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PubMed1991

Comparison of traditional gas chromatography (GC), headspace GC, and the microbial identification library GC system for the identification of Clostridium difficile.

Three gas chromatography (GC) methods were compared for the identification of 52 clinical Clostridium difficile isolates, as well as 17 non-C. difficile Clostridium isolates. Headspace GC and Microbial Identification System (MIS) GC, an automated system which utilizes a software library developed at the Virginia Polytechnic Institute to identify organisms based on the fatty acids extracted from the bacterial cell wall, were compared against the reference method of traditional GC. Headspace GC and MIS were of approximately equivalent accuracy in identifying the 52 C. difficile isolates (52 of 52 versus 51 of 52, respectively). However, 7 of 52 organisms required repeated sample preparation before an identification was achieved by the MIS method. Both systems effectively differentiated C. difficile from non-C. difficile clostridia, although the MIS method correctly identified only 9 of 17. We conclude that the headspace GC system is an accurate method of C. difficile identification, which requires only one-fifth of the sample preparation time of MIS GC and one-half of the sample preparation time of traditional GC.

Bacteriological Techniques↗

PubMed2002

Analysis of acute myelogenous leukemia: preparation of samples for genomic and proteomic analyses.

During the last decade, several large clinical studies have demonstrated that analysis of chromosomal abnormalities is an essential basis for therapeutic decisions in patients with acute myelogenous leukemia (AML), and cytogenetic studies should now be regarded as mandatory both for routine treatment and as a part of clinical investigations in AML. However, new techniques for detailed genetic characterization and analysis of gene expression as well as protein modulation will become important in the further classification of AML subsets and the development of risk-adapted therapeutic strategies. In this context, we emphasize the importance of population-based clinical studies as a basis for future therapeutic guidelines. Such studies will then require the inclusion of patients at small clinical centers without specialized hematological research laboratories. To document a high and uniform quality of the laboratory investigations, it will be necessary to collect material for later analysis in selected laboratories. In this article, we describe current methods for collection of biological samples that can be used for later preparation of DNA, RNA, and proteins. With the use of gradient-separated AML cells, it should be possible to establish the necessary techniques for collection and handling of biological samples even at smaller centers, and complete collections from all included patients should then be possible even in population-based clinical studies.

Cytogenetic Analysis↗

PubMed2001

Evaluation of aluminium concentrations in samples of chocolate and beverages by electrothermal atomic absorption spectrometry.

Samples of chocolate, cocoa, tea infusions, soft drinks and fruit juice have been examined by, electrothermal atomic absorption spectrometry (ETA-AAS) for the presence of aluminium (Al). Fruit juices and chocolate were analysed after an adequate sample preparation; the other products were evaluated directly. Sampling was performed in duplicate for 248 independent samples. The mean Al concentration in chocolate was 9.2 +/- 7.5 mg kg(-1), and individual values were correlated with the per cent of cocoa in samples (Y = 0.63 + 0.27X, r = 0.78, p < 0.0001). Al concentration in commercial tea infusions ranged from 0.9 to 3.3 mg l(-1) (mean = 1.80 +/- 65 mg l(-1), whereas in laboratory-prepared samples it was 2.7 +/- 0.93 mg l(-1). In soft drinks, the concentrations of Al were lower, ranging from 9.1 to 179 microg l(-1); the highest values were observed in samples of orange squash (mean = 114 +/- 56 microg l(-1)). Apricot juice showed the highest Al level (mean = 602 +/- 190 microg l(-1)), being statistically, different from that of pear (mean = 259 +/- 102 microg l(-1)), but not different from that of peach juice (mean = 486 +/- 269 microg kg(-1)). Toxicologically, the amount of Al deriving from the consumption of these products is far below the acceptable daily intake of 1 mg kg(-1) body weight indicated by the FAO/WHO, and it is a verv low percentage of the normal Al dietary intake.

Aluminum↗

PubMed2003

Sonication effect on cellular material in sedimentation and gravitational field flow fractionation.

