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At least 451 records · Page 25Linked to original sources
PubMed2004

Quantification of parvovirus B19 DNA using COBAS AmpliPrep automated sample preparation and LightCycler real-time PCR.

The COBAS AmpliPrep instrument (Roche Diagnostics GmbH, D-68305 Mannheim, Germany) automates the entire sample preparation process of nucleic acid isolation from serum or plasma for polymerase chain reaction analysis. We report the analytical performance of the LightCycler Parvovirus B19 Quantification Kit (Roche Diagnostics) using nucleic acids isolated with the COBAS AmpliPrep instrument. Nucleic acids were extracted using the Total Nucleic Acid Isolation Kit (Roche Diagnostics) and amplified with the LightCycler Parvovirus B19 Quantification Kit. The kit combination processes 72 samples per 8-hour shift. The lower detection limit is 234 IU/ml at a 95% hit-rate, linear range approximately 10(4)-10(10) IU/ml, and overall precision 16 to 40%. Relative sensitivity and specificity in routine samples from pregnant women are 100% and 93%, respectively. Identification of a persistent parvovirus B19-infected individual by the polymerase chain reaction among 51 anti-parvovirus B19 IgM-negative samples underlines the importance of additional nucleic acid testing in pregnancy and its superiority to serology in identifying the risk of parvovirus B19 transmission via blood or blood products. Combination of the Total Nucleic Acid Isolation Kit on the COBAS AmpliPrep instrument with the LightCycler Parvovirus B19 Quantification Kit provides a reliable and time-saving tool for sensitive and accurate detection of parvovirus B19 DNA.

Antibodies, Viral↗
PubMed2002

Quantification of bcl-2/JH fusion sequences and a control gene by multiplex real-time PCR coupled with automated amplicon sizing by capillary electrophoresis.

Follicular lymphoma is characterized by the presence of the t(14;18)(q32;q21) chromosomal translocation which juxtaposes the bcl-2 gene at 18q21 with the immunoglobulin heavy chain locus at 14q32. Quantification of t(14;18) carrying cells in FL patients can be achieved by real-time PCR, a highly sensitive technique for evaluating treatment efficacy and minimal residual disease. Despite the many advantages of real-time technology for this purpose, one disadvantage is that current real-time t(14;18) PCR assays amplify a control gene as a normalizer in a separate reaction. Since each PCR reaction has its own kinetics, separate PCR assays for target and control sequences can potentially result in inaccurate quantification of t(14;18)-positive cells. In addition, the real-time t(14;18) PCR assays do not determine the size of the amplified fusion sequence, which is helpful for excluding contamination and is commonly used to demonstrate clonal identity between pre- and post-treatment specimens from a patient. To address these limitations, we designed a multiplex real-time PCR protocol that allows amplification of control and target genes in the same reaction and precise size determination of bcl-2/JH fusion sequences by capillary electrophoresis. This multiplex PCR assay is equally sensitive to previous assays, allows more accurate quantification of bcl-2/JH fusion sequences, and is more convenient.

Chromosomes, Human, Pair 14↗
PubMed2002

Screening, library-assisted identification and validated quantification of oral antidiabetics of the sulfonylurea-type in plasma by atmospheric pressure chemical ionization liquid chromatography-mass spectrometry.

An atmospheric pressure chemical ionization liquid chromatographic-mass spectrometric (APCI-LC-MS) LC-MS assay is presented for fast and reliable screening and identification as well as precise and sensitive quantification of oral antidiabetics of the sulfonylurea-type (OADs) in plasma. It allowed the specific diagnosis of an overdose situation or a Munchausen syndrome caused by ingestion of OADs. After liquid-liquid extraction, the OADs glibenclamide, glibornuride, gliclazide, glimepiride, glipizide, gliquidone, glisoxepide, tolazamide and tolbutamide were separated using fast gradient elution. After screening and identification in the scan mode using our new LC-MS library, the OADs were quantified in the selected-ion mode. The quantification assay was validated according to the criteria established by the Journal of Chromatography B. All validation data were inside the required limits. The assay is part of a general LC-MS procedure for fast screening, identification and quantification of different toxicologically relevant compounds in plasma and has proven to be appropriate for OADs.

Administration, Oral↗
PubMed2003

Simple liquid-chromatographic method for Nile Red quantification in cell culture in spite of photobleaching.

