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PubMed2003

Single-sample preparation for simultaneous cellular redox and energy state determination.

A simple and reliable method for the preparation of biological samples for the evaluation of biochemical parameters representative of the redox and energy states, such as glutathione (GSH), oxidized glutathione (GSSG), oxidized nicotinamide adenine dinucleotide (NAD+), reduced nicotinamide adenine dinucleotide (NADH), oxidized nicotinamide adenine dinucleotide phosphate (NADP+), reduced nicotinamide adenine dinucleotide phosphate (NADPH), coenzyme A (CoASH), oxidized CoASH, ascorbate, malondialdehyde, oxypurines, nucleosides, and energy metabolites, is presented. Fast deproteinization under nonoxidizing conditions is obtained by tissue homogenization in ice-cold, nitrogen-saturated CH3CN + 10 mM KH2PO4 (3:1; v:v), pH 7.40. After sample centrifugation to pellet precipitated proteins, organic solvent removal is performed on clear supernatants by three washings with large volumes of high-performance liquid chromatography (HPLC)-grade chloroform. The remaining aqueous phase, free of solvent and any lipid-soluble substances that may interfere with the further metabolite analysis, is used for the simultaneous ion-pairing HPLC determination of 39 compounds by means of a Kromasil C-18, 250 x 4.6-mm, 5-microm-particle-size column with tetrabutylammonium hydroxide as the pairing reagent. Results obtained by using the present method to prepare different rat tissue extracts demonstrate that it is possible to perform a single tissue preparation only for monitoring, in the same sample, compounds representative of the redox state (through the direct determination of GSH, GSSG, NAD+, NADH, NADP+, NADPH, CoASH, and oxidized CoASH) and of the cell energy state (by the analysis of oxypurines, nucleosides, and energy metabolites). Applicability of this sample processing procedure to quantify variations of the aforementioned compounds under pathological conditions was effected in rats subjected to moderate closed-head trauma.

Animals↗
PubMed2004

Miniaturized sample preparation method for determination of amphetamines in urine.

A simple and miniaturized sample preparation method for determination of amphetamines in urine was developed using on-column derivatization and gas chromatography-mass spectrometry (GC-MS). Urine was directly applied to the extraction column that was pre-packed with Extrelut and sodium carbonate. Amphetamine (AP) and methamphetamine (MA) in urine were adsorbed on the surface of Extrelut. AP and MA were then converted to a free base and derivatized to N-propoxycarbonyl derivatives using propylchloroformate on the column. Pentadeuterated MA was used as an internal standard. The recoveries of AP and MA from urine were 100 and 102%, respectively. The calibration curves showed linearity in the range of 0.50-50 microg/mL for AP and MA in urine. When urine samples containing two different concentrations (0.50 and 5.0 microg/mL) of AP and MA were determined, the intra-day and inter-day coefficients of variation were 1.4-7.7%. This method was applied to 14 medico-legal cases of MA intoxication. The results were compared and a good agreement was obtained with a HPLC method.

Amphetamines↗
PubMed1992

Solid-phase extraction with supercritical fluid elution as a sample preparation technique for the ultratrace analysis of flavone in blood plasma.

A new sample preparation technique, solid-phase extraction with supercritical fluid elution, was developed for the selective isolation of ultratrace levels of drugs from plasma. Plasma samples spiked with a drug were applied to octadecylsilane cartridges and the cartridges were then washed, briefly dried and directly fitted into cells for subsequent supercritical fluid elution. The absolute recovery was studied by using a radiolabeled model compound. The extraction selectivity was examined by chromatographing the extracts with a reversed-phase high-performance liquid chromatographic method with ultraviolet detection. The effects of extraction pressure and the length of capillary restrictors on drug recovery were examined in order to determine the optimal conditions for supercritical fluid elution. The performance of the method was compared to that of conventional solid-phase extraction in terms of recovery, selectivity, precision and accuracy of analysis. Flavone was used as the model compound and dog plasma as the biological matrix for these studies.

Animals↗
PubMedDate not supplied

Sample preparation bias in carbon stable isotope ratio analysis of fruit juices and sweeteners.

Two sample preparation methods are commonly used for carbon stable isotope ratio analysis (SIRA). One involves combustion of the sample with oxygen at 850 degrees C; the other involves combustion of the sample with CuO in an evacuated glass tube at 550 degrees C. I observed in our laboratory that these 2 methods yield different results for sugar-based products such as fruit juices, sweeteners, and vanillin. The CuO method yields results approximately 1%. more positive than the oxygen combustion method. This bias is also observed in other laboratories, as shown in an analysis of the results of the AOAC collaborative studies of carbon SIRA of maple syrup, orange juice, honey, and honey protein. The oxygen combustion method is the AOAC method for honey, apple juice, and orange juice; both methods are incorporated into the AOAC method for maple syrup. I recommend that data generated by the CuO combustion method be appropriately corrected to yield results concordant with the official oxygen combustion method.

