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PubMed1991

Microbial stabilization of antibiotic-containing urine samples by using the FLORA-STAT urine transport system.

The FLORA-STAT Urine Transport System (Wadley Biosciences Corp./Lymphokine Partners Ltd., Dallas, Tex.) was evaluated for its efficacy in maintaining organism count and in effectively blocking the bactericidal action of therapeutic antimicrobial agents in urine samples when the urine samples were held at room temperature. Reconstructions with 53 organism-antimicrobial combinations were performed at 0, 4, 8, and 24 h in which the FLORA-STAT system was compared with two boric acid-based systems (Urine C&S Transport Kit [Becton Dickinson VACUTAINER Systems, Rutherford, N.J.]; Sage Urine Collection Kit for Culture [Sage Products, Inc., Cary, Ill.]) and untreated urine. At 24 h, less than 1-log-unit changes in organism counts were found in 100, 92, and 10% of the urine samples without antimicrobial agents and in 97, 65, and 16% of the urine samples with antimicrobial agents for FLORA-STAT-treated, boric acid-treated, and untreated urine samples, respectively. The FLORA-STAT system was further evaluated by sending split samples prepared from laboratory-inoculated patient urine samples (57 without and 50 with antimicrobial agents) to four commercial laboratories by using their respective transport devices and procedures. Samples were also sent to a local reference laboratory which provided prompt processing. Each laboratory received independently labeled transport devices containing untreated, FLORA-STAT-treated, and preservative-treated (if provided by the commercial laboratory) samples prepared from the same urine specimen of a patient. Average estimated transport times ranged from 13 to 24 h for the commercial laboratories; the transport time was less than 4 h for the local reference laboratory.(ABSTRACT TRUNCATED AT 250 WORDS)

Anti-Bacterial Agents↗
PubMed1990

Design of a photoionization detector for high-performance liquid chromatography using an automated liquid-to-vapor phase interface and application to phenobarbital in an animal feed and to amantadine.

An automated liquid-to-vapor phase interface system forms the basis for a new high-performance liquid chromatography (HPLC)-photoionization detection (PID) system. The system incorporates a six-valve interface enabling peak trapping, solvent switching and thermal desorption of the solute of interest into a vapor phase PID. For reversed-phase HPLC, the eluted solute peak is isolated on a Tenax trap after dilution of the effluent with water; the water is then evaporated, following which the trapped solute is flash-evaporated into the PID system. For normal-phase HPLC, the column effluent is diluted with hexane, the solute peak is concentrated on a short column packed with a propyl-amino/cyano bonded phase and the solvent is evaporated. The solute is then eluted with water onto the Tenax trap, and the above procedure for reversed-phase HPLC followed. All operations are controlled with a microcomputer. The advantages of the new detector system include completely automated operation, fast sample preparation, high sensitivity, and inherent selectivity. The system was applied to phenobarbital, which was extracted with acetonitrile from spiked laboratory animal feed, and to amantadine. The phenobarbital assay used a normal-phase separation with hexane-methyl tert.-butyl ether-methanol eluent. The manual sample preparation time was 5 min and the limit of detection was 2 ng phenobarbital injected; a conventional HPLC assay with UV detection required a longer sample preparation time and had a detection limit of 700 ng. Amantadine was assayed using a reversed-phase HPLC system with a water-methanol-triethylamine-orthophosphoric acid mobile phase. The detection limit was 25 ng injected.

Amantadine↗
PubMed1998

On-line cation exchange for suppression of adduct formation in negative-ion electrospray mass spectrometry of nucleic acids.

One major difficulty in the analysis of nucleic acids by electrospray mass spectrometry is represented by the affinity of the polyanionic sugar-phosphate backbone for nonvolatile cations, especially ubiquitous sodium and potassium ions. A simple on-line sample preparation system comprising a microflow pumping system and 45 x 0.8-mm-i.d. microcolumns packed with weak or strong cation-exchange resins is described for the efficient removal of cations from nucleic acid samples. Samples were analyzed by flow injection analysis at a 3-5 microL/min flow of 10 mM triethylamine in 50% water-50% acetonitrile. After on-line desalting, mass spectra of oligonucleotides revealed no significant sodium adduct peaks. Moreover, signal-to-noise ratios were greatly enhanced compared to direct injection of the samples. Electrospray mass spectrometry with on-line sample preparation allowed accurate molecular mass determinations of picomole amounts of crude oligonucleotide preparations ranging in size from 8 to 80 nucleotides within a few minutes. The good linearity of the calibration plot (R2 = 0.9988) over at least 2 orders of magnitude and a relative standard deviation in peak areas of less than 9% permitted the sensitive quantitative measurement of oligonucleotides in a concentration range of 0.2-20 microM with selected-ion monitoring. Finally, the on-line sample preparation system was evaluated for the mass spectrometric analysis of complex oligonucleotide mixtures.

