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At least 1,333 records · Page 74Linked to original sources
PubMed2005

Natural sample fractionation by FlFFF-MALLS-TEM: sample stabilization, preparation, pre-concentration and fractionation.

Two flow field flow fractionation (FlFFF) systems: symmetrical (SFlFFF) and asymmetrical (ASFlFFF) were evaluated to fractionate river colloids. Samples stability during storage and colloids concentration are the main challenges limiting their fractionation and characterization by FlFFF. A pre-fractionation (<0.45 microm) and addition of a bactericide such as NaN3 into river colloidal samples allowed obtaining stable samples without inducing any modification to their size. Stirred cell ultra-filtration allowed colloidal concentration enrichment of 25-folds. Scanning electron microscope (SEM) micrographs confirmed the gentle pre-concentration of river samples using the ultra-filtration stirred cell. Additionally, larger sample injection volume in the case of SFlFFF and on channel concentration in the case of ASFlFFF were applied to minimize the required pre-concentration. Multi angle laser light scattering (MALLS), and transmission electron microscope (TEM) techniques are used to evaluate FlFFF fractionation behavior and the possible artifacts during fractionation process. This study demonstrates that, FlFFF-MALLS-TEM coupling is a valuable method to fractionate and characterize colloids. Results prove an ideal fractionation behavior in case of Brugeilles sample and steric effect influencing the elution mode in case of Cézerat and Chatillon. Furthermore, comparison of SFlFFF and ASFlFFF fractograms for the same sample shows small differences in particle size distributions.

Colloids↗
PubMedDate not supplied

Storage and preservation of whole blood samples for use in detection of human immunodeficiency virus type-1 by the polymerase chain reaction.

Methods used in the diagnosis of human immunodeficiency virus type-1 (HIV-1) infection by the polymerase chain reaction (PCR) usually require the separation of lymphocytes from a whole-blood sample within 24 hours of patient sampling. A method is described in which blood samples are mixed with a cryopreservative ('Glycigel'), stored frozen, and DNA suitable for use in an HIV PCR recovered. Samples can be stored at -20 degrees C for up to 3 months and still give positive results with all samples from infected patients; storage at -80 degrees C for at least 3 months shows no loss of titre. The method shows no loss of sensitivity compared to previously described sample preparation methods. Deglycerolised Glycigel supernatants were found to be suitable for conventional anti-HIV-1 serological studies and loss of sensitivity only represented the dilution effect due to sample preparation. Application of the method as a means of storing samples frozen at the point of sampling and transporting them to a central laboratory for processing is demonstrated using samples taken from HIV-1-infected mothers and their babies.

Adult↗
PubMed2003

Automated multiple-layer spotting for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of synthetic polymers utilizing ink-jet printing technology.

Recently, a new multiple-layer matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) sample spotting technique for poly(ethylene glycol), offering improved analysis possibilities, was described. In this contribution the application of ink-jet printing to automated, multiple-layer MALDI-TOFMS sample preparation of synthetic polymers is presented, allowing accurate deposition of matrix, additive and analyte solutions. The new sample preparation technique was evaluated for poly(ethylene glycol) as well as poly(methyl methacrylate) standards, and optimized settings for both synthetic polymers have been obtained.

Journal Article↗
PubMed1996

Determination of the novel topoisomerase I inhibitor NU/ICRF 505 and its major metabolite in plasma, tissue and tumour by high-performance liquid chromatography.

A high-performance liquid chromatographic technique is presented for the determination of the novel topoisomerase I inhibitor NU/ICRF 505 (a tyrosine conjugate of anthraquinone), its major metabolite (NU/ICRF 505/M) and an internal standard (NU/ICRF 513, dihydroxyphenylalanine conjugate). The method uses a reversed-phase (Apex ODS-2) stationary phase and a mobile phase consisting of 0.25 M ammonium acetate adjusted to pH 3 with 25% (v/v) trifluoroacetic acid and methanol with gradient elution. Between-day variation in retention times were less than 1% for NU/ICRF 505 and 513 and 2.4% for the metabolite. Selective detection was achieved at a wavelength of 545 nm giving a limit of detection of 2 ng on column and 50 ng/ml after sample preparation for all three components. Chromatograms were free from interfering peaks even at very high detector sensitivity. Sample preparation was based on incubation of biological specimens (0.5 ml plasma or homogenate) with dimethylsulphoxide and acetonitrile at 4 degrees C for 30 min followed by centrifugation. Liver and tumour were homogenised in phosphate buffered saline. Recoveries were consistently high (81.7-106.7% for NU/ICRF 505; 88.7-103.3% for NU/ICRF 513 and 83.7-98.7% for NU/ICRF 505/M) with between day coefficients of variation of normally less than 10%. The method will contribute significantly to the preclinical evaluation of NU/ICRF 505.

