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PubMed2002

Improved sample preparation of biomaterials for in vitro genotoxicity testing using reference materials.

New biomaterial related reference materials with known genotoxic properties were produced in order to study the sample preparation and in vitro genotoxicity testing of biomaterials. We incorporated genotoxic substances like benzo[a]pyrene into the biomaterial Tecoflex, a polyurethane frequently used for catheters and other applications. We demonstrated that the model compound benzo[a]pyrene is sufficiently extracted by organic solvents, whereas cell culture medium only extracts very limited quantities. By changing the medium several times during extraction the extracted amount was augmented. Using higher amounts of organic solvent in relation to the reference material's surface led to a higher recovery of extracted benzo[a]pyrene. For the in vitro genotoxicity testing using the Mammalian Cell Gene Mutation Test (HPRT test), Mammalian Chromosome Aberration Test, and bacterial umu- and SOS-tests, concentration of extracts is a prerequisite because of the low sensitivity of the test systems. Often cytotoxicity interferes with the evaluation of genotoxic effects. We demonstrated that some recommendations of the ISO 10993-Part 3 and 12,(1),(2) dealing with the biological evaluation of medical devices, seem to be insufficient, and new rules for the in vitro genotoxicity testing of biomaterials have to be established.

Animals↗
PubMed2002

Solid-phase microextraction as a sample preparation strategy for the analysis of seleno amino acids by gas chromatography-inductively coupled plasma mass spectrometry.

Solid-phase microextraction (SPME) is used as a sample preparation strategy for gas chromatographic (GC) analysis of the seleno amino acids, selenomethionine (SeMet), selenoethionine (SeEt) and selenocystine (SeCys). Acylation of the amino group and esterification of the carboxylic group in these compounds was performed with isobutylchloroformate to increase volatility. The amino acid derivatives were then extracted by silica fibers with polydimethylsiloxane (PDMS) coatings prepared by the sol-gel process. Investigations of extraction time, acid and salt addition, and polymer length (for the sol-gel process) were conducted with the goal of procedural optimization. Initial characterizations were conducted using gas chromatography with flame ionization detection (GC-FID). Inductively coupled plasma mass spectrometric detection was employed for final selenium detection. Sub-ppb detection limits were obtained for all analytes although relative standard deviations were higher than those typically obtained in solid-phase microextraction.

Amino Acids↗
PubMedDate not supplied

Diagnostic PCR: validation and sample preparation are two sides of the same coin.

Increased use of powerful PCR technology for the routine detection of pathogens has focused attention on the need for international validation and preparation of official non-commercial guidelines. Bacteria of epidemiological importance should be the prime focus, although a "validation infrastructure" once established could easily be adapted for PCR-based detection of viruses and parasites. The aim of standardization should be the widespread adoption of diagnostic PCR for routine pathogen testing. European experience provides the impetus for realization of this vision through preparation of quantitative reference DNA material and reagents, production of stringent protocols and tools for thermal cycler performance testing, uncomplicated sample preparation techniques, and extensive ring trials for assessment of the efficacy of selected matrix/pathogen detection protocols.

Humans↗
PubMedDate not supplied

Specific sample preparation in colorectal cancer.

Large tissue samples from ten patients operated for colorectal cancer were prepared in the operating room in iced phosphate buffered saline, containing ethylene diaminetetraacetic acid and protease inhibitors. After cutting the specimens into small fragments, the tissues were gently pressed through a steel mesh. Membranes were permeabilized in chilled ethanol 70% to allow cytosolic fluoresceine isothiocyanate labeling, performed with anti-cytokeratin (CAM 5.2) antibodies. Samples were quantitatively sorted with a fluorescence activated cell sorter (FACS) and denatured before processing separation by two-dimensional electrophoresis on polyacrylamide gels. This procedure made it possible to sample about 4 x 10(7) viable normal and tumoral cells before fixation, and up to 4 x 10(6) cells after FACS. The gels run before and after fixation showed no major differences. The rate of cytokeratin-positive cells in the samples was the following (mean, CI 5-95%): mucosa 29.5% (8.9-66.7%), tumor 44.3% (6.6-94.8%). The epithelial cell content in colorectal cancer and normal mucosa shows important intersample variations. This is important for any comparison of fresh samples, whether at DNA, RNA, or at the protein level. We propose a method allowing the preparation of pure epithelial cell samples from normal and tumoral colonic fresh mucosa.

Colorectal Neoplasms↗
PubMed1978

Quantitative analysis of steroid profiles from urine by capillary gas chromatography. I. Accuracy and reproducibility of the sample preparation.

