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At least 793 records · Page 44Linked to original sources
PubMed2006

Rapid identification of Escherichia coli and Helicobacter pylori in biological samples by capillary zone electrophoresis.

The differences in surface charge of different bacteria can be exploited for their separation by capillary electrophoresis. However, this method of separation of microorganisms is beset with various drawbacks such as adhesion of bacteria to the fused silica surface or cluster formation. To overcome these phenomena we investigated the addition of poly(ethylene oxide) as a focusing agent to the running buffer and used calcium and myoinositol hexakisphosphate as specific ions that interact with the bacterial surface, changing its electrical properties and electrophoretic mobilities. In the present work, we applied CZE to identification of E. coli in infected urine (direct injection) from patients with urinary tract infections and to identification of Helicobacter pylori, which is a gram-negative bacillus responsible for one of the most common infections found in humans worldwide. Helicobacter pylori colonize the stomach and are responsible for severe diseases of the gastric tract, ranging from chronic gastric ulcer to gastric cancer.

Electrophoresis, Capillary↗
PubMed2004

The reproducible acquisition of comparative liquid chromatography/tandem mass spectrometry data from complex biological samples.

An in-depth study of the reproducibility of data acquired for comparative proteomics analysis using a prototype two-stage heated laminar flow chamber fitted to a commercial high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) instrument was undertaken. The study is based on 24 replicate samples from four independent membrane preparations derived from two matched breast cancer cell lines. Variation and reproducibility in the data were evaluated at several levels highlighting the relative efficiency and variability of the acquisition routines used. Specifically, variation in the number and relative intensities of chromatographic peaks eluted from the LC column, precursor ion selection and sequence identification were evaluated. On average, approximately 6500 chromatographic peaks were generated for each acquisition with a corresponding coefficient of variance (CV) of less than 20%. Precursor ion selection and sequence identification averaged 1380 and 780 events per acquisition sample, respectively, with corresponding CVs of less than 10% for each. The reproducibility in the precursor ion selection was typically better than 60% between similar replicates. Using protein and peptide internal standards, it was found that the CV in retention time across the gradient between two acquisition pairs was typically less than 5%, whereas the average intensity ratio was 1.0 (expected) with a CV approaching 20%. An evaluation of the intensity ratios calculated from endogenous peptide sequences, identified across the acquisition set, indicated a CV of approximately 30%. Similarly, the CV associated with the top 1000 peptides indicated a mean and median of 28.4 and 26.95%. For a given acquisition pair it was also found that approximately 11% of the chromatographic peaks eluting from the column were linked to a sequence or identified. For these experiments, less than 10% of the peak pairs had absolute ratios greater than 2.0 and of those only approximately 10% had sequences linked to them. For each matched acquisition set on average 406 proteins were identified with a CV of less than 10%. Of the proteins that were identified approximately 30% had at least one predicted trans-membrane domain, indicating a four-fold increase over a crude homogenate sample with only minor enrichment. During these experiments it was found that the interface did not significantly alter the relative charge state distribution of ions, nor did it introduce significant interference from background ions. The interface was capable of 24-hour acquisition cycles.

Amino Acid Sequence↗
PubMed2005

The use of turbulent flow chromatography and the isocratic focusing effect to achieve on-line cleanup and concentration of neat biological samples for low-level metabolite analysis.

The use of turbulent flow chromatography in conjunction with column switching isocratic focusing was used to perform on-line sample cleanup and concentration of neat rat plasma for the identification of low-level metabolites. The concentration was achieved by focusing multiple injections, which were cleaned by a turbulent flow column, onto an analytical column prior to elution into the mass spectrometer. In addition, the first application of turbulent flow chromatography for on-line sample cleanup of neat bile samples is reported. The on-line cleanup and concentration method extracts and concentrates a sample 20-fold in 1 h, and is completely automated.

Anatomy, Comparative↗
PubMed2005

High-throughput biological sample analysis using on-line turbulent flow extraction combined with monolithic column liquid chromatography/tandem mass spectrometry.

