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 415 records · Page 23Linked to original sources
PubMed2005

Sample preparation and mass-spectrometric characterization of crystal-derived protein samples.

Mass spectrometry is often used to ascertain the accurate mass of purified protein samples prior to crystallization screening. However, in many cases data regarding the form of the protein crystallizing can also be useful, as this may differ from the original sample. Development of a simple method for the preparation and mass spectrometry of crystal-derived protein samples is described. The method is exemplified by the determination of the phosphorylation state of protein in a crystal derived from a mixture of phosphorylated and unphosphorylated protein.

Chromatography, High Pressure Liquid↗
PubMed1999

Two-layer sample preparation: a method for MALDI-MS analysis of complex peptide and protein mixtures.

The analytical performance of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry for direct analysis of peptide and protein mixtures is strongly dependent on the sample and matrix preparation. A two-layer sample preparation method is demonstrated to be very effective for analyzing complex mixtures. In this method, the first layer on the MALDI probe is the densely packed matrix microcrystals formed by fast solvent evaporation of a matrix solution. A mixture solution containing both matrix and sample is then deposited onto the first layer to form uniform analyte/matrix micrococrystals. It is found that the addition of matrix to the second-layer sample solution proves to be critical in analyzing mixtures of peptides and proteins covering a broad mass range. The effect of solvent conditions for preparing the second-layer solution is discussed. The application of this method is demonstrated for the analysis of cow's milk where milk proteins as well as peptide fragments produced from proteins by indigenous proteinases are detected. Direct analyses of peptides and proteins from a bacteria extract and crude egg white are also illustrated.

Amino Acid Sequence↗
PubMed2004

Sample preparation followed by HPLC under harmless 100% aqueous conditions for determination of oxytetracycline in milk and eggs.

A simple and hazardous chemical-free method for the high-performance liquid chromatographic determination of oxytetracycline (OTC) residues in milk and eggs has been developed. Sample preparation consists in homogenization with an aqueous solution by means of a handheld ultrasonic homogenizer followed by centrifugal ultrafiltration. HPLC is performed with an isocratic aqueous mobile phase and a photodiode array detector. Average recoveries of OTC (0.05, 0.1, and 0.2 microg mL(-1) for milk; 0.1, 0.2, and 0.4 microg mL(-1) for eggs) were > or =84% with relative standard deviations of < or =2.3%. The total time required for the analysis of one sample and LOQs were <30 min and <0.1 microg mL(-1), respectively. In all the processes, no organic solvents or hazardous reagents were used.

Animals↗
PubMed2000

A new sample preparation technique for organochlorines in cod liver oil combining SPE and NP-HPLC with HRGC-ECD.

The analysis of semivolatile organochlorines (polychlorinated biphenyls and chlorinated pesticides) in less polluted biomaterials requires specific strategies in controlling the blank in sample preparation. The procedure described here allows to decrease significantly the level of contamination during the clean-up step of fish oil. Solid-phase-extraction (SPE) on LiChrolut EN and normal phase HPLC in the normal- and the backflush-mode were used to reduce the amount of solvents needed and the analysis time compared to established clean-up procedures. With a certified reference material (BCR-CRM 349; Cod Liver Oil) the precision and effectiveness of the new method were validated. Recovery rates of the Internal Standards (PCB 103 and TCN) lay between 75% and 90% at the microg/kg lipid level. The quantitative analyses were carried out by high resolution gas chromatography with electron capture detector (HRGC-ECD).

Animals↗
PubMed1991

Determination of biocatalyst consumption in an aminopeptidase process using automated sample preparation and high-performance liquid chromatography.