A method is described for the determination of steroid profiles from urine by means of gas chromatography using high-efficiency glass capillary columns. The accuracy and reproducibility of essential steps in the sample preparation (extraction of steroids and steroid conjugates by means of XAD-2, enzymatic hydrolysis with Helix pomatia juice, solvolysis in acidified ethyl acetate and alkali wash) are established using different endogenously labelled urine samples, obtained from normal subjects to whom labelled steroids had been administered. Preliminary results are given on the reproducibility of the derivatization procedure (formation of methoxime-trimethylsilyl (MO-TMS) ethers), the gas chromatographic analysis and the whole method. Two procedures for the purification of MO-TMS steroid derivatives are compared. Application of the method to urine samples of patients with various endocrine disorders is included.

Adult↗
PubMed1981

Analytical methodology for sample preparation, detection, quantitation, and confirmation of N-nitrosamines in foods.

Significant advances have been made over the past decade in methodology for the analysis of foods and other samples for volatile N-nitrosamines. Procedures for the isolation, cleanup, and concentration of N-nitrosamines were developed and applied to a broad range of food types. Several chromatographic techniques and systems have also been developed which are capable of resolving N-nitrosamines in complex mixtures in a single run, and the use of highly selective detectors has decreased sample preparation time while increasing sensitivity and precision. Unequivocal confirmation of N-nitrosamines in foods can now be achieved by mass spectrometry at low to sub-microgram/kg levels. However, further development of methodology for nonvolatile N-nitroso compounds is needed. This review paper discusses these and other topics related to the analysis of foods for N-nitrosamines.

Food Analysis↗
PubMed1987

Effect of sample preparation and liquid chromatography column choice on selectivity and precision of plasma catecholamine determination.

Research into the role of the catecholamines has often depended on the reliable determination of plasma catecholamine concentrations, which present a challenge since they are normally in the low pg/ml range. Most methods employ liquid chromatography, with variations in sample preparation, the separation mechanism, and detection. We tested a new approach to sample clean-up using boric acid gel instead of alumina. No advantage was found. We also compared cation-exchange separation with ion-pair chromatography. Several improvements are possible with the former, most notably greater precision, better specificity, and increased throughput.

Aluminum Oxide↗
PubMed2000

Sample preparation based on dynamic ion-exchange solid-phase extraction for GC/MS analysis of acidic herbicides in environmental waters.

The newly established enrichment technique, dynamic ion-exchange solid-phase extraction (DIE-SPE), was studied for sample preparation for GC/MS analysis of 16 acidic herbicides in environmental waters. C18 bonded silica was the solid-phase material used. The optimal sample pH was weakly acidic to neutral. However, for common tap water and surface water, which run pH 6-9, all the acidic herbicides except for Chloramben could be effectively extracted from a sample of 1,000-mL volume without pH adjustment. The humic acid could be concurrently extracted from water, but most of it was separated from the sample by using 3 mL of 10% methanol in acetone as the eluent, which would completely elute the analytes and leave a large part of the humic acid on the cartridge. The selective elution reduced the interference of humic acid and made the DIE-SPE an effective approach for the analysis of the acidic herbicides in surface water. Comparing DIE-SPE with conventional reversed-phase SPE (RP-SPE), the former gave higher recoveries for the acidic herbicides and was less affected by sample matrixes. A tandem-cartridge system combining RP- and DIE-SPE in sequence was set up for the simultaneous isolation of the acidic herbicides and removal of the interfering substances. Despite some minimal retention on the upper RP-SPE cartridge, most of the acidic herbicides could be extracted on the lower DIE-SPE cartridge with recovery over 80% except for Chloramben (50%), fenoprop (73%), MCPB (67%), and 2,4-DB (70%) when a 500-mL aqueous sample of pH 9.5 was percolated through the tandem-cartridge system. The effectiveness of the system in removing the long carbon chain fatty acids as well as the basic and neutral organic interfering substances from the sample was also demonstrated.

Acids↗
PubMed2003

Imaging of fullerene-like structures in CNx thin films by electron microscopy; sample preparation artefacts due to ion-beam milling.

The microstructure of CN(x) thin films, deposited by reactive magnetron sputtering, was investigated by transmission electron microscopy (TEM) at 200kV in plan-view and cross-sectional samples. Imaging artefacts arise in high-resolution TEM due to overlap of nm-sized fullerene-like features for specimen thickness above 5nm. The thinnest and apparently artefact-free areas were obtained at the fracture edges of plan-view specimens floated-off from NaCl substrates. Cross-sectional samples were prepared by ion-beam milling at low energy to minimize sample preparation artefacts. The depth of the ion-bombardment-induced surface amorphization was determined by TEM cross sections of ion-milled fullerene-like CN(x) surfaces. The thickness of the damaged surface layer at 5 degrees grazing incidence was 13 and 10nm at 3 and 0.8keV, respectively, which is approximately three times larger than that observed on Si prepared under the same conditions. The shallowest damage depth, observed for 0.25keV, was less than 1nm. Chemical changes due to N loss and graphitization were also observed by X-ray photoelectron spectroscopy. As a consequence of chemical effects, sputtering rates of CN(x) films were similar to that of Si, which enables relatively fast ion-milling procedure compared to carbon compounds. No electron beam damage of fullerene-like CN(x) was observed at 200kV.