A high-throughput liquid chromatography/tandem mass spectrometry (LC/MS/MS) method, which combines on-line sample extraction through turbulent flow chromatography with a monolithic column separation, has been developed for direct injection analysis of drugs and metabolites in human plasma samples. By coupling a monolithic column into the system as the analytical column, the method enables running 'dual-column' extraction and chromatography at higher flow rates, thus significantly reducing the time required for the transfer and mixing of extracted fraction onto the separation column as well as the time for gradient separation. A strategy of assessing and reducing the matrix suppression effect on the on-line extraction LC/MS/MS has also been discussed. Experiments for evaluating the resolution, peak shape, sensitivity, speed, and matrix effect were conducted with dextromethorphan and its metabolite dextrorphan as model compounds in human plasma matrix. It was demonstrated that the total run time for this assay with a baseline separation of two analytes is less than 1.5 min.

Analgesics, Opioid↗
PubMed2006

Solid-phase microextraction for gas chromatographic/mass spectrometric analysis of dimethoate in human biological samples.

A new, simple and rapid procedure for the determination of dimethoate in urine and blood samples was developed using direct immersion solid-phase microextraction and gas chromatography/mass spectrometry. This technique required only 0.1 mL of sample, and ethion was used as internal standard. Two types of coated fibre were compared (100 microm polydimethylsiloxane, and 65 microm Carbowax/divinylbenzene). Other parameters, such as extraction temperature, adsorption and desorption time, salt addition, agitation and pH, were optimized to enhance the sensitivity of the method. Limits of detection (LODs) and quantitation (LOQs) were 50 and 100 ng/mL for urine and 200 and 500 ng/mL for blood, respectively. The method was found to be linear between the LOQ and 40 microg/mL for urine, and between the LOQ and 50 microg/mL for blood, with correlation coefficients ranging from 0.9923-0.9996. Precision (intra- and interday) and accuracy were in conformity with the criteria normally accepted in bioanalytical method validation. The mean absolute recoveries of dimethoate were 1.24 and 0.50% for urine and blood, respectively. Because of its simplicity and the fact that small volumes of sample are used, the described method can be successfully used in the diagnosis of poisoning by this pesticide, namely in those situations where the sample volume is limited, as frequently occurs in forensic toxicology.

Dimethoate↗
PubMed2006

Ultra-performance liquid chromatography coupled to linear ion trap mass spectrometry for the identification of drug metabolites in biological samples.

The coupling of ultra-performance liquid chromatography, operating at elevated pressures, to a linear ion trap mass spectrometer provides a high-performance system suitable for drug metabolite characterisation. This system demonstrates improved chromatographic efficiency and sensitivity and at the same time provides diagnostic MSn data often critical for metabolite structural assignment. The linear ion trap was capable of dealing with the high chromatographic efficiencies and hence narrow peak widths associated with 1.7 microm particle-packed column separations. Polarity switching and data-dependent MSn data were generated with ease, and applied to the identification of metabolites found in human plasma.

Biotransformation↗
PubMed2006

An improved analytical method for the determination of urea nitrogen isotopomers in biological samples utilizing continuous flow isotope ratio mass spectrometry.

Over the past few years numerous dual inlet isotope ratio mass spectrometry (IRMS) applications have been adapted to continuous flow systems which allow the automation of sample admission and a higher throughput. The isotopomer analysis of urea nitrogen by IRMS requires the offline conversion of urea into nitrogen gas before analysis. The oxidation of urea with LiOBr results in the monomolecular degradation of urea, which preserves the identity of the parent urea molecule, and has to be conducted under vacuum to prevent contamination with atmospheric nitrogen. We have developed an offline system of urea degradation utilizing disposable Exetainers, in which atmospheric nitrogen is displaced by helium. Recovery of urea nitrogen was linear within the range of the standards tested (0 to 420 microg nitrogen) and standard curves for 15N15N-urea standards showed high coefficients of determination (R2 > 0.9998). A small portion of urea degrades in a non-monomolecular fashion and has been shown to depend on the concentration of urea in the sample. Long-term storage of prepared samples showed a decline in 15N15N enrichment, suggesting air contamination. However, samples were stable for 24 h, which allows for the analysis of large sample batches. Interest in urea metabolism, particularly in ruminant species, has increased recently due to the environmental implications of urea and nitrogen excretion by farm animals. This novel analytical method will allow for accurate measurements and the rapid throughput needed in order to support these field studies.

