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At least 1,225 records · Page 68Linked to original sources
PubMed1996

Fully automated determination of selective retinoic acid receptor ligands in mouse plasma and tissue by reversed-phase liquid chromatography coupled on-line with solid-phase extraction.

A fully automated reversed-phase HPLC method was developed for the quantitative assay of three retinoids (Am-580, CD-2019 and CD-437) which selectively activate the retinoic acid receptors RAR alpha, RAR beta and RAR gamma, respectively. Mouse plasma, embryo and maternal tissues were prepared for injection by on-line solid-phase extraction (SPE) and valve-switching techniques. Following automatic injection, the sample was loaded on preconditioned disposable cartridges, cleaned-up and then transferred onto the analytical column to be eluted in the backflush mode, separated by gradient elution and detected by UV, while a new cartridge was concomitantly conditioned. The overall recovery was quantitative allowing for external standardization. The calibration curves were linear in all biological samples tested so far, with a correlation coefficient (r) >0.99. The intra-day precision was < or = 7.8% (n = 5-6) and the inter-day variability was < or = 9.4% (n = 3). The lower limit of detection was 2.5 ng/ml or ng/g for CD-2019 and CD-437, and 5 ng/ml for Am-580 with a S/N ratio of 5 using a sample weight of 25 microliters or mg. The method is now in routine use in our laboratory for the assessment of the pharmacokinetic profiles of these retinoids. The small sample size required, the simple sample preparation and the rapid analysis with high degree of automation make this method convenient for microanalysis of biological samples both in animal and human studies.

Animals↗
PubMed1982

Purification of acute phase anti-hepatitis A virus (HAV) IgM and development of an IgM solid-phase radioimmunoassay for the detection of HAV.

A simple two-step procedure for the purification of IgM from acute phase hepatitis A infected chimpanzee serum has been developed. The procedure involves the precipitation of the euglobulin fraction with boric acid and the fractionation of the resuspended material on Sephacryl S-300 (Pharmacia Fine Chemicals, Piscataway, NJ). The purified IgM was used to develop a solid-phase radioimmunoassay (SPRIA) in which IgM was used as capture antibody and iodinated IgM was used as a probe. This assay was compared with a conventional IgG-based SPRIA. The IgM-based assay resulted in a superior response (P/N) for the detection of antigen in crude preparations; however, endpoint analyses demonstrated similar sensitivities.

Acute Disease↗
PubMed1990

Measurement of association constants in ELISA. Reactions between solid-phase antibody and fluid-phase biotinylated antigen.

An ELISA procedure which utilized an avidin-peroxidase amplification reaction to detect and quantify the amount of fluid-phase biotinylated antigen bound to solid-phase antibody was developed to determine antibody association constants. The methodology required is uncomplicated, avoids the use of radioisotopes, and is theoretically amenable for use with any protein which can be biotinylated, and any receptor protein which can be immobilized on plastic wells. A plot of known immobilized biotinylated antigen concentrations, which ranged from approximately 100 ng/ml to 1000 ng/ml, versus avidin-peroxidase conjugate product formation, was used to establish a standard curve from which the amount of antibody-associated antigen in binding assays could be determined. Antibody association constants obtained with this method ranged from 10(5) to 10(9) M-1.

Animals↗
PubMed1993

The value of indirect immunofluorescence and solid phase techniques for ANCA detection. A report on the first phase of an international cooperative study on the standardization of ANCA assays. EEC/BCR Group for ANCA Assay Standardization.

This study describes the results of phase I of an international effort to develop and standardize assays for the detection of anti-neutrophil cytoplasmic antibodies (ANCA). 12 sera, four of which were selected for their potential to cause problems in the detection of various ANCA specificities, were analyzed in the standard indirect immunofluorescence (IIF) test and in ELISAs for ANCA routinely performed in the seven participating laboratories. The IIF methodology differed with respect to the dilution of the serum being screened and the concentration of the conjugate used. Results from sera with high ANCA titers were similar, although the quantitative values could not be compared. In sera containing rheumatoid factor and anti-nuclear antibodies (ANA), ANCA-unrelated staining patterns were observed. Six antigen preparations were used in ELISA for the detection of cANCA. In ELISA with purified proteinase-3 all three cANCA sera were positive, but not anti-myeloperoxidase (MPO) or anti-lactoferrin (LF) positive sera. The other assays were less sensitive or gave inconsistent results. Various preparations of purified MPO and LF used in ELISA were readily recognized by anti-MPO and anti-LF positive sera. From this study it can be concluded that the IIF test, although performed with different methods, shows comparable results using strongly positive sera. In general solid phase assays for cANCA detection are not well standardized and need improvement although the purified proteinase-3 ELISA is possibly an exception. MPO and LF can be used in ELISA procedures for the detection of pANCA-related antibodies.

