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At least 415 records · Page 23Linked to original sources

PubMed2000

[Gas chromatography with dual nitrogen-phosphorus detection and flame ionization detection for screening abuse drugs].

A dual-detection Gas chromatographic method for screening out abuse drugs is described. A single capillary separation column is connected to a nitrogen-phosphorus detection (NPD) and a flame ionization detection (FID) by an Y-connector. The ratio responded by NPD/FID is influenced by operational conditions. But the relative ratio is not influenced when carbetane is used as a reference. The ratio and the relative ratio can be used to screening abuse drugs.

Chromatography, Gas↗

PubMed2000

Detection of a urinary biomaker for oxidative DNA damage 8-hydroxydeoxyguanosine by capillary electrophoresis with electrochemical detection.

8-Hydroxydeoxyguanosine (8-OHdG) is present in urine as a result of oxidative DNA damage associated with age-related diseases such as cancer. In this report a method is presented for the detection of 8-OHdG in human morning urine utilizing capillary electrophoresis with electrochemical detection (CEEC). The limit of detection for a aqueous standard of 8-OHdG is 50 nM (signal to noise ratio S/N = 3). A single solid-phase extraction (SPE) step with a C18 column is used for sample cleanup and 20-fold preconcentration of the urine before analysis by CEEC. Optimized conditions for analysis of extracted urine are E(app) = 0.5 V vs. Ag/AgCl with 20 mM sodium borate/20% MeOH v/v, pH 9, as the background electrolyte, and a separation voltage of 22 kV. The concentration of 8-OHdG varied from 6 to 86 nM with an average value of 42 +/- 26.9 nM for four healthy female and four healthy male subjects between the ages of 23 and 43.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed2000

Detection of neuropeptides using on-capillary copper complexation and capillary electrophoresis with electrochemical detection.

Capillary electrophoresis with electrochemical detection using a carbon fiber electrode in conjunction with on-capillary copper complexation was evaluated for the determination of peptides in standard and biological matrices. Peptides composed of 2-10 amino acids were investigated. A comparison was made between the responses obtained for peptides containing the oxidizable residue tyrosine and those obtained for their respective copper complexes. Electrochemical detection of non-tyrosine-containing peptides and a cyclic peptide was also demonstrated. A separation of leucine (Leu)-enkephalin and five metabolites was developed and then used for the investigation of Leu-enkephalin metabolism in plasma. The appearance of the des-tyrosine (des-Tyr) Leu-enkephalin, which cannot be detected directly at a carbon electrode, was monitored using the on-capillary complexation technique. Direct injection of the plasma sample was possible using this methodology.

Angiotensins↗

PubMed1993

Femtogram on-column detection of nicotine by isotope dilution gas chromatography/negative ion detection mass spectrometry.

A gas chromatographic method, utilizing detection by negative chemical ionization (NCI) mass spectrometry, is presented for the detection of nicotine. The method incorporates derivatization with heptafluorobutyric anhydride/pyridine to yield the ring-opened addition product, 1-[4-(3'-pyridyl)-4-heptafluorobutyryloxybutyl]-1- methylheptafluorobutanamide. The resulting heptafluorobutanamide is both relatively stable and an excellent electron scavenger. This methodology provides for the detection of nicotine at femtogram on-column levels, the measurement of nicotine over three orders of magnitude, and the minimization of contamination due to environmental nicotine.

Chromatography, Ion Exchange↗

PubMed1996

Rapid detection of sequence polymorphisms in the human mitochondrial DNA control region by polymerase chain reaction and single-strand conformation analysis in mutation detection enhancement gels.

The article describes a rapid approach for the detection of sequence polymorphisms in the mitochondrial (mt)DNA control region that involves enzymatic amplification of each entire mtDNA control region (HV1 and HV2) and the subsequent analysis of the PCR products by single-strand conformation analysis (SSCA) in mutation detection enhancement (MDE) gels, followed by silver stain detection. HV1 and HV2 SSC reference ladders were developed to standardize the classification of the different mtDNA types. Twenty-five mtDNA types were observed among the 45 Spanish individuals analyzed: 11 types were observed in the HV1 region as compared with 10 types in the HV2 region. This mutation scanning strategy could be a promising method of potential use not only in forensic genetics but also in population and evolutionary studies.