Animals↗
PubMed2001

High-performance liquid chromatography/tandem mass spectrometry for the quantitative analysis of a novel taxane derivative (BAY59-8862) in biological samples and characterisation of its metabolic profile in rat bile samples.

A sensitive, specific, accurate and reproducible high-performance liquid chromatography (HPLC) analytical method was developed and validated for the quantification of the novel oral taxane analogue BAY59-8862 in mouse plasma and tissue samples. A fully automated solid-phase extraction procedure was applied to the plasma after internal standard (IS) addition, with only 0.2 mL volume of the sample loaded on a CN-Sep-pak cartridge. In the case of the tissues a very simple acetonitrile extraction was used to recover BAY59-8862 and its internal standard from liver. The procedure for the quantification of BAY59-8862 and its IS (IDN5127) is based on high-performance liquid chromatography/ion spray-tandem mass spectrometry, operating in selected ion monitoring mode. The retention times of BAY and IS were 7.21 and 10.36 min, respectively. In both plasma and tissue specimens the assay was linear in the range 50-5000 ng/mL (ng/g). The overall precision and accuracy were assessed on three different days. The results for plasma were within 6.1% (precision) and between 99 and 112% (accuracy), and for the liver samples within 7.3% and between 104 and 118%, respectively. The LOD was 5 ng/mL and 20 ng/g in the plasma and liver, respectively. In addition, the biliary excretion of the compound in rats was studied. The study showed that an oxidative chemical reaction was the preferred metabolic pathway for biliary excretion, and two sets of mono- and dihydroxylated metabolites were detected by LC/ISP-MS/MS experiments. With this method, BAY59-8862 pharmacokinetic was determined in mice. The combined results demonstrate that the methodology can be considered a valid approach to conduct pharmacokinetic and metabolic studies during preclinical and clinical investigations.

Animals↗
PubMed2002

Identification of omega hydroxy fatty acids in biological samples as their pentafluoropropyl derivatives by gas chromatography/mass spectrometry with positive and negative ion detection.

A simple one-step procedure for derivatization of the omega hydroxy fatty acids 20-hydroxyeicotetraeonic acid and 12-hydroxylauric acid is presented. The procedure involves acylation of the terminal hydroxy group and esterification of the carboxylic acid with a mixture of pentafluoropropionic anhydride and pentafluoropropanol. Positive and negative ion spectra for the derivatives are presented. The procedure was used to demonstrate conversion of arachidonic acid to 20-hydroxyeicosatetraeonic acid and lauric acid to 12-hydroxylauric acid in kidney microsomal incubations. The reaction appears to be specific, since derivatives of subterminal fatty acids (secondary alcohols) could not be detected.

Animals↗
PubMed1993

A fluorometric assay for the measurement of nitrite in biological samples.

The increasing importance of nitric oxide synthase has been underscored by the elucidation of its role in a growing number of normal and pathophysiological processes. Therefore, techniques for detection of nitrite/nitrate, oxidation products of the enzymatic conversion of arginine to citrulline and nitric oxide, should serve as useful tools in defining the contribution of NO synthase to these processes. We have developed a rapid and sensitive fluorometric assay for quantification of nitrite/nitrate based upon the reaction of nitrite with 2,3-diaminonaphthalene to form the fluorescent product, 1-(H)-naphthotriazole. The assay can be used to detect 10 nM nitrite, making it 50-100 times more sensitive than the well-known Griess assay. Moreover, the assay is adaptable to a 96-well plate format, facilitating the handling of a large number of samples including conditioned media from cell culture or the nitrite generated by the purified enzyme. Nitrite/nitrate levels in blood can also be monitored using this assay when it is combined with a filtration step (to remove hemoglobin) followed by conversion of the nitrate to nitrite by nitrate reductase. Thus, this fluorometric method combines speed and sensitivity with the handling of a large number of samples for the quantification of nitrite generated from in vivo and in vitro sources.

2-Naphthylamine↗
PubMed1994

Detection of HIV1 DNA in biological samples by an homogeneous assay: fluorescence measurement of double-stranded RNA synthesized from amplified DNA.