A rapid and sensitive method has been developed for the determination of the biocatalyst consumption in the chemo-enzymic production of optically pure natural and synthetic alpha-H-amino acids. It is based on automated sample preparation from an enzymic reaction mixture, reversed-phase high-performance liquid chromatographic separation, post-column reaction and fluorimetric detection. The assay procedure has been applied to the enzymic conversion of racemic norvaline amide into L-norvaline, catalysed by an L-specific aminopeptidase from Pseudomonas putida. Both norvaline amide and norvaline can be analysed in a single assay in the low nanogram range. The method yields reproducible results and requires 30 min from the time of sampling the enzymic reaction mixture to quantitation. The reaction mixture is automatically sampled and analysed several times during the course of the reaction. With the results obtained a conversion curve can be constructed from which the exact biocatalyst consumption can be calculated. By adaptation of the mobile phase, the method can also be applied to other amino acid amides used as substrates in the aminopeptidase reaction.

Aminopeptidases↗
PubMed2002

Development of a sample preparation method for the analysis of oxidized flavonols in onions and leek.

A method is presented for the determination of oxidized flavonols (i.e., hydroxybenzoylbenzofuranones) in plant material. Onions and leek are investigated using a newly developed solid-phase extraction procedure for sample preparation prior to high-performance liquid chromatography (HPLC). It is shown that the addition of ascorbic acid as antioxidant is necessary to prevent the generation of the researched analytes during this procedure. Nevertheless, the plant extracts do under certain conditions catalyze the oxidation of flavonoles resulting in benzofuranones.

Antioxidants↗
PubMed1975

[Polysaccharide additives in foods rich in proteins. i Sample preparation for estimation, especially in fresh cheese (author's transl)].

Published procedures for the isolation of polysaccharides from foods rich in proteins, especially fresh cheese, proved not very useful for the concentration in question (0,01--0.2%). We, instead, propose the following sample preparation: 1. Removal of lipids by an ethanol--ethyl ether--petrol ether mixture; 2. complete hydrolysis of proteins with pepsin, pronase E and a mixture of aminopeptidase M and prolidase; 3. separation of the polysaccharides from the low molecular substances and from enzyme residues by gel filtration on Biogel P-2; 4. concentration of the polysaccharides containing fraction by freeze drying, ultrafiltration or evaporation. --The method was tested with quarg and double cream fresh cheese, it can, however, be adopted to use in other products.

Aminopeptidases↗
PubMed1991

Improved sample preparation for accurate determination of low concentrations of lead in whole blood by graphite furnace analysis.

The effect of low concentrations of lead on pre- and post-natal growth and development is a current concern. We describe a simple method of sample preparation for direct determination of lead in whole blood by Zeeman graphite-furnace atomic absorption spectrometry. This procedure improves analytical precision and accuracy of lead determinations at low concentrations as compared with published furnace data. At blood lead concentrations of 0.25, 1.98, and 3.76 mumol/L, within-run CVs were 3.2%, 1.8%, and 1.4% respectively; between-run CVs were 7.3%, 2.9%, and 2.2%. Accuracy, as demonstrated by analytical recovery, ranged from 99% to 102%. Our reproducibility/accuracy score in the 1989 Quebec interlaboratory comparison program was 96% compared with the target, second best of 66 participating laboratories.

Adult↗
PubMed1994

Comparison of sample preparation methods for detection of Chlamydia pneumoniae in bronchoalveolar lavage fluid by PCR.

Amplification inhibitors can lead to false-negative results for PCR. In order to evaluate the reliability of PCR for the detection of Chlamydia pneumoniae, the presence of PCR inhibitors in 75 bronchoalveolar lavage specimens was assessed after treatment by various sample preparation methods. Specimens were collected from patients with acute respiratory infections, including four cases of proven C. pneumoniae infection. Substances inhibitory to the amplification of chlamydial DNA continued to be present in 12% of the samples treated according to the commonly used single-step proteinase K digestion and in 31% of the samples processed by heat treatment. However, the complexing of DNA-contaminating proteins and polysaccharides from digested specimens to cetyltrimethylammonium bromide (CTAB) followed by DNA extraction efficiently removed inhibitors from all experimental samples and provided subsequent identification of all positive clinical samples by PCR. The CTAB method and proteinase K treatment had comparable detection limits of approximately 0.01 inclusion-forming units. CTAB-based DNA purification of respiratory specimens is recommended to increase the diagnostic sensitivity of PCR and confidence in negative results.

