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PubMed1979

Scanning electron microscope surface morphometry of type II alveolar lung cell response to urethane in strain A mice.

Quantitative scanning electron microscope (SEM) examination of the time course of type II cell hyperplasia in mice exposed to a single i.p. injection of urethane is presented. Counts of type II cells from random alveoli suggest that, compared to controls, a significant depression in type II cell numbers occurs during the first week, followed by a doubling in the next 3 to 4 week period. This supports conclusions of earlier studies done with thin sections and light morphometric techniques. The use of a SEM as the morphometric tool is statistically more efficient, however, since initial sample preparation is easier, samples are larger in area, resolution is higher and, most importantly, the amount of surface area available for examination is many times greater than that presented by light microscope examination of cross sections with equivalent magnification.

Animals↗

PubMed1993

A review of the capabilities of ICP-MS for trace element analysis in body fluids and tissues.

ICP-MS is a powerful analytical technique for the determination of trace and ultra-trace elements in biological materials. Results are given of the analysis of human serum and of several biological reference materials (bovine liver, milk powder, wheat flour and pig kidney). Because concentrations of many trace metals of interest in these materials are low, dilution should be kept as limited as possible, although concentrations of certain concomitant elements (e.g. Na, K) can be high enough to cause significant suppression or enhancement of the ion signal. The result is that the dissolution procedure becomes a critical step in the analysis. Microwave digestion, wet digestion with several acids (HNO3, HClO4, HF) and simple dilution are compared with each other. In addition, three possible approaches to overcome these problems are discussed, namely the internal standardization method, the standard addition method and the isotope dilution method. Furthermore, ICP-MS is also more susceptible than initially expected to isobaric interferences arising from the plasma, the acids used in the sample preparation or the sample itself. These interferences are generally less important above a mass number of 80. Nevertheless, several researchers have investigated the possibility of removing the trace metals from the matrix or of using correction formulae based on the isotopic abundances of the elements. Some of these are evaluated. The need for identifying and quantifying chemical species, not just the elements, is well recognized. One of the reasons why elemental determination may not suffice is that different species of the same element may have a different chemical and toxicological behaviour. Arsenic (As(III), As(V)) and mercury (organomercury) are typical examples.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1996

RNA amplification by nucleic acid sequence-based amplification with an internal standard enables reliable detection of Chlamydia trachomatis in cervical scrapings and urine samples.

In the present study, the suitability of RNA amplification by nucleic acid sequence-based amplification (NASBA) for the detection of Chlamydia trachomatis infection was investigated. When comparing different primer sets for their sensitivities in NASBA, use of both the plasmid and omp1 targets resulted in a detection limit of 1 inclusion-forming unit (IFU), while the 16S rRNA appeared to be the most sensitive RNA target for amplification (10(-3) IFU). In contrast, for DNA amplification by PCR, the plasmid target was optimal (10(-2) IFU), which is 10 times less sensitive than rRNA NASBA. To exclude false negativity in NASBA detection because of inhibition of amplification and/or inefficient sample preparation, an internal standard was developed. The internal control was added prior to sample preparation. This 16S rRNA NASBA with an internal control was compared with a plasmid DNA PCR by using a group of C. trachomatis-negative (n = 41) and -positive (n = 37) cervical scrapings, as determined by enzyme immunoassay (EIA). In addition, urine samples from the EIA-positive women were tested (n = 17). Both NASBA and PCR assays were able to detect C. trachomatis in all EIA-positive cervical scrapings, the corresponding urine samples, and two samples from the EIA-negative group. The internal NASBA standard was found clearly in all EIA-negative samples. In conclusion, these results indicate that detection of C. trachomatis by RNA amplification by NASBA with an internal standard is a suitable and highly sensitive detection method, with potential use in the diagnosis of urogenital C. trachomatis infections with cervical scrapings as well as urine specimens.

Bacterial Outer Membrane Proteins↗

PubMed2000

Automation of the GC/MS analysis of mineral oil contaminations in water.

An automation of the sample preparation and analysis of mineral oil contaminations in water was developed. The automated sample preparation was carried out according to ISO/DIS 9377-4 [1]. The standard is applicable to the determination of hydrocarbons in the boiling range of n-decane (n-C10) up to n-tetracontane (n-C40) by GC. Extraction of the sample and clean-up of the extract were performed by an autosampler with a movable head which is capable of carrying different syringes for gas and liquid handling. A GC/MS-system with a programmed temperature vaporizing (PTV) injector including the possibility of large volume injections (LVI) was employed for the analysis. The recovery of analytes was 101.8%, the repeatability 2.9% relative standard deviation (RSD). The linear range covered 0.3 to 40 mg/L oil but may be larger since no higher concentrations were measured. With an FID, being the detector of choice mentioned in the standard, it should be possible to achieve at least four orders of magnitude in the linear range. The limit of determination was found to be 0.3-0.4 mg/L, the limit of detection 0.1-0.2 mg/L [2]. Measurements of spiked deionized, bidistilled water and spiked water from a lake confirmed the accuracy of the analysis. Due to automation and miniaturization of the analysis it is possible to economize time and chemicals without loss of precision and accuracy.

