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At least 865 records · Page 48Linked to original sources
PubMed1993

Sample preparation for bivariate flow cytometric analysis of breast tumor specimens.

The availability of monoclonal antibodies (MAbs) raised against functional or phenotypic cell markers can improve the suitability of flow cytometry (FCM) in assessing and validating prognostic parameters of human neoplasia. The major problems encountered in this type of study, when performed on solid tumors, are the recovery of a large and representative number of tumor cells and the possibility of performing a DNA analysis on antigen-selected populations. A panel of MAbs was chosen on the basis of the recognized membrane antigens important to the biologic characterization of human breast cancer cell populations. The possibility of performing bivariate FCM analysis of cell surface antigens and DNA content was investigated on 4 breast tumor cell lines and 10 fresh breast biopsies. Different fixations and tumor disaggregation procedures were considered. The results indicate that ethanol fixation is the most effective procedure for performing biparametric FCM analysis and that a combined enzymatic-mechanical disaggregation procedure applied on breast tumor specimens allows great recovery of intact cells without affecting antigen detection or DNA distribution.

Antibodies, Monoclonal↗
PubMedDate not supplied

Automated sample preparation for monitoring groundwater pollution by carbamate insecticides and their transformation products.

We investigated automated on-line solid-phase extraction (SPE) followed by liquid chromatographic (LC) techniques for monitoring carbamates and their transformation products. Analytical determinations were performed by LC with UV or postcolumn fluorescence detection (U.S. Environmental Protection Agency Method 531.1 for carbamate insecticides) after preconcentration with on-line SPE using C18 Empore extraction disks. On-line SPE/LC/thermospray mass spectrometry with time-scheduled selected-ion monitoring was used as confirmatory method. The method was used to determine pesticide traces in well waters of a typical aquifer in the Almeria area (Andalucia, south of Spain) from March 1993 to February 1994. The major pollutants, found in highest amounts, were carbofuran, methiocarb, and methomyl, at levels of 0.32, 0.3, and 0.8 micrograms/L, respectively. According to results of seasonal variation studies, pollution by carbamate insecticides is sporadic and exceeds the limit of 0.5 micrograms/L for total pesticides allowed by the European Economic Community Drinking Water Directive only twice a year. 3-Hydroxycarbofuran and methiocarb sulfone also were detected, showing the importance of including the main toxic break-down products of carbamate insecticides in future monitoring programs.

Autoanalysis↗
PubMed1991

Storage and preparation of samples for erythropoietin radioimmunoassay.

The evaluation of a radioimmunoassay for erythropoietin developed using recombinant material as immunogen and radiotracer is presented. A series of serum samples prepared and stored under varying conditions showed that immunoreactive erythropoietin levels were stable at room temperature for at least 10 days and at -20 degrees C for 5 months. The optimum time for separating sera from samples was between 6 and 24 h after venepuncture. Serum EPO values were significantly higher than those measured in heparin or potassium EDTA plasma.

Antibodies↗
PubMed1999

The catalytic mechanism of EPSP synthase revisited.

Recent analysis of EPSP synthase by solid-state NMR has led to the postulation of a new enzyme reaction pathway and raised once again the question of an intermediate species covalently bound to the enzyme [Studelska, D., McDowell, L., Espe, M., Klug, C., and Schaefer, J. (1997) Biochemistry 36, 15555-15560]. Therefore, we have reexamined the mechanism of the reaction catalyzed by EPSP synthase and analyzed the reaction products formed under the conditions used in preparing samples for solid-state NMR. Single-turnover experiments were carried out using both [1-14C]- and [32P]PEP showing the formation and decay of the previously proposed tetrahedral intermediate species on a time scale comparable with the disappearance of substrate and formation of product, thus unequivocally establishing the kinetic competence. The possible presence of a covalently bound enzyme intermediate species was also investigated, using SDS-PAGE and Centricon concentration analysis of the quenched reaction samples. No covalently bound enzyme intermediates were observed during the reaction. An enzyme assay was also performed repeating the conditions used in sample preparation for the solid-state NMR studies. We show that under these conditions, total turnover of substrates to products was observed within 45 s at -30 degrees C prior to freezing and lyophilization. Following lyophilization, the samples were stored at -20 degrees C and analyzed over a period of 21 days. We observed the conversion of the product EPSP into the side product, a cyclic EPSP ketal, and the breakdown product, pyruvate. Thus, the new species reported by solid-state NMR can be accounted for by previously characterized reaction products and side products formed during sample preparation and upon incubation in the solid-state. Our conclusions are also supported by the solution and solid-state NMR studies recently reported [Jakeman et al. (1998) Biochemistry 37, 12012-12019]. These results once again highlight the importance of kinetic competence as a criterion to be used in defining enzyme intermediates and point to the errors in interpretation of results when the time dependence of formation of the proposed intermediates is not considered.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed2001

Influence of processing conditions on the nutritive value of Ogi-baba, a Nigerian fermented sorghum gruel.