Animals↗
PubMed2003

Detergents destabilize the cubic phase of monoolein: implications for membrane protein crystallization.

The in meso method for membrane protein crystallization uses a lipidic cubic phase as the hosting medium. The cubic phase provides a lipid bilayer into which the protein presumably reconstitutes and from which protein crystals nucleate and grow. The solutions used to spontaneously form the protein-enriched cubic phase often contain significant amounts of detergents that were employed initially to purify and to solubilize the membrane protein. By virtue of their surface activity, detergents have the potential to impact on the phase properties of the in meso system and, by extension, the outcome of the crystallization process. The purpose of this study was to quantify the effects that a popular series of nonionic detergents, the n-alkyl-beta-D-glucopyranosides, have on the phase behavior of hydrated monoolein, the lipid upon which the in meso method is based. Phase identity and phase microstructure were characterized by small-angle x-ray diffraction on samples prepared to mimic in meso crystallization conditions. Measurements were made in the 0-40 degrees C range. Samples prepared in the cooling direction allow for the expression of metastability, a feature of liquid crystalline phases that might be exploited in low-temperature crystallization. The results show that the cubic phase is relatively insensitive to small amounts of alkyl glucosides. However, at higher levels the detergents trigger a transition to the lamellar phase in a temperature- and salt concentration-dependent manner. These effects have important implications for in meso crystallization. A diffraction-based method for assaying detergents is presented.

Bacteriorhodopsins↗
PubMed1997

Determination of opiates in hair. Effects of extraction methods on recovery and on stability of analytes.

In order to evaluate (i) the recovery of extraction of opiates from authentic hair samples and (ii) the extent of hydrolysis of acetylated opiates (6-acetylmorphine, acetylcodeine) occurring during sample preparation, three different methods of extraction commonly used for opiates have been compared. To this purpose a sample consisting of a pool of hair collected from several heroin overdose cases has been submitted alternately to (A) digestion in 2 M NaOH at 80 degrees C for 1 h (n = 5), (B) incubation in 0.1 M HCl at 45 degrees C for 18 h (n = 5) and (C) incubation in methanol at 37 degrees C for 18 h (n = 5). After pH adjustment of the different incubation media to 7-8, analytes have been isolated by means of SPE using Bond Elut certify columns and derivatized with MSTFA. Analyses have been performed by either GC-MS in the selected ion monitoring mode or, omitting SPE, by radioimmunoassay. The extent of hydrolysis of 6-acetylmorphine to morphine and of acetylcodeine to codeine have been determined by submitting blank hair samples spiked with the acetylated analytes to the different extraction methods and measuring the amount of morphine and codeine formed. Both the recovery of extraction of the total morphine fraction (6-acetylmorphine + morphine) and the rate of hydrolysis of 6-acetylmorphine were found to be in the order: A > B > C. Similar results were obtained for the total codeine fraction (acetylcodeine + codeine). These results clearly indicate that: (i) the concentration of opiates measured in hair depends on the extraction method used; (ii) ratios between different analytes (e.g. 6-acetylmorphine vs. morphine) may reflect the rate of hydrolysis during sample preparation rather than different types of exposure to opiates.

Gas Chromatography-Mass Spectrometry↗
PubMed2001

Laser desorption ionization and MALDI time-of-flight mass spectrometry for low molecular mass polyethylene analysis.

Polyethylene's inert nature and difficulty to dissolve in conventional solvents at room temperature present special problems for sample preparation and ionization in mass spectrometric analysis. We present a study of ionization behavior of several polyethylene samples with molecular masses up to 4000 Da in laser desorption ionization (LDI) time-of-flight mass spectrometers equipped with a 337 nm laser beam. We demonstrate unequivocally that silver or copper ion attachment to saturated polyethylene can occur in the gas phase during the UV LDI process. In LDI spectra of polyethylene with molecular masses above approximately 1000 Da, low mass ions corresponding to metal-alkene structures are observed in addition to the principal distribution. By interrogating a well-characterized polyethylene sample and a long chain alkane, C94H190, these low mass ions are determined to be the fragmentation products of the intact metal-polyethylene adduct ions. It is further illustrated that fragmentation can be reduced by adding matrix molecules to the sample preparation.

Indicators and Reagents↗
PubMed1983

A study of various factors affecting digestion of fish tissue prior to mercury determination.