Automation↗

PubMed2003

Preparation of enantiomeric gossypol by crystallization.

Large enantiomorphic crystals of gossypol-acetone (1:3) were grown from acetone solutions of rac-gossypol-acetic acid (1:1) at 4 degrees C. By controlling the initial gossypol concentration, crystallization time, and solution volume, single crystals were grown that weighed >50 mg, equivalent to >37 mg of enantiomeric gossypol. Even larger crystals were possible, but it was difficult to produce these reliably without contamination of the antipode. Essentially all of the acetone within the crystal form was removed by storing the crystals under vacuum for 3-4 days. By employing these techniques, gram quantities of enantiomeric gossypol were prepared in high chemical and optical purity. Based on measured and reported optical rotations, the optical purity of samples prepared by crystallization was greater than the optical purity of samples prepared by chromatographic separation of gossypol-amine diastereomers. The principal limitation of crystallization as a preparative method is the need to determine the chirality and purity of each product crystal. Nevertheless, the method competes favorably with preparative-scale chromatographic procedures.

Chromatography, High Pressure Liquid↗

PubMed2003

Automated solid-phase extraction and liquid chromatographic method for retinoid determination in biological samples.

In this study, a method for partly automated sample preparation and fully automated solid-phase extraction method for plasma, kidney and liver samples for various retinoids like all-trans-4-oxo-retinoic acid, 13-cis-4-oxo-retinoic acid, 13-cis-retinoic acid, 9-cis-retinoic acid, all-trans-retinoic acid, retinol and retinyl palmitate was established. Plasma, embryo-, kidney-and liver-homogenates were automatically mixed and extracted on multiple usage solid-phase (C2) extraction cartridges immediately before HPLC analysis. Automated cleaning, preconditioning and incorporation of the loaded cartridge to fully automated HPLC separation and quantification of the various retinoids in a single HPLC run was established. The recovery of the retinoids was generally between 80 and 90%. Intra-day repeatability was < 11.7%. As little as 1.2 ng/ml could be quantified in lipid-mixture standard samples. This method allows a highly automated sample preparation and a fully automated solid-phase extraction with good selectivity for the study of endogenous retinoids and retinoids after nutritional supplementations and pharmacological applications in several biological samples.

Animals↗

PubMed2006

Evaluation of multidimensional capillary electrophoretic methodologies for determination of amino bisphosphonate pharmaceuticals.

Alendronate and pamidronate are amino bisphosphonate analogues of pyrophosphate used for the treatment of a variety of bone diseases. Analysis of these compounds is problematic due to their polar ionic nature, lack of a suitable chromophore and chelation properties and current analytical approaches involve extensive sample preparation and derivatization procedures. The potential of multidimensional capillary electrophoretic methodological approaches, which eliminate sample preparation have been evaluated for the analysis of these compounds both in aqueous and urinary matrices. Capillary isotachophoresis (cITP) was employed as a pre-separation and on-line sample concentration step prior to analytical determination using either cITP or capillary zone electrophoresis (CZE) with conductivity detection. Single cITP, cITP-cITP and cITP-CZE approaches were partially validated with respect to repeatability, recovery and linearity of response for both compounds. The increases in sensitivity achievable through increasing injection volume from 30 to 300 microL may render such strategies appropriate for determination of these agents at biologically relevant concentrations with minimal sample work-up.

Alendronate↗

PubMed2003

Evaluation of a new multiple-layer spotting technique for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of synthetic polymers.

A new multiple-layer matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) sample-spotting technique is described. This fast and easy technique was evaluated with poly(ethylene glycol) (PEG) standards and optimized conditions for these synthetic polymers were obtained. PEGs up to 35 kDa were detectable with this approach and single monomer resolution was observed up to 20 kDa. The spotting was performed using a multiple-layer approach, which offers the capability of complex sample preparation without the requirement of premixing the different matrix, analyte and doping salt solutions. The technique reduces the time required for sample preparation and offers high flexibility with respect to sample composition and solvents utilized for the crystallization of the compounds. The technique is thus perfectly suited for applications in combinatorial material research.