Bronchoalveolar Lavage Fluid↗
PubMed2000

Liquid chromatographic-tandem mass spectrometric method for the analysis of a neurokinin-1 antagonist and its metabolite using automated solid-phase sample preparation and automated data handling and reporting.

Pharmacokinetic studies play a vital role during the development of new pharmaceutical substances. Data presented demonstrate an accurate, precise and robust assay for a neurokinin-1 receptor antagonist and its metabolite with HPLC-MS-MS. Sample preparation is performed by solid-phase extraction (SPE) in the 96-well plate format. This process is fully automated with a Tecan Genesis pipetting system using its standard robotic manipulator arm (ROMA). All instruments are fully integrated in a study oriented laboratory information system (LIMS) with an Oracle database that communicates bi-directional with the analytical equipment. Finally, the results are reported by push button operation.

Automation↗
PubMed2003

Determination of testosterone metabolites in human hepatocytes. I. Development of an on-line sample preparation liquid chromatography technique and mass spectroscopic detection of 6beta-hydroxytestosterone.

A rapid and sensitive RP-HPLC assay for determination of 6beta-hydroxytestosterone in human hepatocytes with corticosterone as the internal standard is described. The procedure employs on-line sample enrichment using a BioTrap 500 MS (20x4 mm I.D.) extraction pre-column and subsequent gradient separation on a Prontosil 60-5 C(18)-H (250x2 mm I.D., 5 micrometer particle size) analytical column in the back-flush mode using a ternary eluent system composed of methanol, tetrahydrofuran and water. Signal monitoring was done by measurement of the responses from liquid chromatography coupled to mass spectroscopy (LC-MS/MS) using an atmospheric pressure chemical ionization (APCI) source conducted in the selected reaction monitoring (SRM) mode. Mean recoveries of 6beta-hydroxytestosterone from an estimate of the biological matrix, i.e., Dulbecco's modified Eagle medium "High Glucose", ranged from 101.8-104.4% for samples containing the target analyte at the 250, 500 and 1000 ng/ml level. The limit of quantitation (LOQ) was 20 ng/ml at an injection volume of 100 microliter determined in the same matrix. Linearity of signal responses versus concentration for all three analytes was accomplished in the range of 100-4000 ng/ml. Mean values of the coefficients of variation (C.V.) for the target analyte obtained for the concentrations 250, 500 and 1000 ng/ml at 5 different days in quintuplicate ranged from 1.5-7.7% (within-day) and 4.8-7.3% (between-day). The corresponding values for the accuracy ranged from 87.7-106.1% for the within-day and from 98.8-102.5% for the between-day measurements. The target analyte was sufficiently stable at both storage and sample preparation conditions because no substantial deviations between analyte concentrations measured before and after subsequently performed freeze and thaw cycles were observed.

Chromatography, High Pressure Liquid↗
PubMed2003

Recovery of semivolatile organic compounds during sample preparation: implications for characterization of airborne particulate matter.

Semivolatile compounds present special analytical challenges not met by conventional methods for analysis of ambient particulate matter (PM). Accurate quantification of PM-associated organic compounds requires validation of the laboratory procedures for recovery over a wide volatility and polarity range. To meet these challenges, solutions of n-alkanes (nC12-nC40) and polycyclic aromatic hydrocarbons PAHs (naphthalene to benzo[ghi]perylene) were reduced in volume from a solvent mixture (equal volumes of hexane, dichloromethane and methanol), to examine recovery after reduction in volume. When the extract solution volume reached 0.5 mL the solvent was entirely methanol, and the recovery averaged 60% for n-alkanes nC12-nC25 and PAHs from naphthalene to chrysene. Recovery of higher MW compounds decreased with MW, because of their insolubility in methanol. When the walls of the flasks were washed with 1 mL of equal parts hexane and dichloromethane (to reconstruct the original solvent composition), the recovery of nC18 and higher MW compounds increased dramatically, up to 100% for nC22-nC32 and then slowly decreasing with MW due to insolubility. To examine recovery during extraction of the components of the High Capacity Integrated Gas and Particle Sampler, the same standards were used to spike its denuders and filters. For XAD-4 coated denuders and filters, normalized recovery was >95% after two extractions. Recovery from spiked quartz filters matched the recovery from the coated surfaces for alkanes nC18 and larger and for fluoranthene and larger PAHs. Lower MW compounds evaporated from the quartz filter with the spiking solvent. This careful approach allowed quantification of organics by correcting for volatility- and solubility-related sample preparation losses. This method is illustrated for an ambient sample collected with this sampler during the Texas Air Quality Study 2000.