Argon↗
PubMed2001

Sample preparation with fiber-in-tube solid-phase microextraction for capillary electrophoretic separation of tricyclic antidepressant drugs in human urine.

Solid-phase microextraction (SPME) is a solvent-free sample preparation technique using a thin coating attached to the surface of a fused silica-fiber as the extraction medium, which has been successfully applied to the analysis of a wide variety of compounds by coupling to gas chromatography (GC). In recent years, in-tube SPME using GC capillary column as the extraction medium has also been developed and coupled with liquid chromatography (LC) for the preconcentration of nonvolatile compounds. In this study, an on-line interface between the fiber-in-tube SPME and capillary electrophoresis (CE) has been developed, and the preconcentration and separation of four tricyclic antidepressant (TCA) drugs, amitriptyline, imipramine, nortriptyline, and desipramine, were performed with the hyphenated system. Under the optimized condition, a better extraction performance than conventional in-tube SPME was obtained, even the length of the extraction medium was much shorter. The results clearly indicated that the fiber was working effectively as an extraction medium. For the separation of these four TCAs, capillary electrophoretic separation with beta-cyclodextrin as the buffer additive has been employed and the application of the developed system to the analysis of complex sample mixtures in a biological matrix is also demonstrated.

Amitriptyline↗
PubMed2007

Isoprostane measurement in plasma and urine by liquid chromatography-mass spectrometry with one-step sample preparation.

BACKGROUND: Isoprostane F(2alpha) (iPF(2alpha)-III) concentration in plasma and urine is widely accepted as a measure of oxidative stress. Gas chromatography-mass spectrometry (GC/MS) methods for measuring iPF(2alpha)-III involve several steps of sample preparation and are labor-intensive, and ELISA methods, although easier to use, are less reliable. Therefore we developed a simple and sensitive method involving 1-step sample cleanup and HPLC/MS quantification. METHODS: Samples of plasma or urine were enriched with a deuterated (iPF(2alpha)-III-D4) standard, treated with KOH to liberate the bound isoprostanes, then loaded onto an immunoaffinity column, and the bound isoprostane was eluted with 95% ethanol. The concentrated sample was injected onto a C-18 HPLC column, and the isoprostane was eluted with a gradient of acetonitrile in water and analyzed by electrospray negative ionization, selectively monitoring the ions 353.2 (iPF(2alpha)-III) and 357.2 (iPF(2alpha)-III-D4). The amount of isoprostane in the sample was calculated from the ratio of the intensities of the 2 ions. RESULTS: The described method has a detection limit of 0.5 ng/L, with a linear dynamic range of 1-5000 ng/L. The intra- and interassay imprecisions were 4.68% and 3.88%, respectively. The values obtained correlated strongly with the GC/MS procedure (r = 0.80), but the absolute values were approximately 4- to 5-fold lower, because the present method measures specifically 1 isomer of isoprostane, whereas the GC/MS method measures 4 isomers together. CONCLUSIONS: Because of its simplicity and lower limit of quantification, the present method provides a useful noninvasive tool for determining oxidative stress in patients.

Adult↗
PubMedDate not supplied

Sample preparation and determination of ginkgo terpene trilactones in selected beverage, snack, and dietary supplement products by liquid chromatography with evaporative light-scattering detection.

Ginkgo biloba leaf extract has been widely used in dietary supplements and more recently in some foods and beverages. Sample preparation procedures for determination of ginkgo terpene trilactones (including bilobalide and ginkgolides A, B, C, and J) in various sample matrixes were developed in this study. Ginkgo leaves and capsules were extracted with 5% KH2PO4 aqueous solution under sonication. Tea bags were extracted with boiling water, whereas drink samples were taken directly from the bottles. After filtration and the addition of NaCl to approximately 30% (w/v), the terpene trilactones in aqueous solutions were quantitatively extracted with ethyl acetate-tetrahydrofuran (4 + 1, v/v). Puff samples (a cereal-based fried snack item) were first defatted by using hexane or by using supercritical fluid extraction and then extracting under sonication with methanol-acetic acid (99 + 1, v/v). After evaporation of the organic phase, the terpene trilactones were redissolved in methanol and determined on a C18 reversed-phase column by liquid chromatography (LC) with evaporative light-scattering detection. The method of standard additions and gas chromatography with flame ionization detection were used for method validation. For most samples, the relative standard deviation was <10%. The identities of target compounds in ginkgo leaves and drink samples were confirmed by LC/electrospray ionization-tandem mass spectrometry.

