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PubMed1993

1H-NMR study on ganglioside amide protons: evidence that the deuterium exchange kinetics are affected by the preparation of samples.

The kinetics of H/2H chemical exchange of the amide proton has been suggested as one of the tools available for investigating hydrogenbond stabilizing interactions in gangliosides. The amide proton/deuterium (NH/2H) exchange rates in GM2 ganglioside were studied by 1H-NMR spectroscopy on 12 samples prepared following different procedures. In samples passed through a sodium salt Chelex-100 cation exchange resin column prior to being analysed the N-acetylneuraminic acid NH exchange occurred in less than 10 min and that of ceramide NH in 30 min. The N-acetylgalactosamine acetamido NH exchange was slower, the half-life of the signal ranging from 15 min to 3.5 h. Contact of the Chelex-treated GM2 samples with water, through a dialysis process, modified the NH/2H exchange rate values, the N-acetylgalactosamine acetamido NH exchange becoming faster than that of ceramide NH and similar to that of N-acetylneuraminic acid NH. Our results indicate that the deuterium/proton exchange rate strongly depends on sample preparation (ion content and minor contaminants present in water). The three-dimensional model involving the N-acetylgalactosamine acetamido NH and the N-acetylneuraminic acid carboxyl group hydrogen-bonding, which is supported by experimental evidence, cannot be confirmed by NH-exchange measurement.

Amides↗

PubMed1994

Simplified sample preparation for detection of wheat streak mosaic virus and barley yellow dwarf virus by PCR.

A PCR diagnostic procedure for wheat streak mosaic virus (WSMV) was developed using a primer derived from 3'-terminal sequences of five WSMV isolates and an oligo d(T)-based primer. Cereal extracts prepared by digestion with proteinase K and boiling permitted PCR-based detection of both WSMV and BYDV in field samples. This procedure saves time, eliminates multiple liquid transfer steps, and reduces the chances of cross contamination. Sensitivity of such assays is still very good; BYDV could be readily detected in plant sap diluted over 1000-fold. Further, parallel detection of WSMV and barley yellow dwarf virus (BYDV) in the same samples is possible with this method.

Base Sequence↗

PubMed1991

Simplified sample preparation for fluoride determination in biological material.

A simple and rapid preparation method for the determination of fluoride in biological materials (blood and food) of various origins, is described. A homogenized sample was placed in a plastic diffusion cell and calcium phosphate added, it was then dried at 55 degrees C and treated with 70% HClO4 and 40% AgClO4. After digestion for 24 h at 55 degrees C, the fluorides released were fixed on the upper part of a diffusion cell containing a thin layer of NaOH. The analyses of the diffused fluoride were carried out with an ion-selective electrode. The proposed microdiffusion method, without mineralization, enables quantitative separation of the fluoride from the biological samples.

Animal Feed↗

PubMed1986

A new method for using fluorescein to demonstrate oto- and rhinoliquorrhea. I. Sample preparation by electrophoresis and photometric identification of fluorescein.

We have described a new method for the identification of sodium fluorescein in cases of cerebrospinal fluid (CSF) leakage. To take samples, small sponges of Merocel are placed into the nostrils or external auditory canal, as indicated. They are left in situ for 12h after intrathecal injection of 5% sodium fluorescein. Fluorescein is identified by electrophoretic separation on 1% agarose gel and demonstration with the fluorescence photometer. This method of testing has various advantages: the use of small sponges enables collections of minimal amounts of CSF; potential sources of disturbance (e.g. hemoglobin, etc.) are eliminated; false-positive results are avoided; the time needed for sample analysis lasts only 10 min. All necessary materials and equipment, as well as the taking and analysis of samples, are described in detail. The method used was tested both clinically on patients and in the laboratory and its sensitivity is clearly illustrated.

Cerebrospinal Fluid Otorrhea↗

PubMed2002

Miniaturized solid-phase extraction as a sample preparation technique for the determination of phthalates in water.

Miniaturized solid-phase extraction (SPE) has been developed and successfully employed for the determination of organic species in water samples by liquid chromatography (LC). The method is based on the concept of a microscale extraction technique using a fused-silica capillary column for gas chromatography (GC), so-called in-tube solid-phase microextraction (SPME). The extraction conditions, such as the extraction time and flow-rate for the extraction and desorption process, were investigated as well as the effect of the internal structure of the extraction capillary on the efficiency. By inserting a stainless steel wire into the extraction capillary to reduce the internal volume of the capillary with the same surface area of the coating, an improved extraction and pre-concentration effects were obtained. Further pre-concentration was accomplished by the extraction device with a novel fiber-in-tube configuration. The direct coupling of the extraction method with a LC system has made it possible to determine low levels of phthalates in water samples without high consumption of organic solvents. The system developed must have potential applications for the analysis of environmental and biological samples in aqueous sample matrices.