Beverages↗
PubMed1988

Method for evaluating mutagenicity of water. II. Conditions for applying new sample preparation method to the Ames test.

Conditions for the application of a new sample preparation method developed in a previous study using the small particle size and highly porous polystyrene and anion-exchange resins CSP800 and CHPA25 were investigated. The desorbing solvents DMSO and 4 N NaNO3 (0.5 ml), corresponding to 11 of water sample, could be dosed on a plate for the mutagenicity test, the Ames test. It was confirmed that trace impurities from the resins were not mutagenic. The mutagenicity of several drinking water samples was determined by this new method and evaluated quantitatively by converting to the concentrations of the standard mutagens, 4-nitroquinoline-1-oxide and 2-aminoanthracene, without S9 and with S9 respectively. Furthermore, the mutagenicity of the nonionic pollutants adsorbed on CSP800 and that of the anionic pollutants adsorbed on CHPA25 could be evaluated separately.

Dose-Response Relationship, Drug↗
PubMed2006

Trizol-based method for sample preparation and isoelectric focusing of halophilic proteins.

A persisting complication in the development of well-resolved two-dimensional PAGE maps of halophilic proteins is their natural incompatibility with isoelectric focusing (IEF). The complete desalting of samples, which is necessary for IEF, tends to aggregate halophilic proteins, often requires relatively large amounts of starting material due to significant loss of sample, and is relatively time-consuming. Here, we describe a method of preparing protein samples from the haloarchaeon Haloferax volcanii that not only desalts the samples thoroughly but also drastically reduces the amount of protein loss associated with previous sample preparation methods and prevents protein aggregation during the removal of salt. This method of sample preparation, which incorporates Trizol (phenol/guanidine isothiocyanate), can easily be extended to analyze halophilic proteins from other organisms.

Archaeal Proteins↗
PubMedDate not supplied

Improvements in the sample preparation for the determination of ruthenium in catalysts by different spectrometric techniques.

The sample preparation steps of two analytical methods for the determination of ruthenium in carbon supported catalysts with flame atomic absorption spectrometry (FAAS) or UV-vis spectrophotometry, were carefully optimised. As a first step, the carbon support is ignited in air at 450 degrees C then the residue is fused with potassium hydroxide and potassium nitrate to convert ruthenium into ruthenate anion RuO4(2-). The melt is dissolved in water with potassium persulphate and potassium hydroxide as stabilisers. Ruthenium may be directly estimated by measuring the UV-vis molecular absorption of ruthenate at the isosbestic point of the ruthenate-perruthenate mixture or by FAAS. In the latter case, an aliquot of the sample solution is added to the concentrated hydrochloric acid to obtain hexachlororuthenate RuCl6(2-), that is nebulized into the flame after proper dilution. This novel procedure allows to determination of ruthenium without the sensitivity loss (-66%) which occurs in the alkaline media resulting from the fusion. The relative standard deviation is 1.1% for FAAS and 1.3% for UV-vis, at 5% Ru (n = 7). The detection limit (3sB) is 0.07% Ru for FAAS and 0.02% Ru for UV-vis. Both methods were applied to the analysis of a commercial sample and a statistical comparison was carried out.

Catalysis↗
PubMed1992

Sample preparation of animal tissues and cell cultures for secondary ion mass spectrometry (SIMS) microscopy.

Sample preparation is a critical step in the elemental analysis of animal tissues and cell cultures with ion microscopy. Since live cells cannot be analyzed with ion microscopy, a careful sample fixation is necessary which preserves the native structural and chemical integrity of a specimen. The evaluation of morphological and chemical integrity of a fixed specimen is necessary before any physiological explanation of ion fluxes is interpreted based on ion microscopy. For diffusible ion localization studies, strict cryogenic procedures are recommended. Examples are shown for diffusible ion microanalysis in frozen-freeze-dried tissues and cell cultures. Ion microscopy studies of tightly bound elements/molecules may be conducted in chemically fixed and/or plastic embedded specimens. Since it is not generally known which elements/molecules are tightly bound to the tissue matrix, a confirmation of elemental distribution with cryogenic procedures is desirable. A recent approach of combining laser scanning confocal fluorescence microscopy and ion microscopy on the same frozen freeze-dried cell is also discussed for recognizing smaller cytoplasmic structures in ion microscopy images.

