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At least 289 records · Page 16Linked to original sources
PubMed1997

Determination of plasma protein adsorption on magnetic iron oxides: sample preparation.

PURPOSE: The purpose of this study was to investigate the influence of the sample preparation on the plasma protein adsorption pattern of polysaccharide-stabilized iron oxide particles by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). METHODS: The iron oxide particles were incubated in vitro in human plasma for five minutes. Thereafter, four different methods for particle recovery, including adsorbed proteins from surplus plasma, were investigated: centrifugation, magnetic separation, gel filtration and membrane-based static microfiltration. Adsorbed proteins were desorbed from the particle surfaces by surfactants and analyzed by 2-D PAGE, as described elsewhere (1,2). RESULTS: All the techniques investigated were able to separate small-size iron oxides (approx. 110 nm) and adsorbed proteins from excess plasma. The gels obtained by the different separation procedures displayed almost identical adsorption patterns. Major proteins identified were: fibrinogen, IgG, albumin and an unclassified protein of about 70 kDa with a pI value of 6.5-7.5. CONCLUSIONS: Centrifugation was regarded as the most suitable separation method due to its speed and ease of use. In contrast to gel filtration, any washing media can be used. The magnetic separation process is restricted to particles with high inducible magnetic saturation, in particular, to iron oxides with overall sizes > 50 nm.

Adsorption↗
PubMed2001

NH4NO3 extractable trace element contents of soil samples prepared for proficiency testing--a stability study.

In view of its intended use as a sample for proficiency testing or as a reference material the stability of the extractable trace element contents of a soil from an irrigation field was tested using the extraction with 1 mol/L ammonium nitrate solution according to DIN 19730. Therefore, changes of the extractability of sterilized and non sterilized soil samples stored at different temperatures were evaluated over a period of 18 months. Sets of bottles were kept at -20 degrees C, +4 degrees C, about +20 degrees C and +40 degrees C, respectively. The NH4NO3 extractable contents of Cd, Cr, Cu, Ni, Pb and Zn were determined immediately after bottling and then after 3, 6, 12 and 18 months with ICP-AES or ETAAS. Appropriate storage conditions are of utmost importance to prevent deterioration of soil samples prepared for the determination of NH4NO3 extractable trace element contents. Temperatures above +20 degrees C must be avoided. The observed changes in the extractability of the metals (especially for Cr and Cu) most likely could be related to thermal degradation of the organic matter of the soil. There is no need to sterilize dry soil samples, because microbiological activity in soils with a low moisture content appears to be negligible with regard to trace element mobilization.

Environmental Monitoring↗
PubMedDate not supplied

A comparison of sample preparation methods for PCR detection of pathogenic Yersinia enterocolitica from ground pork using swabbing and slurry homogenate techniques.

Two sample preparation methods for multiplex polymerase chain reaction (PCR) for detection of plasmid-bearing virulent Yersinia enterocolitica (YEP(+)) from ground pork were compared. Two sets of ground pork samples were inoculated with 10, 1, and 0.5 CFU/cm(2) of a YEP(+) strain, one set was swabbed and the second set was dispersed into a slurry homogenate. Both swab and slurry homogenate samples were enriched in sterile Whirl Pak bags containing modified trypticase soy broth for 48 h at 12 degrees C. From the enriched swab samples, the bacterial cells were pelleted, washed, boiled in sterile distilled water, and treated with proteinase K to prepare cell lysates to use as a DNA template. Since slurry homogenate samples contained food material, DNA extraction was performed using a commercial kit. The DNA from cell lysates and from extracted slurry homogenate samples were evaluated as templates for multiplex PCR employing primers for the chromosomal ail and plasmid virF genes. The enrichment of the YEP(+) strain was more efficient using the sponge-swabbed samples than the slurry homogenate samples at all three inoculum levels tested. It was necessary to dilute the DNA extracted from slurry homogenate to determine the optimal concentration of each sample for PCR amplification. No amplification signal was detected using undiluted DNA, possibly due to DNA inhibitors present in the slurry homogenate that were not removed in the process of extraction. However, DNA could be detected in undiluted cell lysates from swab samples. Thus, the cell lysates from swab samples are more advantageous than DNA extracted from ground pork slurry homogenate samples for the PCR assay.