Sonication procedures are generally used prior to field flow fractionation (FFF) separation in order to produce suspensions without aggregates. Yeast cells manufactured in active dry wine yeast (ADWY) were placed in an ultrasound water bath in order to disrupt possible clumps and to obtain a single-cell suspension to be used in optimal conditions during fermentation processes. In order to determine whether this sample preparation procedure meets absolute needs, different yeast samples before and after sonication were analysed by two field flow fractionation techniques. It is shown that 2 min of sonication in the sample preparation process is sufficient to obtain an optimal dispersion of the yeast cells, that is, without critical percentage of aggregates. To demonstrate this effect, photographs of the yeast cell suspensions were performed with non-sonicated and sonicated yeast sample dispersion. The resulting data are compared with the elution profiles obtained from the two different FFF techniques. It is demonstrated that fractogram profiles prove the effectiveness of sonication methodologies.

Fractionation, Field Flow↗

PubMed2005

Liquid ultraviolet matrix-assisted laser desorption/ionization -- mass spectrometry for automated proteomic analysis.

We have combined several key sample preparation steps for the use of a liquid matrix system to provide high analytical sensitivity in automated ultraviolet -- matrix-assisted laser desorption/ionisation -- mass spectrometry (UV-MALDI-MS). This new sample preparation protocol employs a matrix-mixture which is based on the glycerol matrix-mixture described by Sze et al. The low-femtomole sensitivity that is achievable with this new preparation protocol enables proteomic analysis of protein digests comparable to solid-state matrix systems. For automated data acquisition and analysis, the MALDI performance of this liquid matrix surpasses the conventional solid-state MALDI matrices. Besides the inherent general advantages of liquid samples for automated sample preparation and data acquisition the use of the presented liquid matrix significantly reduces the extent of unspecific ion signals in peptide mass fingerprints compared to typically used solid matrices, such as 2,5-dihydroxybenzoic acid (DHB) or alpha-cyano-hydroxycinnamic acid (CHCA). In particular, matrix and low-mass ion signals and ion signals resulting from cation adduct formation are dramatically reduced. Consequently, the confidence level of protein identification by peptide mass mapping of in-solution and in-gel digests is generally higher.

Electrophoresis, Gel, Two-Dimensional↗

PubMed2006

Automated sample treatment by flow techniques prior to liquid-phase separations.

Sample preparation (SP) is an integral and important part of an analytical process. Lately, SP has been the topic of increased interest in research and development of novel advanced technologies. Major needs in this aspect are miniaturization, automation, and enrichment. Among other methodologies, flow techniques can be conveniently and effectively coupled to liquid-phase separation technologies for on-line sample preparation. This paper reviews the current trends in on-line automated sample preparation by flow-through techniques prior to liquid-phase separations. Strategies and interfaces developed to couple flow techniques with liquid chromatography, capillary electrophoresis, and mass spectrometry are described. Advantages and limitations of the coupling are discussed. The paper also highlights certain selected applications of these coupled systems.

Chromatography, Liquid↗

PubMedDate not supplied

Enrichment of integral membrane proteins for proteomic analysis using liquid chromatography-tandem mass spectrometry.

An increasing number of proteomic strategies rely on liquid chromatography-tandem mass spectrometry (LC-MS/MS) to detect and identify constituent peptides of enzymatically digested proteins obtained from various organisms and cell types. However, sample preparation methods for isolating membrane proteins typically involve the use of detergents and chaotropes that often interfere with chromatographic separation and/or electrospray ionization. To address this problem, a sample preparation method combining carbonate extraction, surfactant-free organic solvent-assisted solubilization, and proteolysis was developed and demonstrated to target the membrane subproteome of Deinococcus radiodurans. Out of 503 proteins identified, 135 were recognized as hydrophobic on the basis of their calculated hydropathy values (GRAVY index), corresponding to coverage of 15% of the predicted hydrophobic proteome. Using the PSORT algorithm, 53 of the proteins identified were classified as integral outer membrane proteins and 215 were classified as integral cytoplasmic membrane proteins. All identified integral cytoplasmic membrane proteins had from 1 to 16 mapped transmembrane domains (TMDs), and 65% of those containing four or more mapped TMDs were identified by at least one hydrophobic membrane spanning peptide. The extensive coverage of the membrane subproteome (24%) by identification of highly hydrophobic proteins containing multiple TMDs validates the efficacy of the described sample preparation technique to isolate and solubilize hydrophobic integral membrane proteins from complex protein mixtures.

