Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “sample preparation”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 595 records · Page 33Linked to original sources
PubMed2000

Liquid-phase microextraction as a sample preparation technique prior to capillary gas chromatographic-determination of benzodiazepines in biological matrices.

Liquid-phase microextraction (LPME) and gas chromatography were applied to determine diazepam and the main metabolite N-desmethyldiazepam in human urine and plasma. The analytes were extracted from 3.0-3.5 ml sample volumes directly into 25 microl of extraction solvent. The microextraction device consisted of a porous hollow fiber of polypropylene attached to two guiding needles inserted through a septum and a 4 ml vial. The hollow fiber filled with extraction solvent was immersed in sample solution. The extraction device was continuously vibrated at 600 rpm for 50 min. An aliquot (1 microl) of the extraction solvent with preconcentrated analytes was injected directly into the capillary gas chromatograph. Thirty samples were extracted simultaneously on the vibrator, providing a high sample capacity. The limits of detection were from 0.020 to 0.115 nmol/ml for diazepam and N-desmethyldiazepam in plasma and urine using a nitrogen-phosphorus detector (NPD).

Chromatography, Gas↗
PubMed2001

Development and validation of an automated method for the liquid chromatographic determination of sotalol in plasma using dialysis and trace enrichment on a cation-exchange pre-column as on-line sample preparation.

A fully automated method for the determination of sotalol in human plasma was developed, involving dialysis through a cellulose acetate membrane, clean-up and enrichment of the dialysate on a strong cation-exchange pre-column and subsequent liquid chromatographic (LC) analysis with UV detection. All sample handling operations were carried out by means of an ASTED system. Before starting dialysis, the trace enrichment column (TEC) was conditioned. The plasma sample, to which the internal standard (atenolol) was automatically added, was then loaded in the donor channel and was kept static while the dialysis liquid, consisting of 0.017 M acetic acid, was passed through the acceptor channel in successive pulses. After each pulse, the dialysate was dispensed onto the TEC. When dialysis was discontinued, the analytes were eluted from the TEC by the LC mobile phase by rotation of a switching valve and transferred to the analytical column packed with octyl silica. The LC mobile phase was a mixture of methanol and pH 7.0 phosphate buffer containing 1-octanesulfonate at a concentration of 7.5 x 10(-4) M (19:81; v/v). The UV detection was performed at 230 nm. The influence of several parameters of the dialysis and trace enrichment processes on analyte recovery and method selectivity was investigated. The method was then validated. The mean absolute recovery for sotalol was about 60%. The limit of quantitation was 25 ng/ml and R.S.D. for repeatability and intermediate precision obtained at a concentration level of 50 ng/ml were 4.3 and 5.8%, respectively.

Automation↗
PubMed1989

Development of the quick-freeze, deep-etch, rotary-replication technique of sample preparation for 3-D electron microscopy.

The images presented here, though covering a size-range from whole cells to individual molecules, have all be generated by the exact same technique, as was outlined in the introduction. All depend upon the high quality of the initial freezing of the sample, which is accomplished by the abrupt application of an ultra-pure copper block cooled with liquid helium. Copies of the freezing machine we have developed to accomplish this are currently manufactured and supplied to other laboratories at cost, by former university machinists who now operate under the name of Med-Vac, Inc., St. Louis, MO. Any laboratory wishing to generate images of the sort shown here are welcome to obtain one of these machines and to call us for further assistance and instruction.

Animals↗
PubMed1977

Quantitative morphological analysis of early mouse embryogenesis in vitro. I. Perfusion culture system, tissue preparation, sampling.

To facilitate a quantitative morphological analysis of early mouse development under controlled conditions, a perfusion culture system capable of supporting embryogenesis to blastocyst stage has been developed. The use of a mesh system allows identification of individual embryos by position, and control of their orientation during culture and preparation for light and electron microscopy. Quantitative evaluation of tissue-processing procedures has permitted selection of conditions which reduce changes in linear dimensions to -1-6 +/- 1-8% in two-cell embryos. Through the definition of a coordinate system based on mesh structure and the development of a special sectioning procedure, sections can be localized within the intact embryo and three-dimensional coordinates given to any element of embryo volume.