Journal Article↗

PubMedDate not supplied

Sampling plans for the determination of aflatoxin B1 in large shipments of animal feedstuffs.

Incremental samples (50, 100, and 500 g) were systematically collected from large shipments of copra meal pellets, copra cake, and palm kernel cake to study the distribution of aflatoxin B1 and evaluate adherence of distribution to the model, CV(2)is (EQ) = A + B/Mis (where CVis = coefficient of variation of the true concentration of aflatoxin B1 within the incremental samples; Mis = mass of the incremental samples; and A and B are constants). Also evaluated was the distribution of aflatoxin B1 among 1 kg composite samples, produced both by random combination of existing incremental samples and by collection of 1 kg composite samples (composed of 10 x 100 g increments) from additional batches of copra meal pellets and cottonseed cake. The efficiency of selected sample preparation (grinding and subdivision) procedures was compared, culminating in the development and description of a variety of sampling plans. The coefficient of variation (CV) among incremental samples varied from 0 to 38%, and was independent of incremental sample size. No significant difference (F-test, 5% significance level) was found between the efficacy of 4 sample preparation methods when these methods were applied to the commodities described above. Various sampling plans were evaluated with estimated CVs from 4.0 to 12.5%, for the aflatoxin B1 content of the composite samples.

Aflatoxin B1↗

PubMed2005

Evaluation of Western blotting methods using samples with or without sodium phosphotungstic acid precipitation for diagnosis of scrapie and chronic wasting disease.

The purpose of this study was to enhance the sensitivity of the Western blot (WB) test for use as an alternative and confirmatory method for the diagnosis of scrapie and chronic wasting disease (CWD) in Canada by comparing 2 sample preparation procedures: an abnormal prion protein (PrPSc) concentration procedure using sodium phosphotungstic acid (PTA) precipitation and a procedure using crude sample without precipitation. A total of 100 cerebrum samples (52 sheep and 48 elk), including 66 negative (31 sheep, 35 elk) and 34 positive (21 scrapie and 13 CWD positive) samples diagnosed by using immunohistochemistry (IHC) on retropharyngeal lymph node (RPLN) and medulla oblongata at obex, were tested by using WB with the 2 sample preparation procedures. The WB using non-PTA enriched sample (crude extract) detected, on average, only 71.7% (9 of 15, 60.0% for scrapie, 5 of 6, 83.3% for CWD) of the samples that tested positive by using WB with PTA enriched samples. No case was positive by WB using crude extract but negative by WB using PTA enriched sample. No false positive was found. Serial dilution of PTA precipitated samples demonstrated that the technique increases the detection limit approximately 100 fold. Additionally, the comparison of the WB and IHC on cerebrum from all the positive cases demonstrated that WB following PTA precipitation and IHC had 100% agreement by detecting 6 positive for CWD on cerebrum; while IHC detected scrapie in only 14 out of 15 positive cerebrum samples by using WB following PTA precipitation. Phosphotungstic acid precipitation is therefore a useful adjunct to WB analysis of scrapie and CWD and tissues.

Animals↗

PubMed2004

Determination of methylmercury in fish using focused microwave digestion following by Cu2+ addition, sodium tetrapropylborate derivatization, n-heptane extraction, and gas chromatography-mass spectrometry.

The analytical procedure for analysis of methylmercury in fish was developed. It involves microwave-assisted digestion with alkaline solution (tetramethylammonium hydroxide), addition of Cu2+, aqueous-phase derivatization of methylmercury with sodium tetrapropylborate, and subsequent extraction with n-heptane. The methylmercury derivative was desorbed in the splitless injection port of a gas chromatograph and subsequently analyzed by electron impact mass spectrometry. Optimum conditions allowed sample throughout to be controlled by the instrumental analysis time (near 7 min per sample) but not by the sample preparation step. At the power of 15-30, 45, and 60-75 W, sample preparation time is only 3.5, 2.5, and 1.5 min, respectively. The proposed method was finally validated by the analysis of three biological certified reference materials, BCR CRM 464 tuna fish, NRC DORM-2 dogfish muscle, and NRC DOLT-2 dogfish liver. The detection limit of the overall procedure was found to be 40 ng/g of biological tissue for methylmercury. The recovery of methylmercury was 91.2-95.3% for tuna, 89.3-94.7% for marlin, and 91.7-94.8% for shark, respectively. The detected and certified values of methylmercury of three biological certified reference materials were as follows: 5.34 +/- 0.30 microg/g (mean +/- S.D.) and 5.50 +/- 0.17 microg/g for CRM 464 tuna fish, 4.34 +/- 0.24 and 4.47 +/- 0.32 microg/g for NRC DORM-2 dogfish muscle, and 0.652 +/- 0.053 and 0.693 +/- 0.055 microg/g for NRC DOLT-2 dogfish liver, respectively. It indicated that the method was well available to quantify the methylmercury in fish.