Amino Acid Sequence↗

PubMed2006

Ozone in ambient air as a source of adventitious oxidation. A mass spectrometric study.

This paper reports on an oxidation of biomolecules that can occur during sample preparation for analysis by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). In measurements performed over several years, the oxidation artifacts were detected on methionine and tryptophan side chains in peptides containing these residues, as well as at the carbon-carbon double bonds of unsaturated phospholipids. A distinct hallmark of the artifacts is their seasonal nature: the highest levels of oxidation are typically seen in samples prepared for MALDI analysis during daytime hours and warmer months. The artifacts appear strongest when samples are allowed to air-dry in the absence of MALDI matrix and can be minimized when sample preparation is carried out in a nitrogen atmosphere or under vacuum. It is proposed that ambient ozone (originating from outdoor air taken into the laboratory through the air handling system) is acting during the obligate MALDI sample dry-down and matrix crystallization steps. To confirm the hypothesis, ozonolysis experiments as well as direct measurements of ambient ozone levels were performed. The hypothesis is reminiscent of the recent finding that ozone is the source of degradation of self-assembled alkanethiol monolayers that are exposed to ambient air (Schoenfisch, M. H.; Pemberton, J. E. J. Am. Chem. Soc. 1998, 120, 4502-4513). Thus, caution should be taken in handling ozone-reactive substances that are placed thinly on surfaces and exposed to ambient air, whether for MALDI analysis or for other increasingly popular applications involving the microdeposition of liquids.

Amino Acid Sequence↗

PubMed2003

Metabolite characterization in drug discovery utilizing robotic liquid-handling, quadruple time-of-flight mass spectrometry and in-silico prediction.

An assay method for identification of metabolites from in vitro microsomal incubations was developed for use in the early stage of drug discovery. We have developed a practical approach which involves integrated sample generation, sample preparation, bioanalysis, and data handling to maximize sample throughput and speed up the process for identification of metabolites. The assay system consisted of a robotic liquid handler (Genesis workstation) to generate and process samples, PALLAS MetabolExpert software to predict possible metabolites, exact mass measurement via a tandem quadrupole time-of-flight mass spectrometer (QTOF-MS) coupled with liquid chromatography to analyze samples, MetaboLynx software to find potential metabolites and Advanced Chemistry Development/MS (ACD/MS) software to provide guidance to the most likely hypothetical metabolite chemical structures. For purposes of evaluating this new method, dextromethorphan, alprenolol, and propranolol were incubated separately for up to 60 minutes with rat and human hepatic microsomes. The incubation and sample preparation were carried out in 96-well plates using the Genesis workstation. The bioanalysis was performed by LC-MS/MS using QTOF with MetaboLynx software to find metabolites. Metabolic products formed in vitro by rat and human microsomes were separated using an analytical column C18 with gradient elution at flow rate of 250 micro l/min. The internal mass calibration was performed by continuous postcolumn infusion of Haloperidol. The mass spectra from incubations containing NADPH were compared to those without NADPH (control) using the MetaboLynx software to find potential metabolites. Finally, the MS/MS spectra were processed by the ACD/MS software to predict the chemical structure. MetaboLynx software successfully identified metabolites for each of the drugs studied by automatically discerning expected metabolites. Exact differences in masses between each metabolite and parent drug were measured from five replicate sample injections. All measured values are accurate to less than 0.001Da or 3.8 ppm with the standard deviation within 0.0015 Da, which allowed good prediction/confirmation of empirical formulae. Hypothetical chemical structures were achieved by the ACD/MS software and provided a useful tool to assist in prediction of the metabolic pathways of the drugs. The metabolites identified were in good agreement with previously published results for all three compounds. This new method will greatly enhance throughput, which in turn will facilitate our ability to rapidly provide this guidance to the synthetic chemist.