Animals↗
PubMed1997

Sample preparation from paraffin-embedded tissue specimens for laser scanning cytometric DNA analysis.

We have developed a simple, rapid method for isolating cells from a block of formalin-fixed, paraffin-embedded tissue specimen for laser scanning cytometric (LSC) DNA analysis by using a grater. The scraping-like tissue samples were obtained by grating a paraffin-embedded tissue block. The grated samples were collected, put into a small plastic tube, and deparaffinized with xylene. Subsequently, the samples were immersed in 100% ethanol to remove the xylene. After using a syringe with a 26-gauge needle and filtering through 40-microm nylon mesh, the cells suspended in ethanol were dropped directly onto a glass slide. As a result, isolated cells adhered tightly to the glass slide. The slides mounted with isolated cells were treated with 0.1% pepsin in 0.1 N HCl for 1 h at 37 degrees C and then 0.1% RNase for 10 min at room temperature. The slides were dipped in propidium iodide (25 microg/ml) to stain DNA and sealed with nail varnish. The coefficients of variation for histograms were small enough to detect an aneuploid peak close to the diploid peak.

Aneuploidy↗
PubMed2003

High-performance liquid chromatographic analysis of nitrendipine in human plasma using ultraviolet detection and single-step solid-phase sample preparation.

A high-performance liquid chromatographic (HPLC) method was developed for the determination of nitrendipine in human plasma using solid-phase extraction (SPE) and ultraviolet detection. A 30-microl aliquot of methanol (containing 2 microg/ml of the internal standard, nimodipine) was added to a 1-ml aliquot of biological sample. After vortex-mixing, the mixture was loaded on C(18) SPE cartridge which was conditioned with methanol and distilled water. After washing with distilled water, the SPE cartridge was eluted with 1-ml aliquot of diethyl ether. The organic phase was collected and evaporated under nitrogen gas. The residue was then reconstituted with a 100 microl aliquot of mobile phase, and a 50 microl aliquot was injected onto the C(18) reverse-phased column. The mobile phase, 10 mM phosphate buffer (pH 4.5):acetonitrile (50:50, v/v), was run at a flow rate of 1.2 ml/min. The column effluent was monitored using ultraviolet detector at 238 nm. The retention times for nitrendipine and the internal standard were approximately 10.1 and 12.6 min, respectively. The detection limit of nitrendipine in human plasma was 2.0 ng/ml. The coefficients of variation (CV) of the assay were below 16.5% for human plasma, and no interferences from endogenous substances were found. This specific, accurate and precise assay was useful in the study for the pharmacokinetic characteristics of nitrendipine.

Chromatography, High Pressure Liquid↗
PubMed1987

Effect of temperature and sample preparation on performance of ion-moderated partition chromatography of organic acids in biological fluids.

A thorough investigation of the behavior of organic acids on the Bio-Rad Aminex cation exchange resin was prompted by both the limitations of, and a number of inexplicable inconsistencies found in, previously published papers using an identical system. In order to stabilise the elution order of various acids it was necessary to analyse samples at a higher temperature than previously recommended. This temperature (50 degrees C) decreased the retention times of all acids permitting the analysis of both aromatic and aliphatic acids within the same 45-min run. Preparation of an acidic fraction of biological fluids improved specificity, allowed direct comparison of urine and plasma profiles and by control of the conditions interference by urate could be substantially reduced. Retention data are given for more than 90 acidic metabolites including nearly 40 of clinical significance and a number derived from diet and drug therapy.

Carboxylic Acids↗
PubMed1996

Automated on-line dialysis for sample preparation for gas chromatography: determination of benzodiazepines in human plasma.