DNA, Mitochondrial↗

PubMed1996

Linearity evaluation in absorbance detection: the use of light-emitting diodes for on-capillary detection in capillary electrophoresis.

A model which takes into account both stray light and polychromatic light was used to predict and evaluate linearity in on-capillary detection in capillary electrophoresis (CE). According to the model the stray light is the major factor which determines linearity under typical CE operating conditions. By calculating theoretical absorbance versus concentration plots, the influence of different levels of stray light and polychromatic light on linearity is demonstrated. Experimentally, six light-emitting diodes (LEDs) in the range from 563 to 654 nm were examined as light sources for on-capillary detection in CE. Fitting theoretical curves to measured linearity plots enabled determination of the values of both effective path length and stray light for a particular detection system. The detector linearity for the four LEDs was compared to mercury and tungsten lamps used with interference filters. For potassium permanganate as the test compound, the linear range for a 563 nm LED was two times greater than that for a mercury lamp operated at 546 nm. The relatively poor linearity of the mercury lamp detector is explained by its high level of stray light. The noise of the LED563-based detector was the same as for the mercury lamp, whereas the other LEDs of higher light intensity gave approximately half the noise of the mercury lamp. The lowest noise level of 3 x 10(-5) AU was obtained for the LED at 554 nm (determined at a detector time constant of 0.1 s).

Electrophoresis, Capillary↗

PubMed1998

Rapid detection of gammaT cell receptor gene rearrangements in acute lymphoblastic leukemia by electrophoresis and silver staining: implications for detection of minimal residual disease.

Minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL) was studied using polymerase chain reaction (PCR). GammaT cell receptor (TCRG) genes are ideal targets for PCR-based detection of MRD due to their molecular characteristics. Polyacrylamide gel electrophoresis (PAGE) analysis of PCR products followed by silver staining was performed for 72 children with ALL at the onset of disease. Silver staining is an effective technique to detect gene rearrangements without the use of ethidium bromide. Moreover, this method may show heteroduplex bands of a clonal nature when both TCRG alleles are rearranged. PCR products subjected to a rapid staining protocol were recovered from the gel, reamplified by a second PCR and directly sequenced. After sequencing, we identified the junctional region and obtained patient-specific probes. In more than half of the patients we detected TCRG rearrangements that were used as molecular markers for residual disease.

Child↗

PubMed2005

Detection of prion protein using a capillary electrophoresis-based competitive immunoassay with laser-induced fluorescence detection and cyclodextrin-aided separation.

The development of capillary electrophoresis (CE)-based competitive immunoassay for prion protein (PrP) using carboxymethyl beta-cyclodextrin (CM-beta-CD) as a buffer additive is described here. The assay was based on the competitive binding of PrP and a fluorescein-labeled peptide from the prion protein with a limiting amount of specific antibody. The amount of both free and fluorescein-labeled peptide bound to antibody (immunocomplex) were determined by CE with laser-induced fluorescence detection. In the presence of PrP, the peak height ratio of the immunocomplex and the free peptide was altered compared to the control. These changes were directly proportional to the amount of PrP present. The fluorescently labeled peptide spanning amino acid positions 140-158 of the PrP and its corresponding monoclonal antibody is reported here. The reaction times of the antibody with either the peptide or the recombinant PrP was less than 1 min and is a large improvement over the 16-18 h required to achieve equilibrium for polyclonal antibodies. CM-beta-CD was explored as a buffer additive to suppress analyte adsorption and enhance separation selectivity in the CE analysis. A fast (1.1 min), selective (resolution 4.7), and reproducible (relative standard deviations of migration time for free and bound fluorescein isothiocyanate (FITC)-peptide 0.56% and 0.64%, respectively) separation was obtained with 0.6% CM-beta-CD in 25 mM N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS) at pH 8.8. The concentration detection limit of the assay for recombinant PrP was determined to be 80 ng/mL (or mass detection limit 1 pg). When blood samples from scrapie-infected sheep and from normal sheep were tested, the results of the blood assay were consistent with scrapie status of the sheep as determined post mortem by Western blot analysis. Development of this assay will lead to a potentially robust, rapid, and specific preclinical diagnosis for transmissible spongiform encephalopathies (TSEs) in animals and humans.