Alprenolol↗

PubMed2004

Rapid identification of environmental bacterial strains by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

In recent years, various mass spectrometry procedures have been developed for bacterial identification. The accuracy and speed with which data can be obtained by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) could make this a powerful tool for environmental monitoring. However, minor variations in the sample preparation can influence the mass spectra significantly. Therefore, the first objectives of this study were the adjustment and the optimization of experimental parameters allowing a rapid identification of whole bacterial cells without laborious sample preparation. The tested experimental parameters were matrix, extraction solvent, salt content, deposition method, culture medium and incubation time. This standardized protocol was applied to identify reference and environmental bacterial strains of Escherichia coli, Salmonella and Acinetobacter. The environmental bacterial strains were isolated from sewage sludge using an original microextraction procedure based on repeated sonications and enzymatic treatments. The bacterial identification was realized by the observation of the respective genus-, species- and strain-specific biomarkers. This bacterial taxonomy could be completed within one hour, with minimal sample preparation, provided that sufficient bacteria had been collected prior to MALDI-TOF analysis.

Actinobacteria↗

PubMed2001

Quantitative evaluation of the CEEM soil sampling intercomparison.

The aim of the CEEM soil project was to compare and to test the soil sampling and sample preparation guidelines used in the member states of the European Union and Switzerland for investigations of background and large-scale contamination of soils, soil monitoring and environmental risk assessments. The results of the comparative evaluation of the sampling guidelines demonstrated that, in soil contamination studies carried out with different sampling strategies and methods, comparable results can hardly be expected. Therefore, a reference database (RDB) was established by the organisers, which acted as a basis for the quantitative comparison of the participants' results. The detected deviations were related to the methodological details of the individual strategies. The comparative evaluation concept consisted of three steps: The first step was a comparison of the participants' samples (which were both centrally and individually analysed) between each other, as well as with the reference data base (RDB) and some given soil quality standards on the level of concentrations present. The comparison was made using the example of the metals cadmium, copper, lead and zinc. As a second step, the absolute and relative deviations between the reference database and the participants' results (both centrally analysed under repeatability conditions) were calculated. The comparability of the samples with the RDB was categorised on four levels. Methods of exploratory statistical analysis were applied to estimate the differential method bias among the participants. The levels of error caused by sampling and sample preparation were compared with those caused by the analytical procedures. As a third step, the methodological profiles of the participants were compiled to concisely describe the different procedures used. They were related to the results to find out the main factors leading to their incomparability. The outcome of this evaluation process was a list of strategies and methods, which are problematic with respect to comparability, and should be standardised and/or specified in order to arrive at representative and comparable results in soil contamination studies throughout Europe. Pre-normative recommendations for harmonising European soil sampling guidelines and standard operating procedures have been outlined in Wagner G, Desules A, Muntau H, Theocharopoulos S. Comparative Evaluation of European Methods for Sampling and Sample Preparation of Soils for Inorganic Analysis (CEEM Soil). Final Report of the Contract SMT4-CT96-2085, Sci Total Environ 2001;264:181-186. Wagner G, Desaules A, Munatu H. Theocharopolous S, Quevauvaller Ph. Suggestions for harmonising sampling and sample pre-treatment procedures and improving quality assurance in pre-analytical steps of soil contamination studies. Paper 1.7 Sci Total Environ 2001b;264:103-118.

Environmental Monitoring↗

PubMed2000

Transmission electron microscopy of yeast.