Animals↗
PubMed2000

Focused ion beam sample preparation of continuous fibre-reinforced ceramic composite specimens for transmission electron microscopy

The microanalysis of interfaces in fibre-reinforced composite materials is dependent on the successful preparation of specimens suitable for transmission electron microscope (TEM) inspection. Ideal samples should possess large amounts of structurally intact and uniform thin area in the fibre/matrix interface regions of the samples. Because fibre/matrix interfaces in this class of materials are often designed to fail under mechanical stress, conventionally prepared samples are prone to interfacial failure and differential thinning, both of which preclude detailed TEM microanalysis. These effects were seen in a conventionally dimpled and ion-beam-thinned specimen prepared from a continuous fibre reinforced ceramic composite composed of CaWO4-coated Nextel 610TM fibres in an alumina matrix. The dimpled specimen showed large amounts of interfacial failure, with only thick regions of the specimen left intact. To overcome these limitations, a focused ion beam (FIB) technique was applied to this same material. The superiority of the FIB-produced sample is evident in both the morphology and scanning transmission electron microscopy analyses of the sample.

Journal Article↗
PubMed2002

Characterization of an insoluble poly(9,9-diphenyl-2,7-fluorene) by solvent-free sample preparation for MALDI-TOF mass spectrometry.

The application of solvent-free sample preparation for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) allowed the characterization of an insoluble fraction of poly(9,9-diphenylfluorene) that was previously hindered by the lack of suitable characterization methods. The MALDI mass spectrometric analysis gives valuable mechanistic information about the heterogeneous polymerization process of the insoluble high molecular weight fraction of the polymer. The fragmentation appearing even under moderate desorption and ionization conditions of this rigid backbone analyte is identified as a multiple loss of the bulky phenyl side groups and can be avoided by applying the new MALDI matrix 7,7,8,8-tetracyanoquinodimethane. A specialized fragmentation study by postsource decay MALDI-TOF MS reveals a molecular weight dependent change in fragmentation mechanism from an exclusive cleavage of side groups from long polymer chains to an additional cleavage of the polymer backbone of short polymer chains.

Journal Article↗
PubMed2002

Strategy for the development of automated methods involving dialysis and trace enrichment as on-line sample preparation for the determination of basic drugs in plasma by liquid chromatography.

Among the sample preparation techniques, dialysis followed by clean-up and enrichment of the dialysate on a pre-column has proved to be a useful approach for the LC determination of drugs in plasma. By use of sample processors, like the ASTED system, such bioanalytical methods can be fully automated, the dialysis and trace enrichment steps being directly coupled to LC. In order to facilitate the development of such automated methods, a strategy based on a decision tree has been elaborated. After the selection of appropriate conditions for the LC analysis, the decision tree provides information about suggested starting conditions and guidelines for the optimisation of the most important parameters likely to influence analyte recovery and method selectivity. The plasma samples are dialysed on a cellulose acetate membrane in the static-pulsed mode and the dialysate is enriched on a trace enrichment pre-column packed with octadecyl silica or with a strong cation-exchange material. This decision tree is until now restricted to the analysis of basic drugs in plasma. In order to demonstrate the applicability of this method development strategy, an automated procedure based on the coupling of dialysis with trace enrichment has been developed for the LC determination of antifungal agents (clotrimazole, econazole and miconazole) in plasma.

Antifungal Agents↗
PubMed2003

Solventless sample preparation for pesticides analysis in environmental water samples using solid-phase microextraction-high resolution gas chromatography/mass spectrometry (SPME-HRGC/MS).

Solid-phase micro-extraction (SPME) coupled on line with high resolution gas chromatography and mass spectrometric detection is described for the analysis of pesticides in environmental water samples. Experiments were performed in order to optimize the SPME extraction conditions for selected pesticides including tiomethon, trichorfon, dimethoate, diazinon, malathion, dicofol, methidathion, ethion, bromopropylate and pyrazophos from spiked water solutions. To enhance the SPME efficiency, experimental conditions including the fiber composition, stirring rate, temperature, adsorption time, desorption time and salt concentration were optimized. After validation, the SPME-GC/MS methodology was applied to real-world environmental water samples.

Acrylic Resins↗
PubMed2002

[Mode of sample preparation for pseudotuberculosis laboratory diagnosis by polymer chain reaction method].