Animals↗

PubMed1990

Modified dot assay with increased sensitivity: detection of small amounts of immunoglobulin molecules and the importance of different detection systems.

The original method of the dot assay described by Hawkes et al. enabled the detection of 100 pg of immunoglobulin molecules. We compared different enzyme detection systems based on the avidin-biotin complex, in conjunction with different substrates. Three commercial kits (Vectastain-ABC, Streptavidin Bridge, and Streptavidin-Peroxidase Complex) based on the same technique were also included in the comparison. In addition, we tested the Aureo-Probe kit, which is based on gold- and silver-enhanced staining. Various incubation times, blocking solutions (horse serum, bovine serum albumin, and milk), and commercial antihuman Ig antisera or monoclonal antibodies were tested. The greatest sensitivity was achieved by using the peroxidase-avidin-biotin complex together with 4-chloro-1-naphtol as substrate, which detected 1 pg of immunoglobulin molecules. Equal sensitivity was also shown by the Aureo-Probe system.

Avidin↗

PubMed1992

Hepatitis C virus antibody detection by a line immunoassay and (near) full length genomic RNA detection by a new RNA-capture polymerase chain reaction.

A rapid and simple RNA-capture polymerase chain reaction assay (RCPA) for detection of hepatitis C virus (HCV) is described. The assay detects specifically the presence of (near) full length genomic RNA of HCV by capturing HCV-RNA at the 3' terminal end on magnetic beads, followed by cDNA synthesis and PCR with 5' end specific primers. Sera were obtained from 30 chimpanzees inoculated with non-A, non-B hepatitis material from various sources, 28-122 months after infection. The sera were tested for the presence of HCV-RNA by RCPA and for HCV antibodies by a Line ImmunoAssay (Inno-LIA HCV Ab). Both tests were compared and show a high degree of agreement. Screening of 30 chimpanzee sera revealed either clearing of the virus below detection level (22/30) or development of a HCV carrier state (8/30). Only 1 of 11 LIA-indeterminate samples was positive by RCPA. As the RCPA is more sensitive, it can be used to test for the presence of HCV in sera which are classified indeterminate by the LIA. The outcome of the infection seems to be independent of the nature of the inocula, suggesting that the individual immune response could determine either clearing of the virus or the development of chronic infection.

Animals↗

PubMed2006

Detection of DNA-bound advanced glycation end-products by immunoaffinity chromatography coupled to HPLC-diode array detection.

Sugars and sugar degradation products are formed during food processing, but also endogenously in vivo. In vitro, nucleosides and DNA react readily with these carbonyl compounds during the formation of the two diastereomers of N(2)-carboxyethyl-2'-deoxyguanosine (CEdG(A,B)), leading to a loss of DNA integrity. Only little is known about DNA glycation in vivo and about the influence of nutrition on CEdG formation. In this study, we developed a sensitive method to analyze DNA glycation by HPLC. For this purpose, immunoaffinity chromatography (IAC) using a polyclonal antibody against N(2)-carboxyethylguanine (CEguanine) was coupled to HPLC-DAD. In some samples, peak identity was confirmed by LC-MS/MS. The recovery of CEguanine from the IAC columns was 52.5% +/- 3.6 (n = 4). Thus, it was possible for the first time to detect CEdG(A,B), N(2)-carboxyethylguanosine (CEG(A,B)), and CEguanine in 11 human urine samples. However, due to imprecision of IAC, valid quantification of the adducts could not be achieved. Furthermore, CEdG was also detected in the DNA of cultured human smooth muscle cells (SMCs) and bovine aorta endothelium cells (BAECs). In BAECs, CEdG(A,B) were found by HPLC-DAD and LC-MS/MS after immunoaffinity purification, whereas in SMCs DNA-advanced glycation end-products were only detected with the more sensitive LC-MS/MS method.

Animals↗

PubMed1991

Motion-insensitive volume-selective pulse sequences for direct and proton-detected 13C spectroscopy: detection of glycogen in the human liver in vivo.