The effect of germination, cowpea fortification and fermentation on the chemical and amino acid composition of ogi-baba, a Nigerian fermented sorghum gruel was investigated. The lowest protein value of 1.4% was obtained in the traditionally prepared sample, while samples prepared from germinated, fortified sorghum had 13%. The ash ranged from 0.9% to above 2%, while the fat was in the range of 1.2% to 2.8%. Generally, fortification, in addition to germination, improved the chemical composition of ogi-baba relative to the control sample. There was improvement in the amino acid profile of all the ogi-baba samples compared to the unprocessed sorghum grains. Lysine, a limiting amino acid in sorghum, increased more than 50% in ogi-baba. However, samples prepared using germinated sorghum without the addition of cowpeas had a lower lysine content.

Amino Acids↗
PubMed1987

Use of alumina columns to prepare plasma samples for liquid-chromatographic determination of catecholamines.

We describe an improved method of sample preparation for liquid chromatographic determination of plasma catecholamines. The catecholamines are extracted from plasma by using small, cheaply-made columns of alumina, with or without prior clean-up on commercially available ion-exchange columns. Advantages of this technique over the conventional batch-extraction method of using alumina are speed, convenience, and improved sample clean-up. In particular, the one-stage method we describe allows results to be reported within 20 min of receiving the sample.

Aluminum↗
PubMed2006

Quantification of estrogenic mycotoxins at the ng/L level in aqueous environmental samples using deuterated internal standards.

Because of their pronounced estrogenicity, resorcyclic acid lactones (RALs) are of concern in aqueous environments even at the low ng/L level. Therefore, we developed an accurate, precise and sensitive HPLC-MS/MS method to detect these mycotoxins in different aqueous environmental samples. The compounds investigated included zearalenone (ZON), alpha- and beta-zearalenol, zearalanone as well as alpha- and beta-zearalanol. The use of isotope labelled internal standards (in this case deuterated RAL-analogues) ensured an accurate quantification of the target analytes, independent of matrix compounds interfering with the analytes during ionisation and analyte losses occurring during sample preparation. Sample enrichment was carried out by solid-phase extraction (SPE) using Supelclean Envi-18 cartridges. Absolute method recoveries for all analytes ranged from 95 to 108%, 70 to 102%, and 76 to 109%, method detection limits from 0.5 to 2.1 ng/L, 0.4 to 1.1 ng/L, and 0.8 to 12.4 ng/L and precision from 3 to 14%, 2 to 13% and 4 to 16% in drainage water, river water and wastewater treatment plant (WWTP) effluent, respectively. The method was applied to verify the emission of RALs from a Fusarium graminearum infested crop field into the drainage system. Zearalenone was present in drainage water in concentrations up to 30 ng/L. So far, none of the other five investigated compounds have been detected.

Analytic Sample Preparation Methods↗
PubMed1993

Ion-exchange liquid chromatographic analysis of bisphosphonates in pharmaceutical preparations.

A simple, fast and uniform method has been developed for the quantitative determination of bisphosphonates in the quality control of pharmaceutical preparations. The method is based on ion-exchange liquid chromatography with conductivity detection. Separation is performed on a Waters IC-PAK Anion column using 2 mM nitric acid or 25 mM succinic acid as the mobile phase. Retention of the bisphosphonates can be influenced by pH and the anion concentration of the mobile phase. Sensitivity and selectivity are sufficient for the assay of bisphosphonates in bulk drug and pharmaceutical preparations. Sample preparation comprises dissolution or dilution of the sample in the mobile phase followed, if necessary, by filtration prior to HPLC analysis. Since the method is stability indicating, it is also well suited for shelf-life studies of bisphosphonate pharmaceutical preparations. Validation of the analytical method for the assay of pamidronate injection indicated an intra-day reproducibility of 1.7% (n = 6) and an inter-day reproducibility of 2.7% (n = 6). A linear relationship between response and concentration was found in the concentration range studied from 200 ng to 10 micrograms pamidronate per 20 microliters injected. The lower limit of detection (signal-to-noise ratio = 3) of pamidronate was about 100 ng.

Chemistry, Pharmaceutical↗
PubMed1991

Improved sensitivity and resolution in the flow cytometric DNA analysis of human solid tumor specimens. Use of in vitro fine-needle aspiration and uniform staining reagents.