Capillary Action↗

PubMed1999

Supersonic molecular beam-hyperthermal surface ionisation coupled with time-of-flight mass spectrometry applied to trace level detection of polynuclear aromatic hydrocarbons in drinking water for reduced sample preparation and analysis time.

Analysis of sub-ppb levels of polynuclear aromatic hydrocarbons (PAHs) in drinking water by high performance liquid chromatography (HPLC) fluorescence detection typically requires large water samples and lengthy extraction procedures. The detection itself, although selective, does not give compound identity confirmation. Benchtop gas chromatography/mass spectrometry (GC/MS) systems operating in the more sensitive selected ion monitoring (SIM) acquisition mode discard spectral information and, when operating in scanning mode, are less sensitive and scan too slowly. The selectivity of hyperthermal surface ionisation (HSI), the high column flow rate capacity of the supersonic molecular beam (SMB) GC/MS interface, and the high acquisition rate of time-of-flight (TOF) mass analysis, are combined here to facilitate a rapid, specific and sensitive technique for the analysis of trace levels of PAHs in water. This work reports the advantages gained by using the GC/HSI-TOF system over the HPLC fluorescence method, and discusses in some detail the nature of the instrumentation used.

Chromatography, High Pressure Liquid↗

PubMed2000

A simple sample preparation for enhancing the sensitivity of mass spectrometric oligosaccharide determinations through the use of an adsorptive hydrophobic resin.

A simple microadsorption technique is described to remove detergent additives from oligosaccharide samples before their mass spectrometric analysis. The described methodology has been validated with submicrogram quantities of contaminated glycoproteins. This procedure is applicable to investigating minute quantities of glycans in both the positive- and negative-ion mode of matrix-assisted laser desorption/ionization mass spectrometry.

Animals↗

PubMed2003

Direct tissue analysis using matrix-assisted laser desorption/ionization mass spectrometry: practical aspects of sample preparation.

Practical guidelines for the preparation of tissue sections for direct analysis by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry are presented. Techniques for proper sample handling including tissue storage, sectioning and mounting are described. Emphasis is placed on optimizing matrix parameters such as the type of matrix molecule used, matrix concentration, and solvent composition. Several different techniques for matrix application are illustrated. Optimal instrument parameters and the necessity for advanced data analysis approaches with regards to direct tissue analysis are also discussed.

Animals↗

PubMed1991

Fully automated high-performance liquid chromatographic analysis of whole blood and plasma samples using on-line dialysis as sample preparation. Determination of oxytetracycline in bovine and salmon whole blood and plasma.

A fully automated technique for high-performance liquid chromatographic analysis of whole blood and plasma is described. Samples are automatically injected into a dialyser where proteins and blood cells are removed. The dialysates are concentrated on a small column prior to analysis. This technique is used for the determination of oxytetracycline in whole blood and plasma. After dialysis oxytetracycline and the internal standard, tetracycline, are retained on a polystyrene enrichment column and subsequently separated on a polystyrene analytical column by ion-pair chromatography. Using ultraviolet detection 50 ng/ml can be detected. Validation showed good within-day and between-day accuracy and precision. Different oxytetracycline concentrations were found in plasma and whole blood. This difference varied between the species.

Animals↗

PubMed2005

Development of a sample preparation method for fungal proteomics.

Since filamentous fungi including basidiomycetous fungi possess an exceptionally robust cell wall as in microorganisms, effective extraction of intracellular proteins is a key step for fungal proteomic studies. To overcome the experimental obstacle caused by cell walls, we utilized fungal protoplasts, prepared from the brown-rot basidiomycete, Tyromyces palustris. The amount and quality of proteins extracted from the protoplast cells were much higher than that from the mycelial cells. Quantitative comparisons of proteome maps prepared from mycelial and protoplast cells indicated protein spots with a wider range of molecular weights and pIs in the protoplast sample. Furthermore, no streaking or tailing was observed in the protoplasts, suggesting that effective extraction of intracellular proteins from protoplasts might help suppress degradation of proteins during this process. In addition to the efficiency of protein extraction, simple and efficient subcellular fractionation was also achieved using protoplast cells.

Basidiomycota↗

PubMed1989

High-performance liquid chromatographic determination of mitoxantrone in plasma utilizing non-bonded silica gel for solid-phase isolation to reduce adsorptive losses on glass during sample preparation.