A nonisotopic homogeneous detection of nucleic acid sequences after amplification is described. We show that a DNA fragment bearing T7 RNA polymerase promoters on each extremity is able to be transcribed in two complementary RNAs, leading to a high yield direct synthesis of double-stranded RNA (dsRNA). Thus, this dsRNA can be easily detected and quantified in solution by fluorescence in the presence of propidium iodide. This reaction, used as a postamplification step, has been associated with a nested polymerase chain reaction (PCR); the second PCR round allowing the incorporation of the T7 promoters. This leads to a very efficient homogeneous assay. The fluorescence signal is proportional to the concentration of PCR product and is highly specific. This method can be easily carried out with currently available reagents and with unsophisticated instrumentation. This homogeneous procedure has been evaluated for the detection of HIV1 in blood samples; the sensitivity and the specificity appear to be equivalent to that of the radioactive method.

Base Sequence↗
PubMed1994

Spectrophotometric determination of dehydroascorbic acid in biological samples.

We describe a method for accurately and precisely measuring dehydroascorbic acid in perchloric acid extracts prepared from human plasma, lymphocytes, and mammalian cells. Samples were assayed by spectrophotometrically monitoring the kinetics of the concentration-dependent absorbance changes of dehydroascorbic acid with phosphate-methanol-containing buffers. The lowest detectable dehydroascorbate concentration using this assay is estimated to be below 0.1 mumol/liter. Total analysis time is less than 10 min and allows the simultaneous measurement of numerous samples. The calibration curve is linear (r > 0.995) over the range 0-200 mumol/liter. The dehydroascorbic acid concentrations measured in supplemented samples agree with known concentrations. Interference of ascorbic acid and 2,3-diketogulonic acid with this assay was excluded. The correlation with a highly specific chromatographic procedure gave comparable results over the range of physiologically relevant concentrations. The procedure avoids the most commonly applied method of measuring the native ascorbic acid, then reducing the dehydroascorbic acid, and finally measuring the total ascorbic acid and determining dehydroascorbic acid by the difference. Stabilization of ascorbic acid during assay was achieved by addition of desferrioxamine.

3T3 Cells↗
PubMed1995

Quantitative measurement of sphingosine 1-phosphate in biological samples by acylation with radioactive acetic anhydride.

We describe here in detail the development of a method to quantitatively measure sphingosine 1-phosphate (Sph-1-P), a bioactive sphingolipid. Sph-1-P was first extracted from cells into the upper aqueous phase under alkaline conditions by Folch's phase separation and then reextracted into the lower chloroform phase under acidic conditions. This phosphorylated sphingoid base extracted was quantitatively converted to N-[3H]-acetylated Sph-1-P, that is [3H]C2-ceramide 1-phosphate (C2-Cer-1-P), by N-acylation with [3H]acetic anhydride. The [3H]C2-Cer-1-P formed with the acylation was resolved by thin-layer chromatography, detected with autoradiography, and quantitated by scraping the corresponding band and counting its radioactivity with a scintillation counter. This assay allows quantification of Sph-1-P over a range from at least 100 pmol (often 30 pmol) to 10 nmol (the highest level tested). The utility and validity of our assay were demonstrated using human platelets. The amount of Sph-1-P in platelet extracts was proportional to the cell number and calculated as 141 +/- 4 pmol/10(8) cells (mean +/- SD, n = 3), which was about four times higher than that of sphingosine. The potent agonist thrombin did not affect the total Sph-1-P amounts in platelet suspensions but induced the release of Sph-1-P stored in the cells into the medium.

Acetic Anhydrides↗
PubMed1995

Quantitation of platelet-activating factor in biological samples using liquid chromatography/mass spectrometry with column-switching technique.

A procedure is described for the fully automated quantitative determination of platelet-activating factor (PAF) using liquid chromatography/fast atom bombardment mass spectrometry (LC/FAB-MS) with two types of column-switching techniques. Various parameters in LC/FAB-MS were optimized, and the use of a microbore column allowed highly selective detection of PAF. A column-switching system was incorporated to minimize band broadening and to increase the permissible injection volume. Analysis of authentic PAF indicated that the limit of quantitation was at the low picogram level (about 50 pg/ml). Another system using both a strong cation-exchange column and an ODS column in tandem was developed for the analysis of PAF in blood samples. These methods were validated with standard samples and applied to the determination of PAF in human polymorphonuclear neutrophils stimulated by addition of a calcium ionophore and in human blood.