An on-line dialysis-solid-phase extraction-gas chromatographic (GC) approach has been developed for the determination of drugs in plasma, using some benzodiazepines as model compounds. Clean-up is based on performing the dialysis of 100 microliters samples for 7 min using water as acceptor phase and trapping the diffused analytes on a PLRP-S copolymer precolumn. After drying of the precolumn with nitrogen for 15 min, the analytes are desorbed with ethyl acetate (275 microliters) and injected on-line into the GC system via a loop-type interface. The system provides a very efficient clean-up, and offers the possibility of adding chemical agents which can help to reduce drug-protein binding and, thus, increase sensitivity. To demonstrate the potential of the described approach, the determination of benzodiazepines in plasma at their therapeutical levels is used as an example with flame ionization, thermionic and mass-selective detection.

Anti-Anxiety Agents↗
PubMed1996

New polyacrylamide gel-based methods of sample preparation for optical microscopy: immobilization of DNA molecules for optical mapping.

New methods have been developed for rapid immobilization of biological macromolecules and other microscopic objects from aqueous solution at gel/gel, gel/solid and gel/solution interfaces using thin polyacrylamide gels covalently bound to glass surfaces. When quickly spread over a dry gel, an aqueous sample loses most of its water and low-molecular-weight solutes due to migration of these components into the gel. All optically observable objects thus become concentrated at the gel surface and may be easily located by light microscopy. Based on this, a procedure for binding DNA at a positively charged gel/solution interface was developed. A mild immobilization of the DNA molecules was obtained, allowing 'all in focus' observations of DNA digestion by restriction endonucleases with an apparent rate close to that in solution.

Acrylamides↗
PubMed1999

Comparison of turbulent-flow chromatography with automated solid-phase extraction in 96-well plates and liquid-liquid extraction used as plasma sample preparation techniques for liquid chromatography-tandem mass spectrometry.

Turbulent flow chromatography (TFC) combined with the high selectivity and sensitivity of tandem mass spectrometry (MS-MS) is a new technique for the fast direct analysis of drugs from crude plasma. TFC in the 96-well plate format reduces significantly the time required for sample clean-up in the laboratory. For example, for 100 samples the workload for a technician is reduced from about 8 h by a manual liquid-liquid extraction (LLE) assay to about 1 h in the case of TFC. Sample clean-up and analysis are performed on-line on the same column. Similar chromatographic performance and validation results were achieved using HTLC Turbo-C18 columns (Cohesive Technologies) and Oasis HLB extraction columns (Waters). One 96-well plate with 96 plasma samples is analyzed within 5.25 h, corresponding to 3.3 min per sample. Compared to this LLE and analysis of 96 samples takes about 16 h. Two structurally different and highly protein bound compounds, drug A and drug B, were analyzed under identical TFC conditions and the assays were fully validated for the application to toxicokinetics studies (compliant with Good Laboratory Practices-GLP). The limit of quantitation was 1.00 microg/l and the linear working range covered three orders of magnitude for both drugs. In the case of drug A the quality of analysis by TFC was similar to the reference LLE assay and slightly better than automated solid-phase extraction in 96-well plates. The accuracy was -3.1 to 6.7% and the precision was 3.1 to 6.8% in the case of drug A determined for dog plasma by TFC-MS-MS. For drug B the accuracy was -3.7 to 3.5% and the precision was 1.6 to 5.4% for rat plasma, which is even slightly better than what was achieved with the validated protein precipitation assay.

Animals↗
PubMed2000

Comparison of commercial solid-phase extraction sorbents for the sample preparation of potato glycoalkaloids.

