and quantify the target-analyte signal(s). The resolution and quantification of severely unresolved target-analyte signals are demonstrated using GC-SIM data obtained from conventional heart-cut two-dimensional gas chromatography with mass spectrometric detection. In addition, the GC-SIM data is used to demonstrate the result of chemometric analysis when the absence of a target-analyte signal is obscured by interference. Chemometric analysis is shown to unambiguously detect an analyte based on its resolved mass chromatograms in situations where the traditional approach of measuring peak height fails to positively detect it. The predicted analyte concentrations are within 8% of the reference concentrations.

Gas Chromatography-Mass Spectrometry↗
PubMed1999

Quantification of deoxynivalenol in wheat using an immunoaffinity column and liquid chromatography.

A simple and accurate method to quantify the mycotoxin deoxynivalenol (DON) in wheat is described. The method uses immunoaffinity chromatography for DON isolation and liquid chromatography (LC) for toxin detection and quantification. Wheat samples are extracted in water, filtered twice and applied to an immunoaffinity column. Following a water wash, DON is eluted from the column with methanol and injected onto an LC system with a UV detector for quantification. Test performance was evaluated in terms of antibody specificity, limit of detection, percentage recovery, precision, column capacity, assay linearity and comparison with the GC-electron-capture detection (ECD) method of Tacke and Casper. Specificity of the immunoaffinity column cleanup procedure was confirmed with only DON (>80%) and its 15-C derivatives (40-50%) being recognized by the antibody while 3-C DON derivatives, nivalenol, T-2 and fusarenon-X did not bind. The limit of detection is at least 0.10 microg/g. Percentage recovery for the entire assay range averages 90% with an average relative standard deviation of 8.3%. Naturally contaminated samples showed comparable precision. Column capacity was determined to be 3.3 microg. The assay showed a high degree of linearity (r2=0.999) and an optimum assay range of 0.10 to 10.0 microg/g. Comparative analysis of 28 naturally or artificially contaminated wheat samples using DONtest-HPLC and the GC-ECD method of Tacke and Casper showed that DONtest-HPLC is a statistically significant predictor of the GC-ECD method (r2=0.982).

Antibody Specificity↗
PubMed2000

Stereological quantification of carboxyfluorescein-labeled rat lung metastasis: a new method for the assessment of natural killer cell activity and tumor adhesion in vivo and in situ.

The function of natural killer (NK) cells is often studied by assessing in vitro levels of NK cell mediated lysis of target cells, or by assessing in vivo levels of lung tumor cell retention or metastatic colonization of intravenously injected tumor cells. However, these methods do not permit direct quantification and visualization of NK cells and their targets in vivo and in situ. Here, a new approach is described to visualize effector-to-target interactions as well as to estimate total numbers of targets in the lung, in vivo and in situ. MADB106 tumor cells were vitally labeled using carboxyfluorescein (CFSE) and intravenously (i.v.) injected into Fischer 344 rats (10(6) cells/rat). This mammary adenocarcinoma derived cell line is syngeneic to the inbred Fischer 344 rat and highly sensitive to NK cell activity in vivo. Effector-to-target interactions were visualized by immunostaining. Using the optical fractionator method, total numbers of CFSE-labeled MADB106 tumor cells were estimated in the left lung of the animals 5 min after tumor inoculation. To further demonstrate the usefulness of this approach in reflecting in vivo processes, rats were inoculated with MADB106 cells and simultaneously with a single i.v. bolus of either 1 microg/kg adrenaline or saline. Both lungs were removed 5 min later. Adrenaline caused a significant 80% reduction in the total number of lung CFSE-labeled MADB106 tumor cells, suggesting a rapid modulation of metastasis by stress hormones. This new approach facilitates the monitoring of effector-to-target interactions and the quantification of immune cell function or tumor adhesion in vivo and in situ.

Adenocarcinoma↗
PubMed2001

An automated method for the quantification of immunostained human Langerhans cells.