Air Pollutants↗
PubMed2002

Determination of lonazolac and its hydroxy and O-sulfated metabolites by on-line sample preparation liquid chromatography with fluorescence detection.

A reliable method, which can be used for the determination of lonazolac and its hydroxylated and O-sulfated metabolites in cell culture media with methyllonazolac as the internal standard is described. The procedure employs on-line sample enrichment using a BioTrap 500 MS (20 x 4 mm I.D.) extraction pre-column and subsequent gradient separation on an Xterra MS C18-HT (100 x 3 mm I.D., 3.5 microm particles) analytical column in the back-flush mode. Signal monitoring was done by measurement of fluorescence responses at 273 nm for excitation and 385 nm for emission. Structural identity of analyte peaks was confirmed by liquid chromatography coupled to mass spectroscopy (LC-MS-MS) using an electrospray ionization (ESI) source in the selected reaction monitoring (SRM) mode. Mean recoveries of lonazolac, hydroxylonazolac and lonazolac sulfate, respectively, from the biological matrix were 104.2 +/- 3.5, 96.7 +/- 2.2, and 100.9 +/- 3.5%. The limit of detection (LOD) for the three compounds was about 5 ng/ml using a total sample volume of only 50 microl. Linearity of signal responses versus concentration for all three analytes was accomplished in the range 10-600 ng/ml. The mean values of the coefficients of variation (CV) for quality control samples measured in duplicate at three different days at the 10, 40, 100, and 400 ng/ml level were 4.46 +/- 1.15, 3.94 +/- 2.13 and 4.79 +/- 2.07% for lonazolac, hydroxylonazolac and lonazolac sulfate. The target analytes were sufficiently stable at both storage and sample preparation conditions because no substantial deviations between analyte concentrations measured before and after subsequently performed freeze and thaw cycles were observed.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed2003

Simultaneous detection of arginine, asymmetric dimethylarginine, symmetric dimethylarginine and citrulline in human plasma and urine applying liquid chromatography-mass spectrometry with very straightforward sample preparation.

Nitric oxide (NO) is synthesized by NO synthase from L-arginine, which can be competitively blocked by endogenous inhibitors such as asymmetric dimethylarginine (ADMA), but not by symmetric dimethylarginine (SDMA). ADMA is degraded by dimethylarginine dimethylaminohydrolase (DDAH) to dimethylamine and citrulline. A growing number of published clinical studies documented a strong correlation between increased ADMA blood levels and cardiovascular morbidity and mortality. We present here a highly sensitive method for the determination of this compounds in plasma and urine by means of HPLC-MS. The sample preparation is very simple and comprises only protein precipitation and concentration in the case of plasma samples and dilution in the case of urine. The samples are derivatized automatically with orthophthaldialdehyde and 2-mercaptoethanol, are separated on a 250 mm x 4 mm RP18 column by gradient elution with formate buffer/methanol and are detected by ESI-MS. The calibration functions are linear and cover the range from normal to pathologic concentration values of the analytes. The intra-day relative standard deviation (R.S.D.) of the assay for ADMA in plasma is 7.5% and the corresponding inter-day R.S.D. is 5.7%. In urine, these values for ADMA are 3.8 and 6.4%, respectively. All other analytes in plasma as well as in urine exhibit intra-day R.S.D. below 8%. The corresponding inter-day R.S.D. are all below 13%.

Arginine↗
PubMed1997

Determination of heterocyclic aromatic amines in food products: automation of the sample preparation method prior to HPLC and HPLC-MS quantification.