Animals↗

PubMed2004

Determination of azide as the 3,5-dinitrobenzoyl derivative by capillary electrophoresis.

A simple, rapid and reliable capillary electrophoresis method with a photodiode array detector was developed for determination of azide as the 3,5-dinitrobenzoyl derivative in drink samples fortified with sodium azide. Sample preparation was simple and rapid because no more than a simple dilution of samples is needed after quick derivatization. Separation was carried out using a buffer system comprising 25 mM phosphate buffer and 4 mM cetyltrimethylammonium hydroxide at pH 3.0. Methyl benzoate was selected as the internal standard (IS). This study investigated the influence of the concentration of phosphate buffer and electroosmotic flow (EOF) modifier, and the buffer pH on migration time and signal response. The optimized method made it possible to determine azide within 5 min. The limit of detection was determined to be 1.9 microg/ml with SIN > 3. The quantitation range was 6.5-323 microg/ml. By the method recoveries of azide in drink samples fortified with sodium azide were investigated. Mean recovery values ranged from 93.6 to 105.8% and results were satisfactory. In addition, no interference was observed in electropherograms of drink samples fortified with sodium azide. Thus, by this method, azide in drink samples can be determined rapidly with high recoveries and good selectivity despite extremely simple sample preparation.

Azides↗

PubMed1997

Inositol 1,4,5-trisphosphate in blood and skeletal muscle in human malignant hyperthermia.

The in vitro contracture test (IVCT) is the only available diagnostic method at present for evaluation of malignant hyperthermia (MH) susceptibility. However, the disadvantage of the IVCT is that it is invasive. Several studies suggest that an altered inositol phosphate system is involved in the development of MH. A greater concentration of inositol 1,4,5-trisphosphate (1,4,5-IP3) was found in MH susceptible (MHS) than in normal (MHN) skeletal muscles. In this study the concentrations of 1,4,5-IP3 in blood samples and skeletal muscle specimens of identical patients were measured in an attempt to define susceptibility to MH. Muscle biopsies were obtained from 34 patients with clinical suspicion of MH. Patients were first classified as MHS (n = 19), MHN (n = 8) or MH equivocal (MHE; n = 7) by the standard IVCT. For detection of 1,4,5-IP3 concentrations, blood samples were obtained and an additional muscle specimen was excised. After sample preparation, concentrations of 1,4,5-IP3 were measured using radioimmunoassay. In blood samples, concentrations of 1,4,5-IP3 were similar in all individuals tested for MH susceptibility and in control patients not tested for MH susceptibility (n = 44). In skeletal muscle, 1,4,5-IP3 concentrations were significantly higher in MHS than in MHE or MHN patients, respectively. Each MHS sample contained more 1,4,5-IP3 than the highest concentration measured in MHN muscle. Defining arbitrary thresholds for 1,4,5-IP3 concentration in skeletal muscles in order to discriminate between MHS and MHN status, it was possible to assign three MHE patients to MHS and four to MHN. This study supports the hypothesis that an altered inositol phosphate system might be involved in MH. However, measurement of 1,4,5-IP3 concentration in a simple blood sample preparation is not reliable for MH susceptibility screening.

Adolescent↗

PubMed2002

Method for the determination of dialkyl phosphates in urine by strong anion exchange disk extraction and in-vial derivatization.

A method for the determination of four dialkylphosphate metabolites in urine by strong anion exchange disk (SAX) was investigated. Calcium hydroxide was added to a 1-mL urine sample to reduce interference. The aliquot was passed through the SAX disk to accumulate dialkylphosphate metabolites on the disk. The retained dialkylphosphate metabolites were derivatized with methyl iodide in acetonitrile online, and the resulting methyl esters of dialkylphosphate metabolites were directly analyzed by capillary column gas chromatography with flame photometric detection. The recoveries of these dialkylphosphate metabolites were found to be stable. When the intact sample was diluted with deionized water at a 1:1 ratio, the recoveries were both increased and stabilized. The urine samples collected from eight fruit farmers showed that levels of dialkylphosphate metabolites in urine were significantly different before and after pesticide application, indicating the method established in this study is applicable for real sample analysis. Compared with previous studies, this method not only can greatly simplify sample preparation, but it can also significantly reduce the consumption of toxic solvents in sample preparation.