The effect of temperature on digestion, the acid combination and their quantities, and the time required for digestion of fish tissue were investigated using Hamour (Epinephelus tauvina) body tissue. Concentrations of Hg in fish tissue digested for four hours at 80 +/- 2 degrees C and 95 +/- 2 degrees C were statistically similar and significantly higher than in tissue digested at 60 +/- 2 degrees C. Eight acid combinations were investigated as digestion media and a 1:2 mixture of concentrated HNO3:H2SO4 proved to be the best. A quantity of 15 ml of this digestion media were found to be sufficient to digest approximately two grams of wet fish tissue. The use of 25 ml of digestion media resulted in significantly reduced Hg concentration whereas 10 ml was not sufficient to digest two grams of fish tissue. A digestion period of four to six hours at 80 degrees C was sufficient to oxidize the fish tissue. However, a two hour digestion resulted in reduced Hg values. Mercury determinations made from the samples prepared by the best combination of all the experimental conditions showed a good agreement with those of samples prepared in Teflon Acid Digestion Bombs. This study has pointed the necessity of developing a uniform standard procedure for digesting fish tissue prior to Hg determinations.

Acids↗
PubMed2001

Application of a PCR assay to enhance the detection and identification of Tritrichomonas foetus in cultured preputial samples.

The traditional diagnostic test for Tritrichomonas foetus involves collection of preputial or vaginal samples followed by culture in a growth media and microscopic examination. Recently, polymerase chain reaction (PCR) techniques have been described for use as a diagnostic assay. The objective of this study was to evaluate a previously described PCR assay for detecting T. foetus in cultured preputial material. The detection limits of the assay for T. foetus organisms in a growth medium, in samples prepared from washing microscope slides, and in preputial material cultured in a growth medium were determined. Preputial samples were collected from 13 bulls uninfected with T. foetus. The PCR assay was able to detect 5 T. foetus organisms in the growth medium and the cultured preputial material. Amplification products were obtained from samples prepared from washes of microscope slides containing as few as 3 visualized organisms. The PCR assay was able to detect organisms in culture at a lower concentration than was possible by direct microscopic examination. This low detection limit may allow the PCR assay to be used to enhance the sensitivity of the current diagnostic test. In addition, the assay could be used to confirm the identification of T. foetus organisms observed by direct microscopic examination when other confirmation techniques, such as staining and phase microscopy, are not practical.

Animals↗
PubMedDate not supplied

[Comparison of the MTP immunoassay with EMIT in blood screening for drugs].

We compared the MTP immunoassay with EMIT for the screening of drugs of abuse (opiates, cannabinoids, cocaine metabolites and amphetamines) in whole blood samples. These blood samples were obtained from the German police, when driving under the influence of drugs of abuse was suspected. For screening with the MTP immunoassay 25 microliters of serum or blood (without any pretreatment) was pipetted into the wells of the microtiter plates and the procedure was followed as described. Prior to screening with a Cobas Mira and EMIT reagents, the samples were treated with acetone to precipitate serum proteins. The cutoff for all drugs of abuse was set at 10 ng per ml of serum or blood. In most cases there was a good agreement between the negative and positive results of the two screening assays. The agreement between the two assays in the detection of opiates and cocaine was 91% and 93%, respectively, and for cannabinoids and amphetamines approximately 80%. The MTP immunoassay was more sensitive than EMIT for the detection of cannabinoids--but at the same time the MTP immunoassay was less specific. Both screening assays have a sensitivity of 100% for the detection of opiates and cocaine, but the specificity of the EMIT--also for opiates--was substantially lower. The MTP immunoassay has in respect to amphetamines a very high sensitivity, whereas the sensitivity of EMIT for amphetamines is inacceptable due to losses during sample preparation. The specificity of MTP immunoassay for amphetamines is not optimal, because a relatively large amount of samples tested false-positive for amphetamines at the cutoff of 10 ng/ml. In summary the MTP immunoassay, although not automated, performs well in comparison with EMIT, especially if the sample preparation for EMIT testing ist considered.

Enzyme Multiplied Immunoassay Technique↗
PubMed1999

The use of infrared spectrophotometry for measuring body water spaces.