Animals↗
PubMed2002

An automated on-line multidimensional HPLC system for protein and peptide mapping with integrated sample preparation.

A comprehensive on-line two-dimensional 2D-HPLC system with integrated sample preparation was developed for the analysis of proteins and peptides with a molecular weight below 20 kDa. The system setup provided fast separations and high resolving power and is considered to be a complementary technique to 2D gel electrophoresis in proteomics. The on-line system reproducibly resolved approximately 1000 peaks within the total analysis time of 96 min and avoided sample losses by off-line sample handling. The low-molecular-weight target analytes were separated from the matrix using novel silica-based restricted access materials (RAM) with ion exchange functionalities. The size-selective sample fractionation step was followed by anion or cation exchange chromatography as the first dimension. The separation mechanism in the subsequent second dimension employed hydrophobic interactions using short reversed-phase (RP) columns. A new column-switching technique, including four parallel reversed-phase columns, was employed in the second dimension for on-line fractionation and separation. Gradient elution and UV detection of two columns were performed simultaneously while loading the third and regenerating the fourth column. The total integrated workstation was operated in an unattended mode. Selected peaks were collected and analyzed off-line by MALDI-TOF mass spectrometry. The system was applied to protein mapping of biological samples of human hemofiltrate as well as of cell lysates originating from a human fetal fibroblast cell line, demonstrating it to be a viable alternative to 2D gel electrophoresis for mapping peptides and small proteins.

Cell Line↗
PubMed1987

A robotic sample preparation scheme for the high performance liquid chromatographic determination of ivermectin in animal plasma.

A lengthy sample preparation scheme for the high performance liquid chromatographic determination of the antiparasitic agent ivermectin at ppb concentrations in animal plasma is adapted to a laboratory robotic system. Sample treatment involves both liquid-liquid partitioning and solid phase extraction. Specific modifications to the manual procedure include the use of serial vortex mixings in place of batchwise lateral shaking and the substitution of small (6 mL), disposable solid phase extraction columns driven by compressed gas for large (25 mL), gravity-fed, reusable glass columns. Coupling these columns to an automated solvent dispensing device simplifies handling of the large solvent volumes prescribed in the manual procedure. A productivity gain over the manual procedure is realized when operating the robotic system in the single sample mode, and an additional gain is achieved by integrating the system for multiple sample handling. Equivalence to the manual procedure is demonstrated.

Animals↗
PubMed2004

Development of miniaturized sample preparation with fibrous extraction media.

Introducing fine polymeric filaments as the extraction medium, a miniaturized sample preparation technique for micro-column liquid chromatography (micro-LC) has been developed along with the investigation of a reproducible preparation scheme of the extraction capillary. The polymeric filaments were packed longitudinally into either a fused-silica capillary or a polyether ether ketone (PEEK) capillary of appropriate dimensions, and the extraction capillary was installed to the injection valve in micro-LC system. The number of packed filaments should be precisely counted before the packing process to make sure the reproducible preparation of the extraction capillary. With conventional stationary phase materials for open-tubular gas chromatography, polymeric coating to the surface of the filaments was also studied in order to further enhance the extraction performance and selectivity. Coated with the polymeric material suitable for the extraction of particular analyte, a dramatic improvement on the extraction power was obtained. The results suggest that the future possibility of novel tailored fibrous extraction medium with an appropriate coating on it, especially for the analysis of complex sample matrices.

Chromatography, Liquid↗
PubMed2004

Sample preparation of gel electrophoretically separated protein binding partners for analysis by mass spectrometry.

