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PubMed1988

Simple assay procedure for tyrosine hydroxylase activity by high-performance liquid chromatography employing coulometric detection with minimal sample preparation.

A simple assay procedure for tyrosine hydroxylase activity in crude tissue samples was devised that requires minimal sample preparation and use of high-performance liquid chromatography with coulometric electrochemical detection. After incubation of enzyme samples, such as human brain homogenates or rat pheochromocytoma PC12h cells, with L-tyrosine and a tetrahydropterin cofactor, in the presence or absence of p-bromobenzyloxyamine, an inhibitor of aromatic L-amino acid decarboxylase, the reaction was terminated by addition of an equal volume of 0.1 M perchloric acid. For quantitation of L-DOPA produced, the sample was centrifuged, filtered and directly applied to the chromatographic apparatus connected to a coulometric electrochemical detector. This method makes redundant a time-consuming step in the previous methods, purification and concentration of L-DOPA or dopamine using alumina. The reaction conditions for the assay of tyrosine hydroxylase activity in brain homogenates and PC12h cells were re-examined by this method. Both tyrosine hydroxylase samples required a naturally occurring cofactor, (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin [(6R)BH4], catalase and NSD-1055 for the full activity, and tyrosine hydroxylase in human brain homogenates required Fe2+ ions for its full activity. (6R)BH4 proved to be a more effective cofactor than a synthetic cofactor, (6RS)-methyl-5,6,7,8-tetrahydropterin, which is commonly used for this assay.

Adrenal Gland Neoplasms↗

PubMed2003

Investigation of sample preparation and instrumental parameters in the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of noncovalent peptide/peptide complexes.

The application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) to the direct detection and investigation of noncovalent solution-phase complexes is far from being routine and some principal problems and questions still exist. Therefore, this study systematically investigates several main problems, namely, the effect of sample preparation and some instrument-related parameters on the stability of the noncovalent complexes as well as the formation of nonspecific cluster ions in the case of the MALDI-MS analysis of specific peptide/peptide complexes. The complexes formed between biologically active fragments of human gastrin I, which contain the sequence motif EEEEE, and different peptides, which contain the interacting sequence motifs RR and RKR, were chosen as examples. A broad variety of MALDI matrices and sample preparation protocols were screened systematically and evaluated. The two 'less acidic' matrices 2,4,6-trihydroxyacetophenone and 6-aza-2-thiothymine, in combination with carefully selected solvents and additives, turned out to allow the reproducible detection of the solution-phase peptide/peptide complexes with good intensity, whereas the classical MALDI matrices could not be applied with the same success. Because both matrices also tend to induce the formation of nonspecific cluster ions, control experiments using nonbinding peptides were performed to definitely prove the specificity of the detected complexes. In contrast to the sensitivity of the peptide/peptide complexes to solution-phase conditions, the gas-phase stability during desorption/ionization was found to be extraordinary high. Neither the application of high laser fluence nor switching from continuous to delayed extraction mode as well as variation of the delay time up to 520 ns had considerable effect on the relative intensities of the specific peptide/peptide complexes.

Amino Acids↗

PubMed2000

Automated sample preparation of Roxifiban tablets: transfer of a manual method to an automated workstation.

Automation offers obvious advantages for the preparation of tablets prior to analysis by HPLC including unattended operation, minimization of human intervention and an electronic audit trail. However, significant effort has to be put in up front to develop and validate an automated method, particularly if it is required to closely follow an existing manual method. Here, method transfer for Roxifiban, a fibrinogen receptor antagonist, will be discussed. A Zymark tablet processing workstation II (TPWII) was used for all automated sample preparations. Manual methods for composite assay, content uniformity, weight variation and degradation products testing of a tablet formulation were transferred to the TPWII. The method involved weighing of the sample, disintegration of the dosage form by homogenization, extraction of the analyte in the homogenate solution, filtration of the homogenate, dilution of the filtrate and transfer to autosampler vials. Obstacles to a quick transfer included limitations in the volume capabilities of the TPWII, poor analyte solubility and achieving proper conditioning of the transfer lines and filter. After resolving these issues, a validated method was achieved. Spiked recoveries were from 99.4 to 101.1% (RSD's <0.5%). A cross-validation between automated and manual assay methods was compared by Westlake analysis giving a 0.7% calculated interval at the 95% confidence level. Carryover was 0.07% after 20 sample preparations at the highest tablet strength.

Amidines↗

PubMed2003

Effect of sample preparation on the in vitro genotoxicity of a light curable glass ionomer cement.