Antibodies, Antineutrophil Cytoplasmic↗
PubMed1984

Regions in the ribosomal minichromosome of Physarum polycephalum are protected from restriction nucleases; protection is insensitive to high salt in the G phase and sensitive in the M phase of the cell cycle.

In the central spacer region of the extrachromosomal ribosomal DNA of the slime mold Physarum polycephalum, four small regions of related sequence are completely inaccessible to restriction endonucleases (HinfI and MboI). In addition, some sequences neighboring the inaccessible ones, are partially inaccessible to restriction enzymes and micrococcal nuclease. Taking advantage of the natural synchrony of Physarum plasmodia, we found that this protection is present throughout the cell cycle. Treatment with high salt (2.5 M-NaCl) of nuclei from the G2 phase of the cell cycle left the protection essentially unchanged. When nuclei from the M phase were treated with salt, the protection was abolished. The inaccessible sites are located close to the origins of replication of the rDNA.

Base Sequence↗
PubMed1987

Changes in phase I and phase II biotransformation with age in male Fischer 344 rat colon: relationship to colon carcinogenesis.

Using male Fischer 344 rats classified as young (2-4 months), middle aged (12-14 months), and old (22-25 months), the activities of several Phase I and Phase II biotransformation pathways in the large intestine were investigated, including benzo[a]pyrene hydroxylase (BPOH), alcohol dehydrogenase (ADH), glutathione S-transferase (GST), glutathione peroxidase (GSH-PX), beta-glucuronidase (BG), and microsomal and nuclear glucuronyltransferase (UDPGT). Levels of oxidized (GSSG) and reduced (GSH) glutathione and uridine 5'-diphosphoglucuronic acid (UDPGA) were also measured. BPOH increased 33% in old rats, while ADH and BG activity remained unchanged with age. Nuclear UDPGT remained unchanged with age, whereas form I of GSH-PX declined slightly in old rats. GST, microsomal UDPGT, and form II of GSH-PX declined by 38, 37 and 44%, respectively, in old rats. The decrease in GST and microsomal UDPGT was also significant in middle aged rats. Levels of colonic GSH, GSSG and UDPGA were found to be unchanged with age. These in vitro data suggest the possibility that if reactive intermediates are generated to the same extent in old rats as in young rats, decreased detoxification mechanisms in the old rat may increase susceptibility of the colon to actions of chemical carcinogens.

Aging↗
PubMed1992

Alterations in hepatic phase I and phase II biotransformation enzymes by garlic oil in rats.

Studies were conducted to examine the effect of a single and repeated administrations of garlic oil (diallyl sulfide) on Phase I and Phase II biotransformation enzymes in rats. Adult, male Sprague-Dawley rats treated with a single dose of garlic oil (500 mg/kg i.p.) showed a significant depression of hepatic cytochrome P-450, aminopyrine N-demethylase and aniline hydroxylase while microsomal protein content, cytochrome b5, NADPH-cytochrome c reductase, benzphetamine N-demethylase and cytosolic glutathione, S-transferase remained unaffected 24 h following the treatment. Although certain microsomal enzymes were depressed, there was no liver damage caused by garlic oil as judged by the putative serum enzyme test. On the other hand, daily administration of garlic oil (50 mg/kg i.p. for 5 days) produced a significant increase in hepatic cytochrome P-450, aminopyrine N-demethylase and benzphetamine N-demethylase activities, but not in the rest of the aforementioned parameters of biotransformation reactions. These data indicate that the effect of garlic oil on the hepatic drug-metabolizing enzyme system is dose-dependent.