The challenges of sample preparation can limit a researcher's selection of transmission electron microcopy (TEM) for analysis of yeast. However, with the exception of thin sectioning, preparation of well-fixed and infiltrated samples of yeast cells is achievable by any reasonably equipped laboratory. This review presents a general overview of TEM sample preparation methods and detailed protocols for chemical fixation of yeast for ultrastructural analysis and immunolabeling. For ultrastructural analysis, the most commonly used chemical fixation involves treatment with glutaraldehyde followed by either potassium permanganate or osmium. Prior to osmium postfixation, the cell wall must be enzymatically digested to allow optimal fixation and embedding. Freeze substitution methods continue to provide the highest quality of fixation, but equipment needed for these protocols is not generally available to many labs. The low viscosity of Spurr's resin makes it the resin of choice for ultrastructure studies. Immunoelectron microscopy has enjoyed great success in analysis of yeast molecular organization. For immunoelectron microscopy, glutaraldehyde/formaldehyde-fixed cells are embedded in LR White resin. The thin sections are then treated in much the same way as an immunoblot: following blocking, they are incubated in primary antiserum, washed, and then incubated in gold-labeled secondary antiserum.

Microscopy, Electron↗

PubMed1989

Direct determination of unconjugated HVA in human plasma filtrates by HPLC coupled with dual-electrode coulometric electrochemical detection.

We report a procedure for the rapid determination of unconjugated homovanillic acid (HVA) in human plasma by cartridge filtration followed by direct injection into an isocratic HPLC system coupled with dual-electrode coulometric electrochemical detection. Sample preparation is rapid and more than 72 samples can be studied in 24 h using an automated HPLC system. The intra- and interassay precisions of the assay equal or exceed previously reported methods. While this methodology has been employed to study plasma free HVA, the concept of the technique may be applicable to other compounds, as well as different assay procedures, to greatly decrease sample preparation time.

Autoanalysis↗

PubMed1995

Determination of fumagillin in muscle tissue of rainbow trout using automated ion-pairing liquid chromatography.

A high-performance liquid chromatographic assay is described as a routine analytical method for the determination of fumagillin in rainbow trout muscle tissue. Muscle tissue samples (1 g) containing fumagillin were deproteinized with 8 ml of an acetonitrile-water mixture (2:6, v/v). The extracts were purified with a Bond Elut Octyl C8 cartridge column, washed with a water-methanol mixture (95:5, v/v; 4 ml) and fumagillin was eluted with acetonitrile (1 ml). Analytical separations were performed by reversed-phase HPLC with UV detection at 351 nm under gradient conditions. The mobile phase was acetonitrile-0.005 M tetrabutyl ammonium phosphate in water (pH 7.8). The assay is specific and reproducible within the fumagillin range of 20-1000 ng/g and recovery at 20 ng/g was 69.2%. Sample preparation involves the use of a robotic sample preparation system. Gravimetric validation of all operations enabled Good Laboratory Practices to be observed.

Animals↗

PubMed2006

Light scattering measurements on microemulsions: estimation of droplet sizes.

Different scattering methods were used as tools to assess the size of droplets in highly diluted microemulsions. These were obtained after dilution of a self-emulsifying system made up of an oil, a surfactant and ethanol. Typical methods, often used in size and shape determination of particles, such as SAXS and USAXS suffer in the present case from a lack of electrondensity contrast. It becomes clear from our extensive use of dynamic light scattering that one should be careful in interpreting the latter data as well. Sample preparation and the subsequent handling of the samples during the experiments strongly affect reproducibility of the results. There is a need for well-defined protocols at the level of sample preparation and data handling. In the present research one uses extensively dynamic light scattering (DLS) in the back scattering mode and strengths and pitfalls, inherent to the backscattering technique, are discussed. It is crucial to be aware of droplet size distributions (monomodal/bimodal/multimodal) while reporting mean radii (Rh) as this radius is only relevant in the case of well-defined monomodal distributions. Moreover, one should asses the shape of the droplets prior to data interpretation, as usual in scattering methods, by an independent method. Anyway the shape of the time correlation functions of the scattered intensity should be reported or at least inspected as they provide information on the reproducibility of the experiments hence safegarding the value of the physical meaning of the final value of droplet size (Rh). Preferentially static light scattering (SLS) measurements should always support DLS experiments as the angular dependence is very sensitive to the presence of large particles.

Castor Oil↗

PubMed1998

Determination of drugs in biological fluids by high-performance liquid chromatography with on-line sample processing.