In this study five different commercial sorbents C18, SCX, CN, Certify and Oasis HLB were compared for the solid-phase extraction of potato glycoalkaloids. The recoveries were determined using alpha-solanine, alpha-chaconine and alpha-tomatine, which contained dehydrotomatine as an impurity, as standard compounds. The samples were analysed by reversed-phase liquid chromatography under gradient elution conditions using a Zorbax Rx-C18 column and acetonitrile-25 mM triethylammonium phosphate buffer (pH 3.0) as the mobile phase. The highest recovery (approximately 100%) was achieved with Oasis HLB (60 mg) cartridges. An acetic acid extract of wild Solanum brevidens leaf material was used for the testing of a clean-up procedure. The SCX proved to be the most selective and efficient for removing the undesired components from the leaf extract.

Alkaloids↗
PubMed1997

Detection of Coxiella burnetii in cow's milk by PCR-enzyme-linked immunosorbent assay combined with a novel sample preparation method.

The use of an adequate concentration of Triton X-100 enhanced immunomagnetic separation of Coxiella burnetii from milk. PCR-enzyme-linked immunosorbent assay (PCR-ELISA) could detect coxiellas more sensitively than could conventional PCR. PCR-ELISA is therefore thought to be suitable for the simultaneous assay of a large number of samples. However, the number of cows from which raw milk tested positive for coxiellas by PCR-ELISA was inconsistent with that found with the antibody to coxiella by indirect immunofluorescence assay. The inconsistency is thought to be associated with the differences in the infectious route, infectious dose, or the timing of yielding the antibody and the period of duration of the antibody.

Animals↗
PubMed2005

Assessment of the repeatability of a milk Ostertagia ostertagi ELISA and effects of sample preparation.

An indirect Ostertagia ostertagi ELISA on milk is a promising diagnostic tool in bovine parasitology. Interpretation of the test results requires a good knowledge of the test characteristics. In this study, border effects, the repeatability of the ELISA and the effect of different factors such as storage, skimming and freeze-thaw cycles of the milk samples were investigated. The border effects trial showed that significant border effects can occur. The repeatability trial was conducted over 3 days. An alternative graphical technique to assess the repeatability over a large number of ELISA plates measured over different days was developed. From these graphs, it was obvious that the ODR values obtained on the third day were deviating from the values on the first and second day. On the third day, also abnormal control values were observed. When the control values were normal, 94% of the variability was explained by the milk sample and 6% by assay variability. The expected 95% range of the difference of 2 ODR readings of the same sample on the same plate and the same sample on different plates was -0.14 to 0.14 and -0.16 to 0.16. No extra variability was observed when samples were tested on a different day, however these results are based on the measurement of 2 days. Storage for 2-4 days at 4 degrees C, using whole milk instead of skimmed milk and up to 2 extra freeze-thaw cycles of the milk samples did not significantly affect the test results.

Animals↗
PubMed1978

Direct analysis of lead in fruit juice by anodic stripping voltammetry with no prior sample preparation.

A method is presented for determining lead in fruit juice in less than 3 min after instrument calibration. The range examined is 0.01 to 1.3 ppm lead. The method is compared with 3 other methods in general use. Standard error of estimates between the methods compared range from 0.023 to 0.051 ppm for a set of 50 samples and from 0.037 to 0.091 ppm for a set of 9 samples. Regression correlation coefficients between methods range from 0.968 to 0.995. Judged by the comparisons, the direct method is precise and accurate over greater than a 100-fold range of lead concentrations.

Beverages↗
PubMed2006

Methylmercury measurement in whole blood by isotope-dilution GC-ICPMS with 2 sample preparation methods.