Allyl Compounds↗
PubMed1993

On-line fully automated determination of clozapine and desmethylclozapine in human serum by solid-phase extraction on exchangeable cartridges and liquid chromatography using a methanol buffer mobile phase on unmodified silica.

A fully automated method for determination of clozapine and desmethylclozapine in human serum using high-performance liquid chromatography was developed. On-line solid-phase extraction was performed on an exchangeable cyanopropyl cartridge. The analytes were eluted with a methanol-ammonium acetate buffer mobile phase, separated on a silica column, and measured by ultraviolet detection at 261 nm. The total time for one analysis was 13 min. Inter-day variation was < 6% and < 8% for clozapine and desmethylclozapine, respectively. Detector response was linear in the range of 30-300 ng/ml. Comparison with liquid-liquid extraction showed good agreement. The patients had clozapine serum concentrations in the range 40-1500 ng/ml. Desmethylclozapine concentrations were 25% lower and closely related.

Autoanalysis↗
PubMed1993

High-performance liquid chromatographic separation of ondansetron enantiomers in serum using a cellulose-derivatized stationary phase and solid-phase extraction.

R(-)-Ondansetron and S(+)-ondansetron in human serum were resolved and quantified using a stereospecific HPLC method. Each enantiomer and the internal standard prazosin were isolated from serum using a solid-phase extraction procedure on a cyanopropyl column. Recoveries of 97, 96 and 88% were obtained for the R(-)-enantiomer, the S(+)-enantiomer, and the internal standard, respectively. A cellulose-based chiral analytical column (Chiralcel OD) was used with a mobile phase consisting of hexane-95% ethanol-2-propanol-acetonitrile (65:25:10:1, v/v). Linear calibration curves were obtained for each enantiomer in serum in the concentration range 10-200 ng/ml. The limit of quantitation of each enantiomer was 10 ng/ml. The detection limit for each enantiomer in serum using UV detection at 216 nm was 2.5 ng/ml (signal-to-noise ratio of 3).

Cellulose↗
PubMed1995

Improved amphetamine and methamphetamine determination in urine by normal-phase high-performance liquid chromatography with sodium 1,2-naphthoquinone 4-sulphonate as derivatizing agent and solid-phase extraction for sample clean-up.

Solid-phase extraction techniques were evaluated for the treatment of urine samples in the analysis of amphetamine and methamphetamine by normal-phase high-performance liquid chromatography with 1,2-naphthoquinone 4-sulphonate. Six different packing materials were tested, and the results obtained are compared with those obtained in a classical liquid-liquid extraction with n-hexane. Different clean-up eluents and the influence of pH of urine have been tested. The intra-day and inter-day precision, the accuracy of the method and the addition of beta-phenylethylamine as internal standard were also studied.

Amphetamine↗
PubMed1995

Separation and identification of phase I and phase II [14C]antipyrine metabolites in rat and dog urine.

A simple and accurate HPLC procedure was developed to quantify, in a single run, all phase I and phase II [14C]antipyrine metabolites that occur in rat and dog urine. All metabolites were subjected to thermospray-LC-MS and EI-MS in order to establish their structure. The rat metabolizes antipyrine to eight major metabolites, six of which are conjugated; 1.4% of the dose was excreted unchanged, 18.9% in a free form, 30.6% as sulfates and 21.1% as glucuronides. The dog metabolizes antipyrine to four metabolites, all as sulfate (61.0% of the dose) or glucuronide conjugates (16.2% of the dose).

Animals↗
PubMed1996

Theory of phase formation in aqueous two-phase systems.

Currently there are a number of different mathematical models for phase equilibria in aqueous two-phase systems available. This diversity can create some confusion for model users, since most models seem to perform reasonably well. Choosing a model, thus, becomes rather a difficult task. In trying to address this problem, the principal models and the relevant theory available are reviewed. A discussion of osmotic viral expansions, lattice theory, group contribution, scaling ideas, excluded volume, electrostatics and other modeling approaches is presented. The strengths of the different approaches are critically evaluated and suggestions offered. Choosing a model, however, requires sophistication because each model is typically best at representing only a few particular aspects of system behavior, and the intended use of the model must be considered. Some suggestions for future work are also given.