Nile Red fluorescent marker is widely-used for different purposes, such as staining cell structures and for the visualization and localization of colloidal drug carriers. However, when fluorescence-dependent imaging or quantification is performed, the risk of inexact results is increased due to photobleaching. The proposed, simple quantification method of using an HPLC-UV-Vis system allows the determination of Nile Red even in photobleached samples. The intra- and inter-assay accuracies for all analytes were found to be within 94.9 and 100.8%, respectively, of target values. When samples underwent photobleaching by laser, UV-Vis detection varied at around 99+/-5%, whereas fluorescence decreased down to 86%. Such results show this method to be interesting for approaches where quantification should be performed after analysis such as fluorescent imaging.

Calibration↗
PubMed2001

A method for quantification from composite spectra: application to the determination of isomeric DNA photoproducts by tandem mass spectrometry.

Quantification of mixture components from their composite optical or mass spectra is a common need in analytical chemistry. We encountered the need when applying a combination of enzymatic digestion with nuclease P1 and tandem mass spectrometry to a mixture of isomeric photomodified oligodeoxynucleotides. In the procedure, we collisionally activated the [M - H]- or [M + Na - 2H]- ion of trinucleotide triphosphates, which were extricated enzymatically from the larger, damaged oligodeoxynucleotides, and we measured the relative abundances of characteristic fragment ions. The results sometimes yield curved calibrations for plots of the relative fragment ion abundances in the product ion spectra of isomers versus their relative amounts. We developed a normalized linear model, which brings understanding to the nonlinear plots and allows quantification of the mixture components from their composite spectra. The outcome demonstrates a general quantification procedure and shows that different yields for generating fragment ions from different constituents of the mixture cause the curved calibration lines.

DNA↗
PubMed2002

Identification and quantification of feathers, down, and hair of avian and mammalian origin using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

We describe a fast method for the identification of the origin of native and chemically processed feathers, down, and hair and for distinguishing closely related species using enzyme digestion and MALDI-TOF mass spectrometry. Additionally we present two methods for the quantification of different identified bird and mammalian samples, respectively, in binary species mixtures. Without any prior cleaning or isolation of single proteins, enzymatical digests of feathers, down, and hair are performed. Fragments generated are analyzed by MALDI-TOF mass spectrometry, and peak groups of different selectivity are established for every animal species. For the identification of individual animal species, only unique species-specific peaks are accepted while for species classification of tinted or bleached hair, the group of semispecific peaks (SEMPs) is used. Samples from native animal species show a higher coincidence of digest peak masses of SEMPs than far-related species, indicating a phylogenetical relationship of the investigated structure proteins. Quantification of mixed binary avian samples is performed by identification of preweighed single feathers and down, followed by calculation of the gravimetric mass fractions. The composition of mixed binary mammalian samples is calculated from correlation of the quotient of the relative peak intensities or areas of these species and the quotient of corresponding gravimetric mass fractions. High accuracy is achieved by both quantification methods. The fast methods are well suited for industrial quality control for example.

Animals↗
PubMed2004

Absolute quantification of proteins in solutions and in polyacrylamide gels by mass spectrometry.

A combination of nanoelectrospray tandem mass spectrometry and (18)O-labeled peptide internal standards was applied for the absolute quantification of proteins from their in-solution and in-gel tryptic digests. Although absolute quantification from in-solution digests was accurate, we observed that in-gel digestion compromised the quantification accuracy by affecting the recovery of individual peptides and, therefore, the provided estimates might be strongly influenced by the selection of reference peptides. Under optimized experimental conditions, it was possible to provide a semiquantitative estimate of the absolute amount of gel separated proteins within better than 50% error margin.

Amino Acid Sequence↗
PubMed2004

Confirmation and quantification of hemoglobin-based oxygen carriers in equine and human plasma by hyphenated liquid chromatography tandem mass spectrometry.

Oxyglobin (OXY) and Hemopure (HMP) are produced from bovine hemoglobin (Hb) and were developed for the treatment of anemia in animal and human patients, respectively. Hemolink (HML) is a blood substitute of human Hb origin under development. The ability of these agents to carry oxygen in circulating blood and their promise to improve oxygen delivery to tissues supports the potential for their abuse in equine and human athletes. To deter athletes from abuse of these agents, a method has been developed for the detection, confirmation and quantification of OXY, HMP, and HML in equine and human plasma. OXY, HMP, and HML were extracted from equine or human plasma by solid-phase extraction using Bond Elut ENV cartridges and were digested by trypsin at 37 degrees C for 3 h. The tryptic digests were analyzed by LC-MS/MS, and tryptic peptides specific for bovine and human Hbs were targeted. OXY and HMP were detected, quantified, and confirmed using the y14 ion and b8 ion of the tryptic peptide from bovine Hb alpha chain residues 69-90, and HML was quantified using the tryptic peptide from human Hb alpha chain residues 63-91. The limit of detection for OXY in equine plasma and HML in human and equine plasma was 50 and 250 microg/mL for HMP in human and equine plasma. The limit of confirmation was 250 microg/mL for OXY in equine plasma, 500 microg/mL for HML in human and equine plasma, and 1000 microg/mL for HMP in human and equine plasma. The linear range for quantification was 50-5000 microg/mL for OXY in equine plasma and for HML in human and equine plasma, and 250-5000 microg/mL for HMP in human and equine plasma. The intraday and interday CV were less than 17% for quantification of OXY in equine plasma with external calibration. OXY was stable for more than 30 days at -20 and -70 degrees C. OXY was detected and quantified in equine plasma up to 24 h following administration of a very low dose of OXY (32.5 g in 2 x 125 mL per horse), and its presence in equine plasma was confirmed up to 12 h postadministration.