An automated two column HPLC system with the new packing material LiChrospher RP-18 ADS (alkyl-diol-silica) was tested for the determination of several drugs and metabolites (talinolol, celiprolol, metoprolol, oxprenolol, triamterene, trimethoprim, tiracizine, articaine, detajmium, ajmaline, lamotrigine) in various biological fluids (serum, urine, intestinal aspirates, supernatants of cell cultures and supernatants after protein denaturation). The method allows the direct injection of biological fluids into a reversed-phase HPLC system and on-line clean-up and sample enrichment by a column-switching technique. Precision, accuracy and sensitivity were similar to conventional assays as described in the literature. With this new method it was possible to measure drug concentrations in various biological fluids without changing the sample preparation procedure. In some cases an additional sample preparation like protein denaturation or solid-phase extraction was advantageous to enhance the sensitivity of the method and the life-time of the ADS column.

Autoanalysis↗

PubMed2004

Measuring seven endogenous ketolic estrogens simultaneously in human urine by high-performance liquid chromatography-mass spectrometry.

A rapid, sensitive, and specific high-performance liquid chromatography-electrospray ionization-multistage mass spectrometry (MS) method for measuring endogenous ketolic estrogen metabolites in human urine has been developed. The method requires a single hydrolysis/extraction/derivatization step and only 2.5 mL of urine, yet is able to simultaneously quantify estrone and its 2-methoxy and 2-, 4-, and 16alpha-hydroxy derivatives, 16-ketoestradiol, and 2-hydroxyestrone-3-methyl ether metabolites. The combination of a simple hydrazone derivatization step with multistage MS greatly enhances the sensitivity and specificity of the analysis of endogenous estrogen within human urine. Standard curves are linear over a 100-fold concentration range with linear regression correlation coefficients typically greater than 0.99. The lower limit of quantitation for each ketolic estrogen is 0.2 ng/2.5-mL urine sample (10 pg on column), with an accuracy of 93-103% and an overall precision, including the hydrolysis, extraction, and derivatization steps, of 1-13% relative standard derivation (RSD) for samples prepared concurrently and 8-16% RSD for samples prepared in separate batches. This method also allows for the identification of 2-hydroxyestrone-3-methyl ether in urine obtained from both pre- and postmenopausal women. This potentially protective estrogen metabolite has been previously reported only in the urine of pregnant women. Since individual patterns of estrogen metabolism may influence the risk of breast cancer, accurate and specific measurement of estrogen metabolites in biological matrixes will facilitate future research on breast cancer prevention, screening, and treatment.

Adult↗

PubMed2005

Measuring fifteen endogenous estrogens simultaneously in human urine by high-performance liquid chromatography-mass spectrometry.

A sensitive, specific, accurate, and precise high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry method for measuring the absolute quantities of 15 endogenous estrogens and their metabolites in human urine has been developed and validated. The method requires a single hydrolysis/extraction/derivatization step and only 0.5 mL of urine, yet is capable of simultaneously quantifying estrone and its 2-, 4-methoxy and 2-, 4-, and 16alpha-hydroxy derivatives, and 2-hydroxyestrone-3-methyl ether; estradiol and its 2-, 4-methoxy and 2-, 16alpha-hydroxy derivatives, 16-epiestriol, 17-epiestriol, and 16-ketoestradiol in pre- and postmenopausal women as well as men. Standard curves are linear over a 10(3)-fold concentration range with the standard error of the estimate (SEE) and the relative standard error of the estimate (RSEE) for the linear regression line ranging from 0.0131 to 0.1760 and 1.2 to 7.3%, respectively. The lower limit of quantitation for each estrogen is 0.02 ng/0.5 mL urine sample (2 pg on column), with the percent recovery of a known added amount of compound (accuracy) of 96-107% and an overall precision, including the hydrolysis, extraction, and derivatization steps, of 1-5% relative standard deviation (RSD) for samples prepared concurrently and 1-12% RSD for samples prepared in separate batches. Since individual patterns of estrogen metabolism may influence the risk of breast cancer, accurate, precise, and specific measurement of endogenous estrogen metabolites in biological matrixes will facilitate future research on breast cancer prevention, screening, and treatment.

Adult↗