Cations↗
PubMed2002

Laser capture microdissection and two-dimensional polyacrylamide gel electrophoresis: evaluation of tissue preparation and sample limitations.

Laser capture microdissection (LCM) is now well established as a tool for facilitating the enrichment of cells of interest from tissue sections, overcoming the problem of tissue heterogeneity. LCM has been used extensively in combination with analysis at the DNA and RNA levels, but only a small number of studies have employed LCM with subsequent protein analysis, albeit with promising results. This study focuses on the potential of LCM in combination with two-dimensional polyacrylamide gel electrophoresis. The effects of tissue section preparation and sample type were evaluated to fully determine the suitability of using LCM in global protein profiling. The effects of several histochemical stains (hematoxylin and eosin, methyl green and toluidine blue) and immunolabeling on subsequent two-dimensional polyacrylamide gel electrophoresis were investigated. Quantitative analysis was performed to establish the extent of changes in the relative intensity of protein species and their reproducibility. All staining protocols tested were found to be compatible with protein analysis although there was variation in protein recovery and the quality of the protein profiles obtained. LCM of renal and cervix samples indicated that protein yield after dissection was acceptable, although the extent of enrichment and dissection time was tissue-dependent, which may preclude the use of this approach with some tissue types. These results indicate that LCM has potential as a tool in proteomic research.

Electrophoresis, Gel, Two-Dimensional↗
PubMed1985

Analytical strategies for therapeutic monitoring of drugs in biological fluids.

Therapeutic drug monitoring can involve quantitation in either microgram, nanogram or picogram concentrations present in a complex biological matrix (whole blood, urine or tissue). The chemical structure of a compound influences not only the analytical method best suited to its quantitation, but also its acid/base character (pKa) and its extractability. The dose administered, the bioavailability of the dosage form, and the pharmacokinetic profile of the drug govern the circulating concentrations of either the parent drug and/or its metabolites present in vivo, and dictate the ultimate sensitivity and specificity required of the analytical method. The degree of sample preparation required is dependent on the analytical method used (gas--liquid chromatography, thin-layer chromatography, high-performance liquid chromatography) and on the tolerance of the specific type of detection system to contamination. Factors leading to compound losses during sample preparation (adsorption, stability) are critical at low concentrations and can adversely affect the reliability of an assay, therefore maximizing the overall recovery of the assay is essential not only for high sensitivity but also for good precision and accuracy. Therefore, the criteria to be used in sample preparation should aim to optimize all of the above factors in the overall development of a reliable and validated method for the compound suitable for use in clinical therapeutic monitoring.

Biotransformation↗
PubMed1983

Analytical strategies for therapeutic monitoring of drugs and metabolites in biological fluids. Plenary lecture.

Therapeutic drug monitoring can involve quantitation in either microgram, nanogram or picogram concentrations present in a complex biological matrix (whole blood, urine or tissue). The chemical structure of a compound influences not only the analytical method best suited to its quantitation, but also its acid/base character (pKa) and its extractability. The dose administered, the bioavailability of the dosage form, and the pharmacokinetic profile of the drug govern the circulating concentrations of either the parent drug and/or its metabolites present in vivo, and dictate the ultimate sensitivity nd specificity required of the analytical method. The degree of sample preparation required is dependent on the analytical method used (gas--liquid chromatography, thin-layer chromatography, high-performance liquid chromatography) and on the tolerance of the specific type of detection system to contamination. Factors leading to compound losses during sample preparation (adsorption, stability) are critical at low concentrations and can adversely affect the reliability of an assay, therefore maximizing the overall recovery of the assay is essential not only for high sensitivity but also for good precision and accuracy. Therefore, the criteria to be used in sample preparation should aim to optimize all of the above factors in the overall development of a reliable and validated method for the compound suitable for use in clinical therapeutic monitoring.

Benzodiazepines↗
PubMed2004

PLE in the analysis of plant compounds. Part I. The application of PLE for HPLC analysis of caffeine in green tea leaves.

A broad spectrum of sample preparation methods is currently used for the isolation of pharmacologically active compounds from plant and herbal materials. The paper compares the effectiveness of infusion, microwave assisted solvent extraction (MASE), matrix solid-phase dispersion (MSPD) and pressurised liquid extraction (PLE) as sample preparation methods for the isolation of caffeine from green tea leaves. The effect of PLE variables, such as extraction temperature, pressure and time, on the yield of caffeine from the investigated matrix is discussed. The obtained results revealed that PLE, in comparison with other sample preparation methods applied, has significantly lower efficacy for caffeine isolation from green tea leaves. The evaluation of PLE conditions leads to the conclusion that elevated pressure applied in the PLE process is the factor hindering the extraction.