Chromatography, Liquid↗
PubMed1997

Trace quantification of the oxidative damage products, meta- and ortho-tyrosine, in biological samples by gas chromatography-electron capture negative ionization mass spectrometry.

Oxygen radicals damage biomolecules and may contribute to cellular aging and degenerative disease. We describe a sensitive method for the quantification of two endogenous biomarkers of oxidative damage: meta-tyrosine (m-Tyr) and ortho-tyrosine (o-Tyr). The assay can be applied to direct analysis of free amino acids or protein-bound amino acids following hydrolysis. The assay involves derivatization with pentafluorobenzyl bromide and extraction into n-decane, followed by gas chromatography-mass spectrometry. Stable isotope labeled m- and o-Tyr (2H4) and phenylalanine [i.e., Phe (2H5)] were added as internal standards to improve analytical accuracy. Quantification of as little as 50 pg of m- and o-Tyr in 100 micrograms protein is possible and the data are expressed as a molar ratio of m- and o-Tyr to native Phe. The assay was used to determine the levels of m- and o-Tyr in freshly isolated human plasma protein (4.05 +/- 0.67 m-Tyr per 10(4) Phe, 0.35 +/- 0.07 o-Tyr per 10(4) Phe). Exposure of human plasma to reactive oxygen species significantly increased the levels of m-Tyr (56.4 +/- 1.1 m-Tyr per 10(4) Phe, P < 0.0001) and o-Tyr (48.9 +/- 1.3 o-Tyr per 10(4) Phe, P < 0.0001). The mild hydrolysis and derivatization conditions caused no artifactual formation of either m- or o-Tyr.

Chemical Phenomena↗
PubMed1997

Analysis of 12-oxo-phytodienoic acid enantiomers in biological samples by capillary gas chromatography-mass spectrometry using cyclodextrin stationary phases.

The octadecanoid plant growth regulator 12-oxo-phytodienoic acid (12-oxo-PDA), which is also an intermediate in the biosynthesis of jasmonic acid, is obtained from 13-hydroperoxylinolenic acid via an unstable allene oxide generated by the enzyme allene oxide synthase. Recombinant, bacterially expressed and purified allene oxide synthase from Arabidopsis thaliana yields racemic 12-oxo-PDA as a mixture of 94:6 cis:trans diastereomers. In the presence of allene oxide cyclase from Solanum tuberosum, a product of high enantiomeric purity was obtained, which was shown to be (+)-cis-12-oxo-PDA (98:2 cis:trans diastereomers). Based on this coupled reaction, a preparative procedure was developed that yields pure (+)-cis-12-oxo-PDA in milligram quantities. Furthermore, an analytical technique employing capillary gas chromatography-mass spectrometry and beta- or gamma-cyclodextrin stationary phases was developed that enables the direct analysis of nanogram amounts of enantiomers of 12-oxo-PDA, as their methyl esters, in plant tissues. In the species analyzed, endogenous cis-12-oxo-PDA is the (+)-enantiomer.

Cyclodextrins↗
PubMed1998

Measurement of hydrogen peroxide in biological samples containing high levels of ascorbic acid.

The physiological concentration of hydrogen peroxide in the aqueous humor was reported to range between 25 and 60 microM, and conditions leading to elevated levels could have important damaging effects such as cataract formation. However, the high concentration of ascorbic acid in aqueous humor, which is 20 times that of plasma, was recently shown to interfere in the dichlorophenol-indophenol assay for hydrogen peroxide. The actual concentration of hydrogen peroxide in this fluid has become a controversial issue. In the present study, we used the method of ferrous oxidation of xylenol orange (FOX1 assay) performed in a nitrogen atmosphere to accurately measure low levels of hydrogen peroxide, even in the presence of ascorbic acid at concentrations normally found in aqueous humor. Contrary to values reported in the literature, we observed that the concentration of hydrogen peroxide in the rabbit aqueous humor is less than 5 microM, which is the detection limit of the method.

Air↗