Heat-processing protein-rich foods may cause the formation of heterocyclic aromatic amines (HAAs), all of which have mutagenic and some also carcinogenic potential. Accurately measuring HAA levels in food products is therefore a necessary to realistically assess this risk factor. A solid-phase extraction method for quantitative HAA analysis has been developed by us over the last few years. This method has recently been automated using a robotic workstation and now allows almost unattended sample preparation, a process which saves a human operator about five hours of benchwork. Cleaned-up samples were analyzed by high performance liquid chromatography (HPLC) and ultraviolet (UV) or mass spectrometric (MS) detection. While HPLC-UV remains the daily tool to quantify HAAs, we found HPLC-electrospray-MS to be an alternative detection method with unique advantages, suited for both HAA identification and quantification.

Amines↗
PubMed2004

Combination of a new sample preparation strategy with an accelerated high-performance liquid chromatography assay with photodiode array and mass spectrometric detection for the determination of destruxins from Metarhizium anisopliae culture broth.

A method is presented allowing the qualitative and quantitative analysis of destruxins (dtxs) in fungal culture broth. Sample preparation was carried out by ultrafiltration over a commercially available acetylated cellulose (CTA) membrane with a Mr 10000 cut-off. The developed high-performance liquid chromatography assay with diode array detection (HPLC-DAD) cuts down the analysis time by 50% compared to most of the currently applied methods (retention times: dtx A = 8.3 min, dtx B = 8.9 min, dtx E= 7.5 min) and enables dtx detection down to sub-ppm range (limits of detection: dtx A = 0.19 mg/l, dtx B = 0.41 mg/l, dtx E = 0.10 mg/l). Stability of dtx E in filtrated culture broth was found to be much lower than anticipated (half-life time = 64.5 +/- 1.7 h). Thus, the detoxification of this metabolite is an abiotic process. Coupling of the HPLC-DAD system to an ion trap mass spectrometer with an electrospray ionization source operating in the positive mode allowed identification of most dtxs encountered by utilizing multiple stage MS-MS experiments and retention time rules.

Calibration↗
PubMed2004

Integration of surface plasmon resonance with mass spectrometry: automated ligand fishing and sample preparation for MALDI MS using a Biacore 3000 biosensor.

Integrating surface plasmon resonance analysis with mass spectrometry allows detection and characterization of molecular interactions to be complemented with identification of interaction partners. We have developed a procedure for Biacore 3000 that automatically performs all steps from ligand fishing and recovery to sample preparation for matrix-assisted laser desorption/ionization (MALDI) mass spectrometry including on-target digestion. In the model system used in this study a signal transduction protein, calmodulin, was selectively captured from brain extract by one of its interaction partners immobilized on a sensor chip. The bound material was eluted, deposited directly onto a MALDI target, and analyzed by mass spectrometry both as an intact protein and after on-target tryptic digestion. The procedure with direct deposition of recovered material on the MALDI target reduces sample losses and, in combination with automatic sample processing, increases the throughput of surface plasmon resonance mass spectrometry analysis.

Amino Acid Sequence↗
PubMed2003

Sample preparation techniques for the determination of natural 15N/14N variations in amino acids by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS).

In order to identify natural nitrogen isotope variations of biologically important amino acids four derivatization reactions (t-butylmethylsilylation, esterification with subsequent trifluoroacetylation, acetylation and pivaloylation) were tested with standard mixtures of 17 proteinogenic amino acids and plant (moss) samples using GC-C-IRMS. The possible fractionation of the nitrogen isotopes, caused for instance by the formation of multiple reaction products, was investigated. For biological samples, the esterification of the amino acids with subsequent trifluoroacetylation is recommended for nitrogen isotope ratio analysis. A sample preparation technique is described for the isotope ratio mass spectrometric analysis of amino acids from the non-protein (NPN) fraction of terrestrial moss. 14N/15N ratios from moss (Scleropodium spec.) samples from different anthropogenically polluted areas were studied with respect to ecotoxicologal bioindication.

Amino Acids↗