The identification and characterization of binding partners from protein complexes is increasingly undertaken by mass spectrometry because of its high sensitivity and expedient elucidation of protein structure by accurate mass measurement. A variety of affinity purification methods including immunoprecipitation and glutathione-S-transferase (GST) pull-downs are commonly employed for the isolation of protein complexes and coupled to gel electrophoresis for further separation and basic information with regard to their constituents. For the successful analysis of gel-separated proteins by mass spectrometry, additional sample preparation steps involving sample clean-up, proteolysis, and peptide recovery are essential. This chapter describes the important procedure of in-gel digestion with particular emphasis on maximum peptide recovery and compatibility for subsequent mass spectrometric analysis.

Electrophoresis, Polyacrylamide Gel↗
PubMed2003

Reagentless mechanical cell lysis by nanoscale barbs in microchannels for sample preparation.

A highly effective, reagentless, mechanical cell lysis device integrated in microfluidic channels is reported. Sample preparation, specifically cell lysis, is a critical element in 'lab-on-chip' applications. However, traditional methods of cell lysis require purification steps or complicated fabrication steps that a simple mechanical method of lysis may avoid. A simple and effective mechanical cell lysis system is designed, microfabricated, and characterized to quantify the efficiency of cell lysis and biomolecule accessibility. The device functionality is based on a microfluidic filter region with nanostructured barbs created using a modified deep reactive ion etching process. Mechanical lysis is characterized by using a membrane impermeable dye. Three main mechanisms of micro-mechanical lysis are described. Quantitative measurements of accessible protein as compared to a chemically lysed sample are acquired with optical absorption measurements at 280 and 414 nm. At a flow rate of 300 microL min(-1) within the filter region total protein and hemoglobin accessibilities of 4.8% and 7.5% are observed respectively as compared to 1.9% and 3.2% for a filter without nanostructured barbs.

Animals↗
PubMed2000

Sample preparation by SDS/PAGE and in-gel digestion.

A convenient method for the isolation of samples for microsequencing or mass spectrometry utilizing polyacrylamide gel electrophoresis is described in detail, including pre-requisites for a successful outcome. Briefly, after visualization by e.g. Coomassie or silver, internal peptides are generated by in-gel proteolytic digestion, and after extraction, the corresponding protein is analyzed, i.e. the protein is identified by search in sequence data banks using a peptide sequence or a peptide mass map. Alternatively, a novel protein is detected, and an oligonucleotide probe for subsequent molecular biology work is synthesized, based on the obtained peptide sequence. One obvious advantage is that the procedure allows practically any protein to be studied. In-gel digestion has become the fundamental means of preparing samples in the many ongoing proteome projects. A few representative examples are given.

Chromatography, Liquid↗
PubMed1986

Microwave extraction. A novel sample preparation method for chromatography.

The applicability of microwave irradiation to the extraction of various types of compounds from soil, seeds, foods and feeds as a novel sample preparation method for chromatography was investigated. Samples were ground and mixed with an appropriate solvent, methanol or methanol-water for polar compounds and hexane for non-polar compounds. The suspensions were irradiated for 30 s, but they were not allowed to boil. After cooling, the irradiation was repeated several times. The samples were then centrifuged, and aliquots of the supernatant were injected into a chromatographic column. The yields of the extracted compounds obtained by microwave irradiation were compared with those obtained by the traditional Soxhlet or shake-flask extraction methods. The microwave extraction method was more effective than the conventional methods. Due to the considerable savings in time and energy, this novel method is suitable for fast extractions of large sample series.

Chromatography, High Pressure Liquid↗
PubMed1986

Rapid sample preparation and high performance liquid chromatographic determination of total and unbound serum disopyramide.