Twenty-five unselected fresh colon or breast tumors were studied to identify specific components of sample preparation, sample staining, and flow cytometer operation affecting the sensitivity of DNA stemline analysis. Solid tumors were disaggregated using both a published method for mechanical/enzymatic whole cell (M/EWC) isolation and a fine-needle aspiration (FNA) technique. Staining FNA samples with CycleTEST propidium iodide reagents demonstrated improved sensitivity in the recognition of near diploid and near tetraploid aneuploid populations: 9 of 20 resolvable aneuploid DNA stemlines identified in FNA suspensions were not detected or clearly resolved in M/EWC preparations. These results suggest that previously reported discordances between flow and static image cytometry in the recognition of near tetraploid DNA stemlines may be related to inherent limitations of M/EWC. The in vitro FNA technique, when compared to M/EWC, may yield increased sensitivity and precision in clinical DNA stemline analysis when using fresh, unfixed, solid tumor specimens.

Aneuploidy↗
PubMed1997

[Discussion of the validation of thin-layer chromatographic procedure in pharmaceutical analysis].

TLC has become a convenient, fast, robust, and cost efficient technique in pharmaceutical analysis because of its coherent features. The important analytical performance parameters are summarized, such as accuracy, precision, reproducibility, specificity, detection limit, quantitation limit, linearity and range, etc. The validation methods and the acceptance criteria are discussed for sample preparation, sample stability in solution and on plate, sample exposure time, robustness and quality of stationary and mobile phases, application of sample to plate, spot dimension and shape, temperature, humidity and chamber saturation, detection, and quantitation, etc. It is recommended that the definition of the validation parameters and the validation procedures for quantitative TLC should be described whenever the results are reported.

Chromatography, Thin Layer↗
PubMed1993

Carbon-13 "magic-angle" sample-spinning nuclear magnetic resonance studies of human myelin, and model membrane systems.

We have obtained high-field (11.7 Tesla), high-resolution carbon-13 solid-state "magic-angle" sample-spinning nuclear magnetic resonance (NMR) spectra of a variety of phospholipids, sphingolipids, myelin and white matter samples, resolving and assigning over 40 resonances in the spectra of human and bovine myelin. The NMR results indicated no large spectral changes due to sample preparation, sample freezing, or brain location, and also no changes in myelin structure detectable via light microscopy, electron microscopy, thin layer chromatography, or sodium dodecyl sulfate-polyacrylamide gel electrophoresis, attributable to the sometimes lengthy NMR data acquisition process. Human myelin and white matter chemical shift assignments were made based on 13C "magic angle" sample spinning (MAS) NMR spectra of individual model lipids, as well as on spectra of lipid mixtures. In all myelin samples there were essentially no features attributable to membrane proteins, with the exception of one small feature due to C zeta of Arg residues, primarily in the myelin basic proteins. The general similarity between the model lipid and intact myelin spectra suggested no major effects of protein on lipid mobility. We have also investigated human myelin samples as a function of developmental age (4, 15, 48 months and adult), and our results showed only small changes in overall lipid composition, although there were significant decreases in lipid hydrocarbon chain unsaturation with age, as determined by computer line-shape simulations of myelin and model compounds. The spectrum of an infant leukoencephalopathy myelin showed marked decreases in galactocerebrosides. Overall, the ability to resolve and assign over 40 resonances in the 13C MAS NMR spectra of myelin, and to detect changes as a function of development and disease, should provide a useful starting point for further more detailed studies of myelin membrane molecular motions, and function.

Adult↗
PubMed2005

Development and validation of a stability-indicating high-performance liquid chromatographic assay for ketoprofen topical penetrating gel.

An isocratic RP-HPLC procedure has been developed and validated for the quantitative determination of ketoprofen in a topical gel. The HPLC procedure consist of a YMC ODS-AQ, 5-microm particle size analytical column (150 mm x 4.6 mm); Alltech Econosphere C18, 5-microm particle size guard column; detection at 233 nm; 1 ml/min flow rate; 20-microl injection volume. The mobile phase consisted of pH 3.5 phosphate buffer-water-acetonitrile (8:43:49, v/v). Sample preparation was a simple extraction of ketoprofen with mobile phase. The above conditions resolved and eluted ketoprofen, excipients, and potential degradants within 35 min, with ketoprofen eluting at about 6.5 min. The procedure was validated with respect to specificity, accuracy, precision, and linearity. The accuracy of the procedure, determined by spike recovery measurements, was 100.1-100.5%. The intra- and inter-day precisions were demonstrated by the relative standard deviations (RSD) of 0.3-0.6% and 0.5%, respectively. The intermediate precision was determined by comparing the results obtained with four independently prepared samples by two chemists using two columns on different days. The results indicate no significant difference (P = 0.17). The procedure showed linearity over the concentration range 4 x 10(-5) to 1 x 10(-1) mg/ml.

Administration, Topical↗