BACKGROUND: The conventional method of measuring total body water by the deuterium isotope dilution method uses gas isotope ratio mass spectrometry (IRMS), which is both expensive and time-consuming. We investigated an alternative method, using Fourier transform infrared spectrophotometry (FTIR), which uses less expensive instrumentation and requires little sample preparation. METHOD: Total body water measurements in human subjects were made by obtaining plasma, saliva, and urine samples before and after oral dosing with 1.5 mol of deuterium oxide. The enrichments of the body fluids were determined from the FTIR spectra in the range 1800-2800 cm-1, using a novel algorithm for estimation of instrumental response, and by IRMS for comparison. RESULTS: The CV (n = 5) for repeat determinations of deuterium oxide in biological fluids and calibrator solutions (400-1000 micromol/mol) was found to be in the range 0.1-0.9%. The use of the novel algorithm instead of the integration routines supplied with the instrument gave at least a threefold increase in precision, and there was no significant difference between the results obtained with FTIR and those obtained with IRMS. CONCLUSION: This improved infrared method for measuring deuterium enrichment in plasma and saliva requires no sample preparation, is rapid, and has potential value to the clinician.

Body Water↗
PubMedDate not supplied

Rapid determination of aflatoxins in raw peanuts by liquid chromatography with postcolumn iodination and modified minicolumn cleanup.

A method is described for rapid cleanup followed by reverse-phase liquid chromatographic (LC) quantitation of aflatoxins in raw peanuts. A modified minicolumn cleanup is used for sample preparation, and a preliminary estimation of aflatoxin content by minicolumn can be made so that highly contaminated samples can be diluted before LC analysis. The use of the simple, quick minicolumn cleanup eliminates the need for further column or cartridge cleanup, thus greatly reducing sample preparation time. Sensitive quantitation is achieved using a phenyl column, a mobile phase of water-tetrahydrofuran (80 + 20, v/v), and postcolumn derivatization with water-saturated iodine followed by fluorescence detection. The recoveries of aflatoxins B1, B2, G1, and G2 from peanut meal spiked at 3 levels ranged from 71.7 to 88.3% (average 80%) with coefficients of variation from 2.7 to 10.4%.

Aflatoxins↗
PubMed2005

Sensitivity of different Trypanosoma vivax specific primers for the diagnosis of livestock trypanosomosis using different DNA extraction methods.

There are several T. vivax specific primers developed for PCR diagnosis. Most of these primers were validated under different DNA extraction methods and study designs leading to heterogeneity of results. The objective of the present study was to validate PCR as a diagnostic test for T. vivax trypanosomosis by means of determining the test sensitivity of different published specific primers with different sample preparations. Four different DNA extraction methods were used to test the sensitivity of PCR with four different primer sets. DNA was extracted directly from whole blood samples, blood dried on filter papers or blood dried on FTA cards. The results showed that the sensitivity of PCR with each primer set was highly dependant of the sample preparation and DNA extraction method. The highest sensitivities for all the primers tested were determined using DNA extracted from whole blood samples, while the lowest sensitivities were obtained when DNA was extracted from filter paper preparations. To conclude, the obtained results are discussed and a protocol for diagnosis and surveillance for T. vivax trypanosomosis is recommended.

Animals↗
PubMed2005

High-throughput analysis of tetracycline and penicillin antibiotics in animal tissues using electrospray tandem mass spectrometry with selected reaction monitoring transition.

A simple, rapid, and simultaneous analysis method for oxytertracycline, tetracycline, chlortetracycline, penicillin G, ampicillin, and nafcillin in meat has been developed by using electrospray ionization tandem mass spectrometry. The sample preparation was performed by homogenizing with water followed by a centrifugal ultrafiltration, after addition of internal standards (demeclocycline, penicillin G-d5, ampicillin-d5 and nafcillin-d6). The MS/MS analysis involves the combined use of sample enrichment on the short column and a multiple reaction monitoring technique. The overall recoveries from animal (bovine and swine) muscle, kidney, and liver fortified at the levels of 0.05 and 0.1 ppm ranged from 70 to 115% with the coefficients of variation ranging from 0.7 to 14.8% (n = 5). Analysis time, including sample preparation and determination, is only 3h per eight sample and detection limits for all antibiotics are 0.002 ppm. The method is considered to be satisfactory for the rapid screening of the tetracycline and penicillin antibiotic residues in meat.

Animals↗
PubMed2004

Reducing focused ion beam damage to transmission electron microscopy samples.