Amino Acid Sequence↗
PubMed2005

Identification and elimination of polysiloxane curing agent interference encountered in the quantification of low-picogram per milliliter methyl tert-butyl ether in blood by solid-phase microextraction headspace analysis.

Widespread use of the gasoline additive methyl tert-butyl ether (MTBE) and the subsequent human exposure that follows have led to the need to quantify MTBE in a variety of complex biological matrixes. In this work, we demonstrate our latest MTBE quantification assay for whole blood and uncover previously unidentified contamination sources that prevented routine quantification in the low picogram per milliliter (parts per trillion, ppt) range despite a sensitive and selective analytical approach. The most significant and unexpected sources of contamination were found in reagents and laboratory materials most relevant to sample preparation and quantification. In particular, significant levels of MTBE were identified in sample vial septa that use poly(dimethylsiloxane) (PDMS)-based polymers synthesized with peroxide curing agents having tert-butyl side groups. We propose that MTBE is one of the byproducts of these curing agents, which cross-link PDMS via the methyl side groups. Residual MTBE levels of approximately 20 microg/septa are seen in septa whose formulations use these curing agents. Fortunately, these levels can be significantly reduced (i.e., <0.2 ng/septa) by additional processing. Performance achieved with this sample preparation approach is demonstrated using a mass spectrometry-based method to quantify blood MTBE levels in the low-ppt range.

Air Pollutants↗
PubMed2004

Detection and quantification of lanthanide complexes in cell lysates by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Gadolinium (III) complexes are under intense scrutiny as contrast agents for magnetic resonance imaging. Although currently used mainly as extracellular agents, there is a growing interest to exploit their contrast enhancing ability in the intracellular environment. To ascertain the preservation of their chemical integrity upon the intracellular entrapment, it is necessary to have a method for their dosage in the cell lysates. Herein, a mass spectrometric method for detection and quantification of gadolinium complexes in cell lysates is reported. The detection by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was carried out by using a non-acidic matrix (2,4,6-trihydroxyacetophenone), which does not allow any leakage of gadolinium from the complex. Quantification has been possible by using as an internal standard an ytterbium complex with the same ligand of the analyte. Ytterbium was chosen because, among the lanthanides, it is the one with the isotopic distribution pattern the most similar to that of gadolinium. Sensitivity was enough to detect low micromolar quantities of a cationic complex and high micromolar quantities of a neutral complex in cell lysates of rat hepatoma cells. In the case of anionic complexes, sensitivity was too low for quantitative analysis. To the best of our knowledge, this is the first report concerning the quantification of metal complexes by MALDI-TOF-MS.

Animals↗
PubMed2005

Characterization and performance of MALDI on a triple quadrupole mass spectrometer for analysis and quantification of small molecules.

The usefulness of MALDI for small-molecule work has been limited by matrix chemical interference in the mass range of interest, tedious sample preparation, and various crystallization and sample deposition issues. We report instrument characterization and small-molecule quantification performance data from a high repetition rate laser MALDI ion source coupled to a triple quadrupole mass spectrometer. The high repetition rate laser improves sensitivity and precision and allows a proportional increase in sample throughput. Tandem mass spectrometry is used to discriminate the signal from the high chemical background caused by the MALDI matrix. Successful quantification requires use of an internal standard and a means of sample cleanup for typical in vitro sample compositions. This instrument combination and analysis technique is relatively insensitive to sample crystal quality and spot homogeneity. Quantitative performance results are characterized for 53 small-molecule pharmaceutical compounds and compared to those obtained by ESI-MS/MS. Further comparison between MALDI and ESI is examined, and the potential for high-throughput MALDI-MS/MS quantification is demonstrated.

Coumaric Acids↗
PubMed2005

Quantification of relative gene dosage by single-base extension and high-performance liquid chromatography: application to the SMN1/SMN2 gene.