A rapid sample preparation procedure, which requires no solvent extraction or concentration, for the high performance liquid chromatographic (HPLC) determination of disopyramide is described. The chromatography is performed on a C-18 radial compression mu-Bondapak column and detection by absorbance at 254 nm with a run time of 12 min. The mobile phase is 10 mM sodium acetate (pH 4.5)/acetonitrile (3:1 vol/vol). For the total drug assay, 50 microliter 30% (wt/vol) trichloroacetic acid is added to 500 microliter serum, which causes the precipitation of protein. Following centrifugation, 100 microliter of supernatant is mixed with 25 microliters of internal standard (25 micrograms/ml, ethyl p-aminobenzoate), and 50 microliters of this mixture is injected into the HPLC. Unbound disopyramide is separated from protein-bound drug by filtration with an Amicon Centrifree filter, which removes 99.6% of protein and does not retain disopyramide. To 100 microliters of this filtrate is added 25 microliters of internal standard, and 50 microliters is injected into the HPLC. The assay is linear to at least 20 micrograms/ml. The total drug assay shows an average recovery of 93.0% with an average coefficient of variation (CV) of 3.4%. The unbound drug assay shows an average CV of 4.1%. The percentage of free drug in a sample containing 4.85 g/dl protein varies from 68.0 to 83.5% at concentrations of 2.5-10 micrograms/ml, which illustrates the concentration-dependent nature of the protein binding, and the need to measure the unbound fraction of drug. Of 31 drugs tested for interference, none was found to interfere.

Chromatography, High Pressure Liquid↗
PubMed1982

Sample preparation and liquid-chromatographic analysis for tricyclic antidepressants in serum.

We report two separate sample-preparation procedures and a "high-pressure" liquid-chromatographic method for quantitating nine tricyclic antidepressant drugs: trimipramine, doxepin, amitriptyline, imipramine, desmethyldoxepin, nortriptyline, desipramine, maprotiline, and protriptyline. The drugs are extracted from 1 mL of serum by a single manual hexane extraction or by using DuPont's PREP I Automated Sample Processor. The drugs from either process are chromatographed on a mu-Bondapak-CN column with a mobile phase of acetonitrile/methanol/Na2HPO4, 5 mmol/L (60/15/25 by vol), pH 7.0, at a flow rate of 2.0 mL/min. Absorbance at 254 nm is linear with drug concentration from 25 to 1000 mg/L for each drug. With the manual extraction procedure the detection limit was 10 mg/L, absolute recovery 20-44%, within-run precision (CV) 10.1%, and run-to-run precision 11.1%. The performance of automated sample extraction was better, with a detection limit of 5 mg/l, absolute recovery 72-97%, within-run precision 6.1%, and run-to-run precision 6.6%.

Antidepressive Agents, Tricyclic↗
PubMed2006

Sample preparation methods for beeswax characterization by gas chromatography with flame ionization detection.

New and simpler methods of sample preparation to determine several families of compounds in beeswax by conventional and high temperature gas chromatography are proposed. To analyze hydrocarbons and palmitates, a dilution of sample is enough whereas for the total acid content, a hydrolysis and simultaneous methylation with BF3-methanol results more effective than the usual methods; for the total content of alcohols, a further acetylation with acetic anhydride is necessary. Free alcohols are directly acetylated in a sample dissolution but for free acids and monoesterified 1,2,3-propanetriols analysis, a previous extraction with acetonitrile is required. The concentrations of all the compounds studied are expressed in weight percentage referred only to one standard: octadecyl octadecanoate. The precision of the analytical methods has been evaluated showing its importance in the analysis of beeswaxes used in apiculture.

Alcohols↗
PubMed2001

Optimization of sample preparation for peptide sequencing by MALDI-TOF photofragment mass spectrometry.