Two compact pulse sequences are reported for the volume-selective detection of 13C nuclei. 13C signals are either directly acquired after enhancing the amplitude by polarization transfer from the coupled protons or, preferably, indirectly detected by heteronuclear editing of proton signals of 1H nuclei coupled to 13C. In the latter case, the full sensitivity of proton resonance is achieved, and signals from uncoupled protons or protons coupled to non-13C nuclei or 13C nuclei of undesired compounds are suppressed. Both sequences are single-scan procedures and are insensitive to the pulse phases and to motions of the investigated organs. The insensitivity to organ motions is due to the extremely compact character of these sequences avoiding coherence evolution periods as far as possible. This is achieved first by the introduction of double-resonance sandwich (DORSA) pulses accomplishing the polarization transfer slice selectively in a very short time. The second reason is that the initial evolution interval which is usually part of polarization transfer experiments is avoided by the aid of a doublet line-selective inversion pulse. Several test experiments in vivo are reported. In particular, it is demonstrated that glycogen can be detected in natural abundance in the human liver in vivo using a 2-T whole-body tomograph even without the use of heart or respiration triggers. The total acquisition time was 22 min, and a signal-to-noise ratio of 6 was achieved.

Carbon↗

PubMed1994

Electrochemically active DNA probes: detection of target DNA sequences at femtomole level by high-performance liquid chromatography with electrochemical detection.

Electrochemically active DNA probes were prepared by linking a ferrocene unit with 5'-aminohexyl-terminated oligonucleotides. The DNA sequences of probes 5a, 5b, and 5c were 5'-T12-3', 5'-T20-3', and 5'-TGCAG TTCCG GTGGC TGATC-3', respectively. Probe 5a could form a complex selectively with a single-strand poly(A) and a double-strand DNA fragment containing an A13 sequence and these complexes could be detected at femtomole levels by an electrochemical detector (ECD) on HPLC. The observed ECD response was proportional to the amount of the complex over the range 20-100 fmol. Probe 5c was capable of detecting femtomole levels of a restriction DNA fragment having oncogene v-myc. Moreover, probe 5b was able to detect picogram levels of mRNA taken from rat brain or yeast total cellular RNA. This proves that the electrochemically active DNA probes are useful in analyzing traces of DNA and RNA carrying the complementary sequence.

Base Sequence↗

PubMed1998

Detection of 8-oxoguanine in cellular DNA using 2,6-diamino-8-oxopurine as an internal standard for high-performance liquid chromatography with electrochemical detection.

The quantitative aspect of the electrochemical detection method to detect 8-oxo-7,8-dihydroguanine (8-oxoGua) has been improved by using an internal standard. In addition, emphasis was placed on the reduction of artifactual oxidation of DNA during isolation and hydrolysis. Nuclear DNA was isolated from rat organs and purified on an anion-exchange column following treatment with proteinase K and RNase. DNA hydrolysis to nucleobases or nucleosides was performed using either formic acid treatment or enzymatic digestion, respectively. The levels of either 8-oxoGua or 8-hydroxy-7,8-dihydro-2'-deoxyguanosine were comparable. For accurate quantification, 2,6-diamino-8-oxopurine [(NH2)2-OH-Pur], added prior to hydrolysis, was used as an internal standard for the high-performance liquid chromatography with electrochemical detection assay. The baseline level of 8-oxoGua in DNA of Sprague-Dawley rats was estimated to be 2 to 5 8-oxoGua residues per 10(6) DNA bases, with slight differences depending on the tissue origin. In agreement with the results of previous observations, the level of the oxidized base in the kidney of animal treated with iron complexed to nitrilotriacetic acid (Fe-NTA) (15 mg/kg) was three- to fourfold higher than that of untreated rats or animals treated with a saline solution, while there was no change in 8-oxoGua levels in the liver and colon of these treated animals.

2-Aminopurine↗

PubMed1999

The separation and direct detection of ceramides and sphingoid bases by normal-phase high-performance liquid chromatography and evaporative light-scattering detection.