The glass ionomer cement Vitrebond showed a clear genotoxic effect in the in vitro Mammalian Cell Gene Mutation Test (HPRT Test) with CHO cells as well as in the bacterial umu-test with Salmonella typhimurium TA1535/pSK1002. Both DMSO and Ham's F12 cell culture medium extracts according to ISO 10993-12 (Biological evaluation of medical devices-Part 12: sample preparation and reference materials, Geneva, Switzerland) exhibit a clear genotoxic effect in the umu-test. The effect is independent of the extraction volume in a range from 0.5 to 4 ml Ham's F12 cell culture medium. Subsequent extractions of Vitrebond showed no significant difference in the genotoxic response although weight loss and content of 2-hydroxyethyl-methacrylate dropped significantly. In vivo conditions of Vitrebond were simulated by extractions with artificial and collected human saliva. These extracts showed a clear genotoxic effect in the umu-test, even if only a few seconds of extraction time were applied. In conclusion, sample preparations for genotoxicity testing according to ISO 10993-12 reflect the in vivo conditions of Vitrebond applications. This seems to be mostly due to the hydrophilic nature of the genotoxic ingredients.

Animals↗

PubMed1981

Measurements of the distribution of time intervals between cell passages in flow cytometry as a method for the evaluation of sample preparation procedures.

It is expected that the time sequence of cells passing through the sensing region of a flow cytometer should follow the statistics of a Poisson process. Thus, measurement of time intervals between passages of fluorescent microspheres and cells stained with mithramycin demonstrated the negative exponential distribution characteristic of such a process. However, analysis of cells stained with fluorescein-iso-thiocyanate showed marked deviations from a negative exponential distribution of time intervals. The distribution had an additional component due to time intervals with mean value about 60 times shorter than the population mean, and in certain cases more than 50% of the cells were registered with such short time intervals. This percentage could, however, be reduced to acceptable levels by modifications of the sample preparation procedures. Measurement of the distribution of time intervals between cell passages is proposed as a method for the evaluation of possible improvements in sample preparation procedures.

Cell Line↗

PubMed2001

Affinity chromatography as a method for sample preparation in gas chromatography/mass spectrometry.

Analytical chemistry aims at developing analytical methods and techniques for unequivocal identification and accurate quantitation of natural and synthetic compounds in a given matrix. Analytical methods based on the mass spectrometry (MS) technology, e.g., GC/MS and LC/MS and their variants, GC/tandem MS and LC/tandem MS, are best suited both for qualitative and quantitative analyses. GC/MS methods not only serve as reference methods, e.g., in clinical chemistry, but they are now widely and routinely used for quantitative determination of numerous analytes. However, despite inherent accuracy, analytical methods based on GC/MS commonly consist of several analytical steps, including extraction and derivatization of the analyte. In general, unequivocal identification and accurate quantification of an analyte in very low concentrations in complex matrices require further chromatographic techniques, such as high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC) for sample purification. In recent years, affinity chromatography (e.g., boronate and immunoaffinity chromatography) has been developed to a superior technique for sample preparation of numerous classes of compounds in GC/MS. In this article, the application and importance of affinity chromatography as a method for sample preparation in modern quantitative GC/MS method is described and discussed, using as examples various natural and synthetic compounds, such as arachidonic acid derivates, nitrosylated and nitrated proteins, steroids, drugs, and toxins.

Blood Proteins↗

PubMed2006

Sample preparation for serum/plasma profiling and biomarker identification by mass spectrometry.

In this article, we present an overview of the different strategies for sample preparation for identification by mass spectrometry (MS) of biomarkers from serum and/or plasma. We consider the effects of the variables involved in sample collection, handling and storage, and describe different approaches for removal of high abundance proteins and serum/plasma fractionation. We review the advantages and disadvantages of such techniques as centrifugal ultrafiltration, different formats for solid phase extraction, organic solvent extraction, gel and capillary electrophoresis, and liquid chromatography. We also discuss a variety of current proteomic methods and their main applications for biomarker-related studies.

Analytic Sample Preparation Methods↗

PubMed1984

Analysis of sample preparation procedures for enumerating fecal coliforms in coarse southwestern U.S. bottom sediments by the most-probable-number method.