One of the most commonly used techniques for genotyping of single-nucleotide polymorphism (SNP) is detection of single-base extensions (SBEs). We present a new, rapid, simple, and highly reliable method for accurate quantification of SNP variants in a single reaction. Our approach is based on SBE detection coupled with high-performance liquid chromatography (HPLC) quantification. To demonstrate the utility of our approach, we report data to determine the gene dosage for relative amounts of alleles in a homologous gene, allowing detection of mutation causing exon skipping in human SMN genes to determine the ratio between the copy numbers of the SMN1/SMN2 gene. We successfully determined the relative ratio of the SMN1 and SMN2 genes and showed assay characteristics using the SBE reaction coupled with HPLC. This assay approach readily scaled to high parallelization with multiplex SBE reactions in a single sample screened in one analysis. By screening for particular SNP genotypes, this assay can be used to determine the relative gene dosage that correlates highly with the patient's disease state. The next challenge is to apply this novel methodology in a clinical screening and quantification setting for special gene regions within highly homologous genes.

Base Sequence↗
PubMed2006

UV fluorescence lifetime imaging microscopy: a label-free method for detection and quantification of protein interactions.

Due to the ability to detect multiple parameters simultaneously, protein microarrays have found widespread applications from basic biological research to diagnosis of diseases. Generally, readout of protein microarrays is performed by fluorescence detection using either dye-labeled detector antibodies or direct labeling of the target proteins. We developed a method for the label-free detection and quantification of proteins based on time-gated, wide-field, camera-based UV fluorescence lifetime imaging microscopy to gain lifetime information from each pixel of a sensitive CCD camera. The method relies on differences in the native fluorescence lifetime of proteins and takes advantage of binding-induced lifetime changes for the unequivocal detection and quantification of target proteins. Since fitting of the fluorescence decay for every pixel in an image using a classical exponential decay model is time-consuming and unstable at very low fluorescence intensities, we used a new, very robust and fast alternative method to generate UV fluorescence lifetime images by calculating the average lifetime of the decay for each pixel in the image stack using a model-free average decay time algorithm.To validate the method, we demonstrate the detection and quantification of p53 antibodies, a tumor marker in cancer diagnosis. Using tryptophan-containing capture peptides, we achieved a detection sensitivity for monoclonal antibodies down to the picomolar concentration range. The obtained affinity constant, Ka, of (1.4 +/- 0.6) x 10(9) M(-1), represents a typical value for antigen/antibody binding and is in agreement with values determined by traditional binding assays.

Algorithms↗
PubMed2006

Use of elemental analysis to determine comparative performance of established DNA quantification methods.

Quantification of genomic DNA is critical for many analyses in molecular biology. Current methods include optical density (OD) measurements or fluorescent enhancement but both approaches have limitations on achievable accuracy. In this study we performed an elemental analysis to quantify genomic DNA to provide an independent value for comparing the performance of four quantification methods. Specifically ICP-OES (inductively coupled plasma-optical emission spectroscopy) was used to assign a concentration value to a DNA stock solution, based on the stoichiometry of phosphorus within the molecule. Two absorbance- and two fluorescence-based methods were then used to quantify the same DNA solution using replicate analyses. The precision of each method was assessed by measurement of replicate spread (coefficient of variation) and trueness by t-test. Results showed that performance of the methods was variable, both in terms of concordance with the independent ICP-OES value and repeatability of data. While need for expensive equipment and technical expertise may preclude widespread replacement of more traditional methods for DNA quantification, use of primary methods such as ICP-OES analysis for production of accurate calibrants may increase quantitative accuracy and give greater appreciation of the true performance of current methods.

DNA↗
PubMed1999

Quantification and interpretation of total petroleum hydrocarbons in sediment samples by a GC/MS method and comparison with EPA 418.1 and a rapid field method.

Total petroleum hydrocarbons (TPH) as a lumped parameter can be easily and rapidly measured or monitored. Despite interpretational problems, it has become an accepted regulatory benchmark used widely to evaluate the extent of petroleum product contamination. Three currently used methods (GC/MS, conventional EPA 418.1, and a rapid field method PetroFLAG) were performed to quantify the TPH content in samples collected from a site contaminated by transformer oil. To standardize the method and improve the comparability of TPH data, crucial GC-based quantification issues were examined, e.g., quantification based on internal standards (ISTD) vs external standards (ESTD), single vs multiple ISTD, and various area integration approaches. The interpretation of hydrocarbon chromatographic results was examined in the context of field samples. The performance of the GC/MS method was compared with those of EPA 418.1 and PetroFLAG. As a result, it was observed that the ISTD quantification method was preferred to the ESTD method, multiple ISTD might be better than single ISTD, and three different area integration approaches did not have a significant effect on TPH results. Evaluation of the chromatograms between a reference sample and three unknown samples showed that the extent of contamination varied appreciably with sample depth. It was also found that there existed a good positive correlation between GC/MS and both EPA 418.1 and PetroFLAG, and that EPA 418.1 produced the higher overall estimate while GC/MS and PetroFLAG resulted in lower, more statistically comparable TPH values.