Mitoxantrone, a highly active antineoplastic agent, was found to bind strongly to non-bonded silica gel and glassware. When a Hamilton syringe was used to load and inject a mitoxantrone solution (0.4 microgram/ml in water) on to a high-performance liquid chromatographic (HPLC) system, about 95% of the loaded compound was found to bind to the glass surface of the syringe barrel and could not be removed by rinsing with water. It could, however, be removed slowly with an acidic solution and thus a small peak of mitoxantrone was present on the chromatogram whenever a blank acidic solution was injected with the syringe. The bound mitoxantrone could be removed effectively from the syringe surface with a solution of tetramethylammonium chloride, citric acid, methanol and water (elution solvent). This binding introduces a large error in assay results and might be one of the major factors responsible for contradictory pharmacokinetic data that have been reported. A new plasma preparation scheme and an HPLC method for mitoxantrone were developed to address this binding problem. Mitoxantrone was extracted directly from plasma samples with a plastic mini-column packed with non-bonded silica gel and eluted with the above elution solvent. The eluent was analysed by HPLC on an ODS column with an absorbance detector at 658 nm. The mobile phase was 0.1 M triethylamine phosphate (pH 3.0) in water-tetrahydrofuran-methanol (69:1:30) containing 0.02 M tetramethylammonium chloride. Methylene blue was added as an internal standard. Preliminary results showed that mitoxantrone levels in human plasma followed a triphasic decay curve after an intravenous bolus injection. The terminal elimination half-lives measured in three patients (mean t1/2 gamma = 25 min) were all shorter than the published values which ranged from 56 min to 9 days.

Chemical Phenomena↗

PubMed1998

Small-Scale DNA Sample Preparation Method for Field PCR Detection of Microbial Cells and Spores in Soil.

Efficient, nonselective methods to obtain DNA from the environment are needed for rapid and thorough analysis of introduced microorganisms in environmental samples and for analysis of microbial community diversity in soil. A small-scale procedure to rapidly extract and purify DNA from soils was developed for in-the-field use. Amounts of DNA released from bacterial vegetative cells, bacterial endospores, and fungal conidia were compared by using hot-detergent treatment, freeze-thaw cycles, and bead mill homogenization. Combining a hot-detergent treatment with bead mill homogenization gave the highest DNA yields from all three microbial cell types and provided DNA from the broadest range of microbial groups in a natural soil community. Only the bead mill homogenization step was effective for DNA extraction from Bacillus globigii (B. subtilis subsp. niger) endospores or Fusarium moniliforme conidia. The hot-detergent-bead mill procedure was simplified and miniaturized. By using this procedure and small-scale, field-adapted purification and quantification procedures, DNA was prepared from four different soils seeded with Pseudomonas putida cells or B. globigii spores. In a New Mexico soil, seeded bacterial targets were detected with the same sensitivity as when assaying pure bacterial DNA (2 to 20 target gene copies in a PCR mixture). The detection limit of P. putida cells and B. globigii spores in different soils was affected by the amount of background DNA in the soil samples, the physical condition of the DNA, and the amount of DNA template used in the PCR.

Journal Article↗

PubMed2004

Sample preparation and determination of gabapentin in venous and capillary blood using liquid chromatography-tandem mass spectrometry.

An analytical method for the determination of gabapentin in serum obtained from venous blood samples has been developed using high-performance liquid chromatography (HPLC)-tandem mass spectrometry. In addition, a comparative study between capillary plasma samples and venous serum samples was carried out. This demonstrates the potential for the use of the described analytical system using very small amounts of blood. As internal standard (S)-(+)-alpha-amino-cyclohexane-propionic acid hydrate was used. Gabapentin and the internal standard are structural isomers, but have different m/z values for the fragments after collision induced dissolution. Gabapentin has 172-->154 and 172-->136 transitions and amino-cyclohexane-propionic acid hydrate has a 172-->126 transition which can be detected in tandem MS. Analysis of gabapentin was carried out on a C8 HPLC column using an isocratic mobile phase consisting of ammonium acetate (pH 3.0; 5mM)-methanol (96:4, v/v). The analytical method was validated for venous serum samples. Limit of detection was 1.6ng/ml and lower limit of quantification was 7.5ng/ml. R.S.D. values and bias values were within the range of acceptance for all concentration levels. The method developed for venous serum samples is being used in a gabapentin monitoring study using population pharmacokinetic modeling.

Acetates↗

PubMed2002

Two-dimensional gel electrophoresis: recent advances in sample preparation, detection and quantitation.