The determination of bacterial densities in aquatic sediments generally requires that a dilution-mixing treatment be used before enumeration of organisms by the most-probable-number fermentation tube method can be done. Differential sediment and organism settling rates may, however, influence the distribution of the microbial population after the dilution-mixing process, resulting in biased bacterial density estimates. For standardization of sample preparation procedures, the influence of settling by suspended sediments on the fecal coliform distribution in a mixing vessel was examined. This was accomplished with both inoculated (Escherichia coli) and raw, uninoculated freshwater sediments from Saguaro Lake, Ariz. Both test sediments were coarse (greater than 90% gravel and sand). Coarse sediments are typical of southwestern U.S. lakes. The distribution of fecal coliforms, as determined by the most-probable-number method, was not significantly influenced by sediment settling and remained homogenous over a 16-min postmix period. The technique developed for coarse sediments may be useful for standardizing sample preparation techniques for other sediment types.

Bacteriological Techniques↗

PubMed1996

Sample preparation for the determination of purine nucleotide analogues in tissues.

A sample treatment procedure for the determination of thiopurine and ganciclovir nucleotides in human tissues was developed. Owing to the lack of suitable standards for most of the active nucleotide analogues, the procedure was based on two steps: (1) perchloric acid homogenization and deproteinization of the tissue specimen and (2) conversion of purine nucleotides into parent drug or free bases by enzymatic or acid hydrolysis. The parent drug or purine bases formed were then analyzed on a Hypersil ODS column using isocratic elution with dihydrogenphosphate buffer for ganciclovir nucleotides or the gradient elution mode with dihydrogenphosphate buffermethanol for thiopurine nucleotides. The sample treatment procedure was evaluated using guanosine triphosphate (GTP), 6-thioinosinic acid (6TIMP) and 6-thioguanosine monophosphate (6TGMP) as standards. Mean analytical recoveries determined by adding known concentrations of standards to the tissue specimen before sampling processing were higher than 97%. The sample preparation described is simple and represents a suitable method for the investigation of active nucleotide pool in tissues.

Antimetabolites↗

PubMed2001

Representative sampling and sample preparation in biological environmental monitoring using spruce shoots.

The design of sampling and sample processing has a tremendous effect on the analytical results from which conclusions are drawn with respect to the quality of the environment and any possible impact on human health. Large scale environmental surveys need rigorous planning and extensive screening experiments to evaluate their boundary conditions. Representativeness and sample homogeneity are highlighted here in the context of a long-term biological monitoring survey for trace elements using spruce shoots. Several aspects concerning the selection of samples, amount of material, material processing and sample preparation prior to chemical characterisation are illustrated by selected examples.

Environmental Monitoring↗

PubMed1981

Effect of sample preparation on basophilic stippling in bovine blood smears.

The effect of sample preparation on the amount of basophilic stippling of erythrocytes (BSE) was studied using blood from a calf with chronic experimental lead poisoning. The combination of EDTA anticoagulation and rapid drying of the blood smear resulted in the most BSE. Alcohol prefixation reduced BSE. Wright-Leishman stain was better than Wright stain in demonstrating BSE.

Journal Article↗

PubMed2002

Initial study of using a laminar fluid diffusion interface for sample preparation in high-performance liquid chromatography.

This report describes a new microfluidic device called the H Filter for sample preparation prior to HPLC. The H Filters make possible a diffusional transfer of an analyte from a sample stream into a stream of a "receiver" fluid. Existing mathematical models can be used for optimizing experimental conditions. The authors have selected the extraction of the antibiotic cephradine from blood to demonstrate the utility of the new device. The extracts of blood samples spiked with cephradine levels between 0.2 and 100 microg/ml were analyzed using a C8 reversed-phase column and UV detection at 260 nm. The HPLC results were in good agreement with theory. The recovery of 32.2+/-2.8% was uniform over the entire range of cephradine concentrations. The new method completely avoids the use of centrifuges, that is otherwise typical for most current methodologies for the preparation of blood samples prior to HPLC analysis.

Cephradine↗

PubMed2002

PREPmate automated processor: comparison of automated and manual methods of liquid-based gynecologic sample preparation.

The development of procedures for fully automated processing of liquid-based gynecologic samples has been the focus of considerable interest to the cytology laboratory. Liquid-based collection and processing technology has been shown to improve sample adequacy, resulting in an overall improvement in quality of sample preparations. PREPmate, an accessory to the PrepStain slide processor, automates the initial enrichment process of mixing and dispensing the specimen over a density gradient. This report describes a study evaluating cellularity and diagnostic reproducibility in SurePath samples processed using the PREPmate accessory compared to samples processed using a manual technique. Samples processed using the PREPmate accessory contained 8.3% more squamous cells. Exact diagnostic reproducibility between preparation types was 83.3%; when considering negative vs. abnormal (ASCUS+), in adequate samples, reproducibility was 100%.