Algorithms↗
PubMed1998

Comparison of two sample preparation techniques for sniffing experiments with broccoli (Brassica oleracea var. italica Plenck).

The suitability of the headspace solid phase microextraction (HSSPME) for gas chromatography-olfactometry (GC-O) with aroma extract dilution analysis in comparison to the dynamic head space sampling on a Tenax trap was tested exemplarily by the aroma volatiles of fresh broccoli. A high number of odour sensations in qualitative olfactometry was registered with both sample preparation techniques. The key aroma compounds of the fresh broccoli material are represented by high flavour dilution factors with dynamic head space sampling and headspace SPME. The SPME method has found to be a convenient and fast technique suitable especially for qualitative GC-O. The adsorption selectivity of the fiber and the substance discrimination have to be taken into account for quantitative use like aroma extract dilution analysis.

Brassica↗
PubMed1987

Sample preparation for gas chromatography with electron capture detection: determination of total and free thyroxin in serum.

Relatively clean gas chromatography with electron capture detection (GC-ECD) chromatograms are obtained for both total and free thyroxin (T4) in serum by improving sample preparation. This is based on establishing a sequence of steps that cumulatively overcome two classes of interference: those present in the initial sample and those introduced by the procedure. The main source for the latter contaminants is the derivatization step, a problem that was largely overcome by employing HPLC after this step. Also it is helpful to use ion-exchange columns early in the procedure under fast-flow conditions with intermediate flows of air to speed up and enhance their reliability. The work establishes some guidelines for future applications of GC-ECD to the determination of sub-nanogram analytes requiring derivatization, an area in which GC-ECD has been remiss in the past. As a side benefit, total T4 in serum is determined by HPLC for the first time with uv detection.

Chromatography, Gas↗
PubMed2000

Development of a method for sample preparation for subsequent identification and measurement of 1,2,3,4-tetrahydroisoquinolines and other potentially neurotoxic compounds by high-performance liquid chromatography with ultraviolet and fluorescence detection in blood plasma of Parkinson's disease patients.

We developed sample preparation methods for the detection of various biogenic phenylethylamine derivatives such as 3,4-dihydroxyphenylalanine, and their cyclisation products with aldehydes, i.e., 1,2,3,4-tetrahydroisoquinoline derivatives in blood samples. 1,2,3,4-Tetrahydroisoquinolines are considered to play an essential role as neurotoxic compounds in the pathomechanism of Parkinson's disease. We used reversed-phase high-performance liquid chromatography with ultraviolet and fluorescence detection for separation and identification. Ultrafiltration, protein precipitation and solid-phase extraction were investigated for purification of blood samples and enrichment of various compounds with a wide range of hydrophilicity and hydrophobicity. Protein precipitation by methanol and perchloric acid is a fast method to separate the analytes from the plasma matrix. A higher yield of the analytes is attained with prior addition of an alkylsulfonic acid giving a fine-grained precipitate. With the addition of ion pairing compounds into the sample it is possible to enrich not only lipophilic compounds such as norharman, tryptamine and melatonin, but also hydrophilic ones such as 3,4-dihydroxyphenylalanine by reversed-phase solid-phase extraction. Ultrafiltration is not useful as a screening method.

Blood Proteins↗
PubMed1999

An automated sample preparation system for large-scale DNA sequencing.

Recent advances in DNA sequencing technologies, both in the form of high lane-density gels and automated capillary systems, will lead to an increased requirement for sample preparation systems that operate at low cost and high throughput. As part of the development of a fully automated sequencing system, we have developed an automated subsystem capable of producing 10,000 sequence-ready ssDNA templates per day from libraries of M13 plaques at a cost of $0.29 per sample. This Front End has been in high throughput operation since June, 1997 and has produced > 400,000 high-quality DNA templates.

DNA↗
PubMedDate not supplied

Aspects of sample preparation for the determination of actinoids in soil.

In the frame of the re-certification measurements of the IAEA-375 soil reference material the activity concentration of four actinoids (U, Th, Pu and Am) was determined. The method is based on microwave digestion or aqua regia leaching, separation by extraction chromatography and alpha-particle counting source preparation by co-precipitation or electro-deposition. This article describes some of the problems encountered during the validation of the sample preparation method and their solutions.

Actinoid Series Elements↗