A strength of two-dimensional polyacrylamide gel electrophoresis (2D PAGE) is its ability to resolve and investigate the abundance of several thousand proteins in a single sample. This enables identification of the major proteins in a tissue or subcellular fraction by mass spectrometric methods. In addition, 2D PAGE can be used to compare quantities of proteins in related samples, such as those from altered environments or from mutant and wild type, thus allowing the response of classes of proteins to be determined. Those proteins showing a correlated difference in expression may participate in related processes, and this subsequently helps to define protein function. Although there are many limitations of the 2D gel technology that mean it will never be comprehensive in protein coverage, its use for the identification of relatively abundant proteins is now widespread. However, there are still surprisingly few examples of quantitative analysis of changes in protein abundance. In this review we highlight recent advances towards true quantitative analysis of 2D gels that will lead to better prediction of protein function. Despite the development of promising alternatives, 2D PAGE is likely to remain in extensive use for the foreseeable future, because the technology is now simple and readily available to many laboratories.

Animals↗

PubMed1983

Sample preparation with ion-exchange resin before liquid-chromatographic determination of plasma catecholamines.

Estimation of plasma catecholamine concentrations by "high-performance" liquid chromatography, although increasingly used, still presents difficulties. We describe a method for clean-up of plasma samples on cation-exchange resin before they are extracted with alumina. This procedure eliminates many of the interfering compounds often present in plasma extracts prepared with the alumina treatment alone. The method can be adapted to use commercially available disposable cation-exchange columns.

Catecholamines↗

PubMed2003

Dynamic three-phase microextraction as a sample preparation technique prior to capillary electrophoresis.

Dynamic three-phase (liquid-liquid-liquid) microextraction was developed for capillary electrophoresis. Four aromatic amines as model compounds were extracted from 4-mL aqueous samples adjusted to basic condition (donor solution) through a small volume of organic solvent impregnated in a hollow fiber, which was held by the needle of a conventional syringe, and retracted into a 5-microL acidic acceptor solution inside the syringe. A renewable organic film and aqueous sample plug were formed inside the hollow fiber with the repeated movement of the syringe plunger enabled by a programmable syringe pump. This is believed to be the first reported instance of a semiautomated dynamic liquid-liquid-liquid microextraction (LLLME) procedure. Following this microextraction, the 5-microL acceptor solution was analyzed by capillary zone electrophoresis (CE). This new technique provided approximately 140-fold enrichment in 20 min. Utilizing 4-chloroaniline as internal standard, dynamic LLLME could provide good reproducibility (<4.0%). In addition, this method allowed the direct transfer of extracted analytes to a CE system for analysis.

Journal Article↗

PubMed1978

[Microdetermination of glucose using glucose dehydrogenase, with independent sample preparation in the routine laboratory (author's transl)].

A micromethod for the determination of glucose in 20 microliter of capillary blood using glucose dehydrogenase is described. After deproteinisation with uranyl acetate, the samples are analysed by an Autoanalyzer II method or by a manual procedure. Precision and accuracy are well correlated with the hexokinase-glucose-6-phosphate dehydrogenase method. Eleven months experience have shown the practicability and economic advantages of this method.

Autoanalysis↗

PubMed2002

Effects of sample preparation on the measurement of organic carbon, hydrogen, nitrogen, sulfur, and oxygen concentrations in marine sediments.

The elemental composition of marine sediment provides useful information for the study of environmental processes including biogeochemical cycling and contaminant partitioning. It is common practice to acidify marine sediment samples to remove carbonate before measuring the concentrations of organic carbon (C). To date, however the effects of acidification on the concentrations of hydrogen (H), nitrogen (N), sulfur (S) and oxygen (O) in marine sediments have not been explicitly addressed. Acidification may contaminate or alter the sediment samples and create experimental artifacts affecting the validity of resulting H/C, C/N and O/C ratios. The objective of this study was to quantify how various preparation techniques affect the measured concentrations of C, H, N, S and O in marine sediments. Effects of four different pretreatments: unacidified (whole), acidification by HCl vapor, acidification by direct addition of HCl, and combustion were evaluated using five marine sediments and a standard reference material. The magnitude of carbonate loss between the vapor and direct acidification treatments was evaluated using stable C isotope analysis. Carbonates were most effectively removed by direct addition of HCl; and our results agree with findings of other studies which found direct addition of HCl to be the most accurate method for measuring organic C. However, the acid treatments elevated the apparent concentration of H and O; and in a few cases concentrations of N and S were significantly affected by acidification. In general, combustion significantly reduced all elemental concentrations compared to the whole sample. Based on these results, we recommend analysis of the untreated whole sediment for determining N, H, O, and S.

Carbon↗