This paper describes the optimization of sample preparation for MALDI 193-nm photofragment ion time-of-flight mass spectrometry to sequence small to medium-sized peptides from peptide mixtures. We show that matrix additives, such as fructose and phenylbutyric acid have a dramatic effect on the abundance of fragment ions observed in the post-source decay spectra. A dried-droplet MALDI matrix consisting of 1:1 alpha-cyano-4-hydroxycinnamic acid/fructose proves to be an excellent matrix for photodissociation because [M + H]+ ions are formed with low internal energies, and the photofragment ion spectrum contains high abundances of sequence-informative ions. The addition of fructose appears to improve overall sample homogeneity and durability, as compared to conventional alpha-cyano-4-hydroxycinnamic acid dried-droplet preparations. MALDI-TOF photodissociation is then used to selectively sequence the peptides bradykinin (RPPGFSPFR), des-Arg9 bradykinin (RPPGFSPF), and substance P-amide (RPKPQQFFGLM-NH2) from a mixture of five peptides.

Animals↗
PubMed2001

Rapid antibiotic drug monitoring: meropenem and ceftazidime determination in serum and bronchial secretions by high-performance liquid chromatography-integrated sample preparation.

A sensitive and rapid HPLC assay for the determination of the beta-lactam antibiotics ceftazidime and meropenem in serum and bronchial secretions is described. HPLC-integrated sample preparation allows direct injection of serum samples without any pretreatment. Sputum samples need only a simple homogenisation and volume measurement but no liquefying reagents are necessary. The inline extraction technique is realized by automatically switching from the extraction column to the analytical column. After the matrix passed the extraction column, the retained analyte is quantitatively transferred to the analytical column where separation by isocratic HPLC is performed. Ceftazidime and meropenem are detected according to their absorption maxima at 258 and 296 nm, respectively. The detection limit of both antibiotics is estimated to be better than 0.5 microg/ml in serum as well as in sputum samples. The described procedure allows determination of the antibiotics within 30-45 min, thereby facilitating drug monitoring in clinical routine.

Bronchi↗
PubMed2002

Evaluation of an automated sample preparation protocol for quantitative detection of hepatitis C virus RNA.

The COBAS AMPLIPREP instrument for automated sample preparation has recently been introduced. In this study, the COBAS AMPLIPREP/COBAS AMPLICOR HCV MONITOR test, which includes this new molecular device, was evaluated and compared to the COBAS AMPLICOR HCV MONITOR test, which includes a manual extraction protocol. Interassay and intra-assay variation, precision, and linearity were determined, and a total of 130 clinical specimens were investigated. For determination of interassay variation, coefficients of variation were found to be between 9 and 59% for the COBAS AMPLIPREP/COBAS AMPLICOR HCV MONITOR test and between 13 and 69% for the COBAS AMPLICOR HCV MONITOR test. For determination of intra-assay variation, coefficients of variation were found to be between 7 and 13% for the COBAS AMPLIPREP/COBAS AMPLICOR HCV MONITOR test and between 8 and 16% for the COBAS AMPLICOR HCV MONITOR test. When precision of the COBAS AMPLIPREP/COBAS AMPLICOR HCV MONITOR test was tested, all results were found to be within +/-0.5 log of the expected results. Determination of linearity resulted in a quasilinear curve over 3 logs. When clinical samples were tested with the COBAS AMPLIPREP/COBAS AMPLICOR HCV MONITOR test and compared with the COBAS AMPLICOR HCV MONITOR test, all results were found within +/-0.5 log. In conclusion, the assay, which included the new molecular device, proved to be suitable for the routine molecular laboratory. It was found to be laborsaving and easy to handle.

Hepacivirus↗
PubMed1993

Sample preparation technique for cross-sectional transmission electron microscopy of quantum wire structures.

A novel cross-sectional sample preparation technique for quantum wire (QWR) structures is described. By coating a thin layer of Au with a designed pattern on the sample as a marker to indicate the position of the wire pattern, the location of the thinned area can be controlled precisely. An example of applying this technique to an InGaAs/GaAs QWR structure is demonstrated. This technique can also be applied to any other small dimensional structures or devices with specific regions of interest.

Gold↗
PubMed2000

Microscale sample preparation.

The demand for microscale analysis has stimulated the development of new solid phase formats in sample preparation.

Chromatography, High Pressure Liquid↗