Sphingolipids are an important class of lipids due to their role as biologically active molecules and as intracellular second messengers. Sphingolipid metabolites are involved in a wide variety of important biological processes including signal transduction and growth regulation. Simple, quantitative analytical methods are needed to assay these complex lipids, in order to study their biological functions. The current methods used to quantify ceramides and long-chain sphingoid bases are primarily based on derivatization with uv or fluorescent tags and with radioactive-based enzymatic assays. A method was developed to separate ceramides and sphingoid bases by normal-phase high-performance liquid chromatography and detect them directly with evaporative light-scattering detection. Ceramides and the sphingoid bases phytosphingosine, dihydrosphingosine, sphingosine, and sphingosine 1-phosphate were resolved with a rapid and quantitative assay in the nanomole range. Yeast extracts grown to various time points were assayed for ceramide and sphingoid bases using a simple, isocratic HPLC system. Both ceramide and phytosphingosine, the primary sphingoid base present in yeast cell extracts, were detected in yeast cell extracts. Phytosphingosine was resolved as a sharp peak with the addition of triethylamine and formic acid modifiers to a chloroform/ethanol mobile phase. This method demonstrates the first direct assay of both ceramides and sphingoid bases.

Ceramides↗

PubMed2001

Comparisons of different contrast resolution effects on a computer-aided detection system intended to cluster microcalcifications detected in dense breast images.

Clustered microcalcifications, which are frequently an important signal of possible cancer, are usually hidden in dense breast images, adding more difficulty in mammogram medical analysis. In this work we evaluate the performance of a previously developed computer-aided detection scheme, modified for application to dense breast images. The main focus of this investigation was on the effect of different contrast resolutions on the processing performance. We have processed dense breast images digitized with 8 and 12 bits to evaluate the performance of this computer-aided detection scheme with different contrast resolutions. As expected, for most of the 12-bit images, the number of detected signals was greater or at least equal to that of the 8-bit images.

Calcinosis↗

PubMed2006

Detection of legionella species in clinical samples: Comparison of polymerase chain reaction and urinary antigen detection kits.

BACKGROUND: Recently, two excellent methods have been used for the diagnosis of Legionnaires' disease: urinary antigen detection and PCR. The purpose of the present study is to analyze and evaluate the sensitivity and specificity of three different urinary antigen detection kits as well as PCR. MATERIALS AND METHODS: A total of 148 samples were collected from 33 patients between 1993 and 2004. These consisted of 73 urine samples obtained from 33 patients, 57 serum samples provided by 29 patients, and 18 respiratory tract specimens from 13 patients. Three commercially available kits were used to detect urinary antigen. For the 5S PCR reaction, primers L5SL2 and L5SR84 were used. RESULTS: Positive results were shown in all patients' urine (representing 79.5% of total samples) using the Binax EIA kit, in 93.9% patients (representing 75.3% samples) using the Binax NOW immunochromatographic kit, and in 90.9% (representing 72.6% samples) using the Biotest EIA kit. Urine samples from 12.1% patients (representing 6.8% of total samples), serum samples from 41.4% patients (representing 35.1% of total samples), and respiratory samples from 84.6% patients (representing 88.9% of total samples) showed positive results with PCR. CONCLUSION: In testing urine of legionellosis patients, it was suggested that three kits were all valuable tools for diagnosis of legionellosis. Since over one-third of patients' serum samples and most respiratory specimens showed positive results with PCR, the addition of PCR for testing of these samples might be useful, particularly in cases of culture negative and serum antibody negative patients.

Adult↗

PubMed1987

Improved detection limits in an organic mass spectrometer using a combination of matrix free FAB and photodiode array detection.

Scanning mass spectrometers suffer from the disadvantage of monitoring only one mass at any particular time in contrast to mass spectrographs which allow the simultaneous detection of an extended mass range. Historically such mass spectra were recorded by directing the ion beam onto a photographic plate which was subsequently developed and interpreted. The introduction of electronic devices such as the photodiode array enabled the development of imaging detectors capable of real time readout, so that a range of masses can be detected and rapidly processed using a computer system. This work uses such a detector in combination with a double focusing MS50RF mass spectrometer for the parallel detection of a limited mass range.

Mass Spectrometry↗