Chemistry Techniques, Analytical↗
PubMed2000

Detection and quantification of neurotensin in human brain tissue by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

A method was developed for mass spectrometric detection of neurotensin (NT)-like immunoreactivity and quantification of NT in human brain tissue. The method is based on immunoprecipitation followed by analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The identity of the major component of the immunoprecipitates as neurotensin was confirmed by fragment ion analysis on an electrospray ionization quadrupole time-of-flight instrument. MALDI-TOF-MS quantification of NT was achieved using stable-isotope-labeled NT as the internal standard, yielding an error of less than 5%. The method allowed detection of low-femtomole amounts of NT, staring from low-milligram amounts of lyophilized brain tissue. In addition to NT, several other peptides were detected in the purified samples, most of which, according to their molecular masses, corresponded to fragments of NT. The method is demonstrated with quantification of NT from human hypothalamus tissue, and a comparison is made with results obtained from competitive radioimmunoassay.

Brain Chemistry↗
PubMed2000

Simultaneous derivatization and quantification of the nitric oxide metabolites nitrite and nitrate in biological fluids by gas chromatography/mass spectrometry.

Simultaneous quantification of nitrite and nitrate, the major oxidative metabolites of L-arginine-derived nitric oxide (NO), in biological fluids by GC or GC/MS methods is currently impossible. The separate analysis of these anions is associated with severe methodological problems. Therefore, a GC/MS method was developed which allows, for the first time, simultaneous quantification of nitrite and nitrate in various biological fluids. The method involves a single derivatization procedure, by which endogenous nitrite and nitrate and their externally added 15N-labeled analogues are simultaneously converted in aqueous acetone by pentafluorobenzyl bromide to the nitro and nitric acid ester pentafluorobenzyl derivatives, respectively, and a single GC/MS analysis. Nitrite and nitrate concentrations measured in plasma and urine of humans by this method correlated excellently with those from quantification of nitrite and nitrate in these matrixes using a previously reported GC/MS method that, however, requires reduction of nitrate to nitrite. Also, the present method enables discrimination between S-nitro- and S-nitroso-glutathione, which have identical chromatographic and spectrophotometric properties. The method is very useful to routinely study metabolism and reactions of NO and its metabolites in vitro and in vivo. It is accurate, interference-free, sensitive-50 fmol of [15N]-nitrite and [15N]nitrate were detected at signal-to-noise ratios of 870:1 and 95:1, respectively-and should be a reference method for nitrite and nitrate measurements.

Body Fluids↗
PubMed2006

Glycoform quantification of chondroitin/dermatan sulfate using a liquid chromatography-tandem mass spectrometry platform.

Chondroitin sulfate (CS) is a glycosaminoglycan consisting of repeating uronic acid, N-acetylgalactosamine disaccharide units {[HexAbeta/alpha(1-3)GalNAcbeta(1-4)](n)()}. CS chains are polydisperse with respect to chain length, sulfate content, and glucuronic acid epimerization content, resulting in a distribution of glycoforms for a chain bound to any given serine residue. Usually, CS glycoforms exist, differing in sulfation position and uronic acid epimerization. This work introduces a novel LC-MS/MS platform for the quantification of mixtures of CS oligosaccharides. The CS polysaccharides were partially depolymerized and labeled with either the light (d(0)) or heavy (d(4)) form of 2-anthranilic acid (2-AA). Excess reagent was removed, and mixtures of the CS standard (d(0)) and unknown (d(4)) were made. The CS mixture was subjected to size exclusion chromatography (SEC) with on-line electrospray ionization mass spectrometric detection in the negative ion mode. Tandem mass spectra were acquired, and quantification of unknown samples within the mixture was made using relative ion abundances of specific diagnostic ions. The high accuracy and precision of the glycomics platform were demonstrated using glycoform mixtures made from standard CS preparations. The CS glycoform analysis method was then applied to cartilage extract, versican, and several dermatan sulfate preparations. This work presents the first application of a glycomics platform for the quantification of CS oligosaccharide mixtures for obtaining specific information about the positions of GalNAc sulfation and uronic acid epimerization.

Animals↗