A mode of feces sample preparation was developed for polymerase chain reaction (PCR) assay. It was based on alkaline treatment of the material. This treatment killed the most part of indigenous microflora, whereas Yersinia survived, because it was relatively resistant to alkaline. The mode was tested using human feces artificially contaminated with Yersinia pseudotuberculosis. Positive responses in samples containing 10(3)-10(8) microbial cells per ml were obtained by PCR assay with Yersi and Yers2, Invl and Inv2, YP3 and YP4 primers. Diagnostic efficiency of PCR for patients, small mammals, and washings from environmental objects was 4.75, 1.66, and 2.12 times higher than diagnostic efficiency of bacteriological analysis of these samples, respectively. Positive results in PCR were obtained at the day of the material collection and treatment, whereas Y. pseudotuberculosis was isolated only after 8-20 days. Positive samples in PCR and in bacteriological analysis were found to coincide. A brief scheme of the Y. pseudotuberculosis laboratory diagnosis is suggested. According to this scheme, target-oriented bacteriological assay is performed only in those samples, in which preliminary PCR assay after 1-3 days of incubation gave positive results of Y. pseudotuberculosis DNA detection.

Animals↗
PubMed2003

Development of an effective sample preparation method for the proteome analysis of body fluids using 2-D gel electrophoresis.

A sample preparation is still the most critical step in two-dimensional electrophoresis (2-DE) and should be optimized for each type of sample. In this study, a protein extraction method from body fluids was developed using a combined centrifugal filter device and a sample treating buffer. When plasma, amniotic fluid, urine, and tear were tested with this method, the recovery of protein reached almost 90% and high-quality separation of 2-DE gel was obtained.

Amniotic Fluid↗
PubMed2003

Sample preparation, extraction efficiency, and determination of six arsenic species present in food composites.

Several sample preparation techniques were investigated to maximize the efficiency of arsenic species extraction from food composites. The optimized method includes lyophilization of food followed by prewashing with acetone and extraction by sonication with 50/50 methanol/water. Six arsenic species were separated and quantitated using an ammonium carbonate buffer system by ion exchange chromatography coupled to inductively coupled plasma mass spectrometry. The performance of the method for speciated arsenic components was evaluated using a matrix containing high fat food composite fortified with arsenic species. A certified reference material, dogfish muscle, was used to evaluate extraction methods for total arsenic content in food composites. More than 200 food composite samples were analyzed during an 18 month period, demonstrating the reliability of the analytical method over a long time period.

Acetone↗
PubMed2001

Sample preparation for residue determination of gentamicin and neomycin by liquid chromatography.

The effect of sample preparation on the determination of gentamicin and neomycin residues in animal tissues was investigated. The extract was mixed with an ion-pair reagent and applied to an octadecyl cartridge. The cartridges were washed with buffer followed by water, and analytes were eluted with ion-pair buffer-acetonitrile mixture. The aminoglycosides were derivatized with 9-fluorenylmethyl chloroformate prior to liquid chromatography using a reversed-phase column and fluorescence detection. Under the conditions applied neomycin was fully separated from all the gentamicin compounds. The highest recoveries of gentamicin and neomycin from spiked tissues were obtained using trichloroacetic acid after initial extraction with phosphate-buffered saline. No interfering peaks from endogenous compounds of matrix were noted at the elution position of the analytes. An intra-laboratory validation of the whole procedure was performed. The calibration graphs were linear from 0.1 to 1.0 mg/kg for gentamicin, and from 0.2 to 1.0 mg/kg for neomycin. Limits of detection were 0.05 mg/kg and 0.10 mg/kg for gentamicin and neomycin, respectively. Limits of quantitation for gentamicin and neomycin were 0.1 and 0.20 mg/kg muscle, liver or kidney tissue, respectively. Recoveries of gentamicin spiked at levels of 0.1 mg/kg porcine tissues ranged from 76 to 86%. Recoveries of neomycin spiked at levels of 0.2 mg/kg porcine tissues ranged from 77 to 83%. The validated procedure was used to determine gentamicin concentrations in porcine tissue after dosing with gentamicin at a level of 5 mg/kg body mass.

Animals↗
PubMed1998

Performance of modern sample preparation techniques in the analysis of Fusarium mycotoxins in cereals.

The efficiency of modern sample preparation techniques are discussed and compared to well-established techniques with respect to the determination of zearalenone in corn and B-trichothecenes in wheat in the microgram/kg range. This includes the use of immuno-affinity columns and of multifunctional Mycosep columns as well as the employment of supercritical fluid extraction for the trace analysis of these major Fusarium mycotoxins. In addition, the performance of new analytical methods was investigated in an interlaboratory comparison study only recently organized by our laboratory. From both the validation data, and from the results of the intercomparison study, the suitability and competitiveness of the described methods could be clearly demonstrated.

Chromatography, Affinity↗