Allergic contact dermatitis is a frequent and increasing health problem. For ethical reasons, the current animal tests used to screen for contact sensitizers should be replaced by in vitro alternatives. Contact sensitizers have been shown to accelerate Langerhans cell (LC) migration from human organotypic skin explant cultures (hOSECs) more rapidly than non-sensitizers and it has been proposed that the hOSEC model could be used to screen for sensitizers. However, chemically induced decreases in epidermal LC numbers need to be accurately quantified if the alterations in epidermal LC numbers are to form the basis of an alternative system for screening contact sensitizers in vitro. As manual counting of LCs is labour intensive and subject to intra- and inter-personal variation we developed an image analysis routine, using the Leica QWin image analysis software, to quantify LCs in situ using immunohistochemically stained skin sections. LCs can be identified using antibodies against the membrane molecule CD1a or the Lag antibody, which recognises cytoplasmic Birbeck granules. Quantification of epidermal LC number using the image analysis software had a much lower inter-person variation than when the same specimens were counted manually, using both the anti-Lag and CD1a antibodies. The software-aided quantification of epidermal LCs provides an accurate method for measuring chemically-induced changes in LC numbers.

Antigens, CD1↗
PubMed2001

RANTES and MIP-1beta mRNA expression in human peripheral blood mononuclear cells: transcript quantification using NASBA technology.

The importance of chemokines in the immune response, as well as in a range of specific disease states, is becoming increasingly apparent. The role of CC- (or beta-) chemokines and their receptors in the pathology and mechanisms of HIV-1 infection has served to intensify interest in these factors. Although the functionality of these factors resides in their protein forms, assays for the detection and quantification of these protein factors in clinical samples are not readily available. Consequently, we designed NASBA-based assays for the quantification of the mRNA encoding two members of the CC-chemokine family: RANTES and MIP-1beta. The NASBA-based assays are extremely sensitive, accurate, and reproducible across a dynamic range of at least four orders of magnitude. Inter-assay performance is comparable to intra-assay performance. We applied these methods to the analysis of normal human PBMC and PBMC from HIV-1 infected individuals. Although MIP-1beta mRNA levels are higher than RANTES levels in both populations, RANTES levels in HIV-1+ patients are higher than in normal individuals. The utility of these assays in longitudinal studies of specific subpopulations of cells, as well as their potential use in clinical diagnostics, is discussed.

Blood Circulation↗
PubMed2002

Development and evaluation of a sandwich ELISA for quantification of the 20S proteasome in human plasma.

Because quantification of the 20S proteasome by functional activity measurements is difficult and inaccurate, we have developed an indirect sandwich enzyme-linked immunosorbent assays (ELISA) for quantification of the 20S proteasome in human plasma. This sandwich ELISA uses a combination of a monoclonal antibody (mcp 20) recognizing the C2-beta subunit of human 20S proteasome (Mr approximately 30,000) and a polyclonal rabbit anti-20S antibody which labels different subunits of the complex. The detection limit of the assay was established as 10 ng/ml (n=10, mean of zero standard+2 S.D.) and the recovery rate ranged from 96% to 104%. The within-run and between-run coefficients of variation (CV) ranges were 2.8-3.3 and 3.0-3.4, respectively. Using serial dilutions of plasma to which various amounts of purified 20S proteasome were added, a linear dose-response was observed between 102 and 2050 ng/ml with a slope of 1.004 and a coefficient of determination r(2) of 0.99. In a preliminary experiment performed on a limited number of patients, the present assay was used to quantify the 20S proteasome in plasma from healthy subjects (n=11) and from a limited number of patients with various diseases (two patients with each of the following diagnoses: acute myeloid leukaemia, chronic myeloproliferative syndromes, Hodgkin's disease and solid tumors). The average concentration of 20S proteasome in plasma from normal subjects was found to be 2319+/-237 ng/ml (n=11). With reference to this normal range, the plasma proteasome concentration was found to be increased in most of these pathological state and as high as 1200% when solid tumors had been detected. For patients with Hodgkin's disease, the changes were more variable whereas in patients with chronic lymphocytic leukaemia, the proteasome concentration was raised during the acute phase of disease and decreased during therapy. We suggest that this robust, accurate and highly reproducible assay could be used to quantify proteasome in human plasma and investigate its value as a biological marker for various malignant and nonmalignant diseases.

Animals↗