One of the most important applications of focused ion beam (FIB) systems is sample preparation for transmission electron microscopy (TEM). However, the use of the FIB inherently involves changing and damaging the sample, and thereby degrades the TEM resolution. This paper addresses the beam-induced damage and artifacts, particularly in applications involving silicon semiconductors. The damage appears in the form of amorphization on the surface of the TEM foil. The characteristics of this amorphous damage were studied by making TEM observations of cross sections of the affected foil. The damage is typically 20 to 30 nm thick for a 30 keV FIB, which is generally overly thick for modern silicon devices with feature sizes less than 250 nm. This paper reviews the reported damage depths of FIB-prepared samples, which are determined by experiments and calculations. Several damage reduction techniques, such as the use of gas-assisted etching, low energy FIB, cleaning the FIBfabricated cross section by wet or dry etching and cleaning by broad ion beam (BIB) milling have also been reviewed, with emphasis on applicability to silicon devices. We conclude that the use of low energy FIB and cleaning by argon BIB are particularly efficient techniques.

Artifacts↗
PubMed1988

Preparation of serum oxytocin and arginine vasopressin prior to radioimmunoassay: simultaneous extraction and separation on C18 Sep-Pak cartridges.

Oxytocin (OT) and arginine vasopressin (AVP) are often secreted in response to the same stimuli. The hormones are seldom assayed together, however, because of labor intensive sample preparation and the duplicate volumes required. A method has been developed for the simultaneous extraction and separation of OT and AVP from a single serum sample. The method is suited for sample preparation prior to radioimmunoassay (RIA) and reduces sample volume and processing time by 50%. Serum, supplemented with labeled and unlabeled OT and AVP, was adsorbed onto C18 (octadecasilyl-silica, ODS) Sep-Pak cartridges. After washing with phosphosaline and 3% aqueous acetone, OT was eluted with 98% aqueous acetone followed by AVP with 80% acidified (0.02 mol/L HCl) acetone. The recoveries, determined by radioactivity and RIA measurements, were 86 +/- 3% (OT) and 71 +/- 7% (AVP). Cross contamination was less than 10%. Sep-Paks extracted up to 100 pg/mL of the hormones from 10 mL of serum. The method was employed to measure OT and AVP in the pregnant ewe. Both hormones were elevated during salt-loading and dehydration and were decreased by carotid infusions of ethanol.

Animals↗
PubMed1995

Estradiol binding to synaptosomal plasma membranes of rat brain regions.

The equilibrium parameters (Bmax and Km) of estradiol binding to synaptosomal plasma membranes prepared from brain regions of adult female rats were examined. Michaelis-Menten plots of the specific binding in samples prepared from the telencephalon and mesencephalic reticular formation showed only one saturation plateau, indicating the presence of a single population of synaptosomal membrane binding sites for estradiol. According to Bmax and Km values, the binding sites appeared to be similar in the two regions. On the other hand, two distinct populations, one with high capacity and lower affinity and another with low capacity and higher affinity, were detected in samples prepared from the nucleus caudatus, brain stem and hippocampus, as shown by two saturation plateaus on corresponding Michaelis-Menten graphs. The equilibrium parameters of the two populations were different in the three regions. The shapes of the Michaelis-Menten plots were substantially the same for intact and for ovariectomized animals. Scatchard and Hill plots revealed cooperativity for estradiol binding in all the regions investigated. These results suggest that central non-genomic effects of estradiol are mediated via multiple types of neuronal membrane binding sites, which appear not to be under ovarian control.

Animals↗
PubMedDate not supplied

Improved preparation of leukocytes for chemiluminescent study of human phagocytic leukocyte-generated reactive oxygen species.

Leukocyte oxidative function was investigated in a more physiological milieu than currently used in the chemiluminescence (CL) technique. Heparinized blood was mixed with 6% dextran-T70 (9:1) and the leukocyte-rich plasma obtained without centrifugation was used for the CL experiments (phagocyte count was adjusted to 0.7 x 10(6)/mL with Hanks' buffer) (method A). In this medium, phagocytes responded to stimulation by opsonized zymosan, producing strong luminescence in the presence of 0.5 mumol/L MCLA. CL was inhibited by superoxide dismutase, suggesting that the luminescence reaction was attributable to O2-. Granulocytes were also prepared by the usual method involving centrifugation and were then suspended in plasma (method B). Oxidative function of phagocytes prepared by the two methods was studied together with whole blood as aliquots diluted with Hanks' buffer up to a factor of 1000. Luminescence reached a peak value at a dilution factor of 16, but at very high dilutions luminescence decreased sharply. Significantly higher luminescence values were obtained with samples from method A. Luminescence of whole blood peaked at a dilution factor of 248 but it was less than the value obtained using samples prepared by method A or B. As samples prepared by method A contain all the leukocyte populations, platelets, residual red cells and plasma proteins, the assay of leukocyte-generated reactive oxygens using CL is attained in more physiological conditions than method B in which leukocytes may be damaged owing to repeated centrifugation and hypotonic shock.

Azides↗