Computer Simulation↗
PubMed1994

Amrinone and N-acetylamrinone assay in human plasma using solid-phase extraction and reversed-phase chromatography.

A simple and sensitive liquid chromatographic assay for simultaneous quantitation of amrinone and N-acetylamrinone in human plasma was developed. The method involves extraction of samples via activated solid-phase extraction Bond Elut C18 disposable columns, followed by chromatographic separation on a reversed-phase phenyl column using isocratic condition and UV detection. The assay can measure concentrations of both compounds over the range 0.075-10 micrograms ml-1. The injection interval is 11 min. The inter-day relative standard deviation (RSD) for replicate analysis of spiked samples is less than 10% and the accuracy more than 94% for both compounds over the standard curve range. The assay has been successfully applied to pharmacokinetic studies in humans.

Amrinone↗
PubMed2004

UV-visible spectral identification of the solution-phase and solid-phase permanganate oxidation reactions of thymine acetic acid.

Solution-phase and solid-phase permanganate oxidation reactions of thymine acetic acid were investigated by spectroscopy. The spectral data showed the formation of a stable organomanganese intermediate, which was responsible for the rise in the absorbance at 420 nm. This result enables unambiguous interpretation of the absorbance change at 420 nm, as the intermediate permanganate ions could be isolated on the solid supports.

Acetic Acid↗
PubMed2004

Solid-phase extraction versus solid-phase microextraction for the determination of chlorinated paraffins in water using gas chromatography-negative chemical ionisation mass spectrometry.

Solid-phase extraction (SPE) and solid-phase microextraction (SPME) were evaluated for the analysis of short-chain chlorinated paraffins (SCCPs) in water samples using gas chromatography coupled to negative chemical ionisation mass spectrometry (GC-NCI-MS). For SPE optimisation, four commercially available SPE cartridges were tested and several SPE parameters, such as the elution solvent, elution volume and breakthrough volume were studied. The best results were obtained with Varian Bond Elut-C18. In order to achieve a high selectivity in the determination of SCCPs, GC-NCI-MS was used. Quality parameters of the optimised SPE and SPME procedures were determined, and the best results were obtained for the SPE/GC-NCI-MS method with LODs of 5 and 20 ng l(-1) for tap and river water, respectively. This method was successfully applied to the analysis of SCCPs in river water samples at concentrations below the microg l(-1) level.

Chlorine↗
PubMed2004

Optimization strategies for the analysis and purification of drug discovery compounds by reversed-phase high-performance liquid chromatography with high-pH mobile phases.

Careful selection of both high-pH mobile phase as well as organic modifier, was performed in order to develop and optimize HPLC conditions for the separation of drug discovery compounds. High-pH mobile phases provide excellent chromatographic resolution and increased mass loading of basic compounds. The analytical methods so defined have been successfully transferred to preparative automated UV-directed purification, an important fact due to the increasing number of samples requiring purification. It should be noted that, the single prerequisite for this approach is an analytical LC-UV-MS run, therefore the system has the ability to collect only fractions likely to contain the target product. A cost-effective strategy for maximizing the purification of drug discovery compounds is proposed.

Chromatography, High Pressure Liquid↗
PubMed2004

Comprehensive two-dimensional separations of complex mixtures using reversed-phase reversed-phase liquid chromatography.

A comprehensive two-dimensional reversed-phase reversed-phase liquid chromatographic system for the separation of a complex mixture of oligostyrenes was developed using results from a previous theoretical assessment of the informational similarity, percent synentropy, orthogonality and peak capacity of hypothetically coupled systems. The degree of sample attribute order in the first separation dimension was also used in the development of the experimental two-dimensional system. A C18(methanol)/CCZ(acetonitrile) two-dimensional system was chosen for the comprehensive analysis of the oligostyrene mixtures because this system had the lowest solute crowding, highest orthogonality and was observed to have order with respect to a sample attribute in the first separation dimension. The separations achieved were in full agreement with the results from information theory and (a geometric approach to) factor analysis assessments. High sampling rates in the first liquid chromatographic dimension were shown to be impossible or inefficient when the peak capacity and separation time of the second dimension was high or when the aim of the exercise was to isolate individual sample constituents in high yield.

Chromatography, Liquid↗