Chromatography, Gas↗

PubMed2001

Analysis of semivolatile organic compounds in atmospheric aerosols by direct sample introduction thermal desorption GC/MS.

A technique for identifying trace amounts of semivolatile organic compounds in atmospheric aerosols and in the NIST Urban Dust standard (SRM1649a) is presented. The technique is based on direct sample introduction (DSI) of small samples followed by thermal desorption in a conventional GC injector. The method enables injection of both solid and liquid samples. Validation of the method, including quantitative determination of EPA-targeted polycyclic aromatic hydrocarbons (PAHs), as well as the reproducibility and recovery efficiency tests are presented. The advantages of using aluminum foil as sampling filter are also discussed. Determination of different classes of compounds such as quinolines, methylquinoline isomers, PAHs, and n-monocarboxylic acids in the ambient size-segregated aerosol sample is also performed. The method was directly applied to the determination of C6-C16 n-monocarboxylic acids, eliminating the need for a complex sample preparation procedure. The small quantities needed for the analysis as well as the lack of complicated sample preparation steps enable fast characterization of semivolatile organic species present in time-resolved or size-segregated aerosol samples. Thus, this method can potentially be employed for air quality monitoring and field measurements as well as for fast screening of the organic content of ambient particles.

Aerosols↗

PubMed1969

Determination of iodine concentration and distribution in rat thyroid follicles by electron-probe microanalysis.

The concentration and the distribution of iodine in various sized follicles of rat thyroid glands have been analyzed by electron-probe microanalysis. The results of the iodine analysis were grouped according to uncorrected lumen diameter size. No significant differences in iodine concentration were observed among the various size categories. When the results for all follicles from a given sample were pooled, the standard error of the mean was approximately 4%. Usually 40-50 follicles per animal were analyzed. The concentration of iodine ranged from 0.9 to 2.1% by weight among individual animals. Scanning pictures and step-scan analysis showed the iodine distribution to be quite uniform across the colloid area. Several techniques of sample preparation were used; they produced no significant differences in the iodine concentrations observed. Sodium concentration, also determined in all samples, was found to vary from 2 to 9% by weight. Because of the mobility of the sodium ion, its distribution was greatly affected by the method of sample preparation. The technique that best preserved the natural chemistry of the sample was that of freezing the tissue, sectioning, and then freeze-drying.

Animals↗

PubMed1996

Accurate mass measurement of oligonucleotides using a time-lag focusing matrix-assisted laser desorption/ionization time-of-flight mass spectrometer.

A method for accurate mass measurement of oligonucleotides up to a DNA 35-mer based on matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is described. In this method, a time-lag focusing time-of-flight mass spectrometer is used to achieve high mass resolution to resolve adduct ions that often complicate the mass analysis of oligonucleotides. Mass resolutions between 1170 and 1300 (full width at half maximum) for a 17-mer, 23-mer, and 35-mer are obtained using a 1 m linear time-of-flight instrument with a total sample loading of less than 10 pmol. The effects of sample preparation, type of calibrant and matrix used on the accuracy of mass measurement, based on external calibration, are discussed. A sample preparation protocol that forms a thin film of matrix and sample crystals on a MALDI probe is described. It is shown that mass measurement error less than 100 ppm with reproducibility better than +/-60 ppm can be obtained with either proteins or DNA fragments as external calibrants. Accurate mass measurement for a mixture of DNA fragments is also illustrated.

Calibration↗

PubMed1980

Availability of diazepam from plastic containers.

The effect of plastic intravenous containers and volume-control administration sets on diazepam availability was studied. Solutions of diazepam in dextrose 5% in water and in 0.9% sodium chloride were prepared in plastic burette chambers and polyvinyl chloride (PVC) bags. Diazepam concentration was measured spectrophotometrically for (1) samples taken from the burette chamber and from the end of the administration set, (2) samples prepared in glass beakers and then infused through plastic administration sets with 0.5-micron filters, (3) samples prepared in PVC bags, and (4) sections of the PVC bags and volume-control sets in which diazepam solutions were prepared and infused. Two hours after admixture, diazepam concentration was significantly reduced in the burette chamber (p less than 0.05). Diazepam concentration was reduced by about 55% after infusion through administration sets (p less than 0.0005) and after two hours of storage in PVC bags. About 24% of the diazepam concentration was recovered from the volume-control set (none was detected in the filter) and 49-56% was recovered from the PVC bags. Admixture of diazepam solutions in plastic containers and administration through plastic administration sets will significantly reduce their diazepam concentrations.

Diazepam↗