Automation↗

PubMed2002

Inhibition of unwanted proteolysis during sample preparation: evaluation of its efficiency in challenge experiments.

Measures to counteract proteolysis during sample preparation are widely used; among them, protein extraction at a basic pH (Tris pH 11.0), sample boiling in sodium dodecyl sulfate (SDS), extraction in denaturing lysis solutions and the use of proteinase inhibitors combined with some of these approaches. Here, we tested their efficiency under stringent conditions using a high proteinase (trypsin and a mixture of pancreatic proteinases) contamination and as substrate, streptokinase, a protein highly sensitive to proteolytic degradation. Total degradation was observed in Tris pH 11.0. There was an efficient inhibition for the pancreatic proteinases after boiling in 1% SDS, 1% dithiothreitol (DTT), while trypsin inhibition was dependent on the enzyme-to-substrate ratio. A panel of 21 lysis solutions with variable concentrations of urea, thiourea and detergents was essayed for the ability to counteract proteolysis. In all solutions containing 7-9 M urea, detergents and proteinase inhibitors but not containing thiourea, there was a strong proteolysis. However, in all samples containing 2 M thiourea, proteolysis was inhibited. Moreover, inhibition was dependent on the thiourea concentration. According to these results, we are prompted to consider that the well-known benefits of incorporating thiourea into the lysis solution are a result of two factors, its efficiency in solubilizing proteins and the inhibition of the proteolysis of sensitive substrates; both contributing to the detection of a higher number of species in two-dimensional electrophoresis (2-DE) gels.

Animals↗

PubMed2002

Semi-automated liquid--liquid back-extraction in a 96-well format to decrease sample preparation time for the determination of dextromethorphan and dextrorphan in human plasma.

A semi-automated, 96-well based liquid-liquid back-extraction (LLE) procedure was developed and used for sample preparation of dextromethorphan (DEX), an active ingredient in many over-the-counter cough formulations, and dextrorphan (DOR), an active metabolite of DEX, in human plasma. The plasma extracts were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS-MS). The analytes were isolated from human plasma using an initial ether extraction, followed by a back extraction from the ether into a small volume of acidified water. The acidified water isolated from the back extraction was analyzed directly by LC-MS-MS, eliminating the need for a dry down step. A liquid handling system was utilized for all aspects of liquid transfers during the LLE procedure including the transfer of samples from individual tubes into a 96-well format, preparation of standards, addition of internal standard and the addition and transfer of the extraction solvents. The semi-automated, 96-well based LLE procedure reduced sample preparation time by a factor of four versus a comparable manually performed LLE procedure.

Automation↗

PubMed2006

Trends in solventless sample preparation techniques for environmental analysis.

The paper presents recent trends in solventless sample preparation techniques for environmental analysis. First, a general classification of solventless methods is given. Next, three of them, treated as preferable techniques, i.e. SPME, SDME and HS, are presented in detail, with respect to their usability and effectiveness for environmental samples. Examples of all discussed techniques are given in the tables.

Chromatography↗

PubMed1996

Sample preparation for taxol and cephalomannine determination in various organs of Taxus sp.

Solid-phase extraction and preparative thin-layer chromatography were applied as sample preparation techniques for the purification of crude extracts from twigs and needles of various Taxus species as well as for the isolation of taxol and cephalomannine for further reversed phase high performance liquid chromatography analysis. Significant differences in the contents of taxanes examined were found. The preparative chromatographic methods used were compared and evaluated as routine and reproducible procedures for the rapid isolation and determination of taxol and cephalomannine in plant extracts.

Antineoplastic Agents, Phytogenic↗

PubMed1977

Simplified micro-scale procedure for preparing samples for theophylline determination by liquid chromatography.

We describe an improved procedure for the preparation of plasma or serum for determination of theophylline by reverse phase high-performance liquid chromatography. Quantitative results are available in less than 30 min from receipt of sample. The chromatogram is complete in 8 to 16 min, which includes the use of an internal standard. Sample preparation consists of simple solvent denaturation of the sample proteins, and centrifugation to remove protein before chromatography. No precolumn is required to pretect the separating column. No interference was noted when sodium or lithium heparin or ethylenediaminetetraacetate were used as anticoagulants, but citrate treatment proved to be unsatisfactory because of a highly absorbing band that eluted with the same retention volume as theophylline.

Analysis of Variance↗