BACKGROUND: Despite its known toxicity, methylmercury is rarely measured directly in clinical studies; instead, conclusions are based on total mercury measurements. We have developed isotope-dilution-based methods for methylmercury-specific analysis of whole blood by coupled gas chromatography-inductively coupled plasma mass spectrometry (GC-ICPMS). METHODS: We analyzed animal and human blood samples after alkaline digestion or extraction of methylmercury into dichloromethane and back extraction into water. Methylmercury was converted to the volatile ethyl derivative, purged, and trapped on a solid-phase collection medium, and then introduced into the GC-ICPMS system. RESULTS: Limits of quantification were 0.4 and 0.03 microg/L at a signal-to-noise ratio of 10 with the alkaline digestion and extraction methods, respectively. Extraction met our selected acceptable total error criterion, with an SD of 0.58 microg/L at the critical maternal blood concentration of 5.8 microg/L. Results obtained with alkaline digestion indicated the need for improved random analytical uncertainty, which was achieved by increasing the enrichment of the isotope dilution. For 37 blood samples, the mean (SD) proportion of total mercury present as methylmercury was 60 (27)%, range 6%-100%. CONCLUSIONS: The combination of extraction and isotope-dilution GC-ICPMS meets the requirements for use as a reference method for measuring methylmercury in whole blood.

Animals↗
PubMed2005

Fast miniaturised sample preparation for the screening and comprehensive two-dimensional gas chromatographic determination of polychlorinated biphenyls in sludge.

Polychlorinated biphenyls (PCBs) in sludge are usually extracted by a technique such as Soxhlet with subsequent fractionation prior to long GC runs using GC-ECD or GC-HRMS. In this study, the extraction of selected chlorinated biphenyls (CBs) from a spiked sludge sample by three rapid techniques, i.e. ultrasonic (USE), pressurised-liquid (PLE), and microwave-assisted (MAE) extraction using a domestic microwave, was studied, with subsequent direct GC-ECD, GC-MS, or GC x GC-microECD analysis of the extracts. The main goal was to select an appropriate, and miniaturised, extraction method after only a brief optimisation and demonstrate the power of GC x GC analysis of dirty extracts. For PLE similar CB recoveries were found when extracting with either n-hexane or n-hexane/acetone (1/1). For USE and MAE, n-hexane/acetone (1/1) was the preferred extraction solvent. USE gave the best recoveries (80-95%; except 130% for CB 105). The only clean-up needed prior to GC-MS or GC x GC-gECD analysis was the removal of sulphur-containing compounds. GC-ECD was not suitable for these dirty extracts. The lowest LODs for the CBs (20 fg or 0.1 ng/g sludge) were found when combining USE and GC x GC-microECD, because of the powerful extraction, high separation power and excellent detectability provided by this technique.

Calibration↗
PubMed1998

A simplified sample preparation method from various foods for PCR detection of pathogenic Yersinia enterocolitica: a possible model for other food pathogens.

A simplified method for the direct application of multiplex polymerase chain reaction (PCR) was developed to detect plasmid-bearing virulent serotypes of Yersinia enterocolitica (YEP+) in a variety of foods. Strains of YEP+ representing five serotypes were detected in enriched swab samples of artificially contaminated pork chops, ground pork, cheese and zucchini using multiplex PCR analysis. The method was also effective for identifying YEP+ strains in naturally contaminated porcine tongues. The use of swabs eliminated time-consuming extraction of DNA from food, inhibition of PCR by food-derived DNA, interference by background flora and reduced the time needed for processing samples. The detection of other food pathogens should be feasible by this technique.

Animals↗
PubMed2006

Development of a harmonised method for the profiling of amphetamines: IV. Optimisation of sample preparation.

The suitability of liquid-liquid extraction (LLE) and solid-phase extraction (SPE) for the preparation of impurity extracts intended for gas chromatographic profiling analyses of amphetamine were evaluated. Both techniques were optimised with respect to the extraction of selected target compounds by use of full factorial designs in which the variables affecting the performance were evaluated. Test samples consisted of amphetamine synthesised by the Leuckart reaction, by reductive amination of benzyl methyl ketone and by the nitrostyrene route. The performance of LLE and SPE were comparable in terms of repeatability and recovery of the target compounds. LLE was considered the better choice for the present harmonised amphetamine profiling method due to the lack of information on the long-term stability of SPE columns.

Journal Article↗