Caffeine↗
PubMedDate not supplied

Carbamylation of proteins in 2-D electrophoresis--myth or reality?

Carbamylation is widely quoted as being a problem in 2-D gel analysis and the associated sample preparation steps. This modification occurs when iso-cyanate, a urea break-down product, covalently modifies lysine residues, thus inducing a change in isoelectric point. Urea is used at up to 9 M concentrations in sample preparation and 2-D gels because of its ability to disrupt protein structure and effect denaturation without the need for ionic surfactants such as SDS. We have studied carbamylation using 7 M urea and 2 M thiourea, under a range of experimental temperatures to establish when, and if, it occurs and what can be done to minimize the modification. The actual time required for protein extraction from a tissue is usually short compared to the time required for procedures such as reduction and alkylation and IPG rehydration and focusing. Therefore, it is the temperature during these post-extraction procedures that is the most critical factor. Our experiments have shown that carbamylation does not occur during electrophoresis in the presence of urea, even with prolonged run-times. However, under poorly controlled sample preparation and storage conditions, it can become a major event.

Alkylation↗
PubMed2004

Liquid injection field desorption ionization: a new tool for soft ionization of samples including air sensitive catalysts and non-polar hydrocarbons.

Mass analysis of air sensitive samples like organometallic catalysts requires inert sample preparation to avoid degradation of such reactive molecules. Non-polar samples like hydrocarbons are stable but nonetheless need soft ionization to reduce congestion of fragment peaks for analysis of complex mixtures. This paper describes a novel type of probe that combines the advantages of field ionization and field desorption (FI/FD) with an efficient liquid inlet. The new method is called Liquid Injection Field Desorption Ionization (LIFDI). Sample solutions are delivered to the emitter wire inside of the ion source without breaking the vacuum. Sample preparation is reduced to dipping the LIFDI transfer capillary into another sample vial. In case of air sensitive samples kept under inert gas, the preparation is inert without special experimental effort. The new tool provides for a significantly raised sample throughput at excellent sensitivity.

Journal Article↗
PubMed1999

Isolation and quantification of volatiles in fish by dynamic headspace sampling and mass spectrometry.

A dynamic headspace sampling method for isolation of volatiles in fish has been developed. The sample preparation involved freezing of fish tissue in liquid nitrogen, pulverizing the tissue, and sampling of volatiles from an aqueous slurry of the fish powder. Similar volatile patterns were determined by use of this sample preparation method and for samples chewed for 10 s. Effects of sampling time, temperature, and purge flow on level of volatiles were tested. Purging at 340 mL/min for 30 min at 45 degrees C was found to be optimal. Detection limits for a number of aldehydes were 0.2-2.7 microg/kg. Levels of volatiles are given for fresh salmon, cod, saithe, mackerel, and redfish.

Aldehydes↗
PubMed1997

Toward a new reference method for the leukocyte five-part differential.

A flow cytometric method performing a five-part leukocyte differential based on three-color staining with anti-CD45-fluorescein isothiocyanate (FITC), anti-CD-14-phycoerythrin (PE)/Cy5, and a cocktail of PE-labeled anti-CD2, anti-CD16, and anti-HLA-DR antibodies was evaluated. Results obtained by using three different sample preparation procedures and two different flow cytometers were compared with those of a 1,000-cell manual differential for evaluation of accuracy. We observed excellent correlations with the manual differential for all leukocyte subclasses and even higher correlations between the different flow cytometric methods. Flow cytometric basophil results were identical to the manual counts, regardless of which sample preparation technique or flow cytometer was used. Therefore, we propose our flow cytometric method as the first acceptable automated reference method for basophil counting. The flow cytometric results for the other leukocyte subclasses were apparently influenced by the sample preparation, which could not be explained by cell loss during washing steps. Moreover, a small influence of the flow cytometer was also observed. Assessing the influence of sample storage, we found only minimal changes within 24 h. In establishing reference values, high precision of flow cytometric results facilitated detection of a significantly higher monocyte count for males (relative count: 7.08 +/- 1.73% vs. 6.44 +/- 1.33%, P < 0.05; absolute count: 0.536 +/- 0.181 x 10(9)/liter vs. 0.456 +/- 139 x 10(9)/liter, P < 0.01). Our data indicate that monoclonal antibody-based flow cytometry is a highly suitable reference method for the five-part differential: It also shows, however, that studies will have to put more emphasis on methodological issues to define a method that shows a high interlaboratory reproducibility.

Antibodies, Monoclonal↗
PubMed2006

Improved matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry of carboxymethyl cellulose.

A refined sample preparation procedure for matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS) was developed for the evaluation of the degree of substitution (DS) in partially depolymerised carboxymethyl cellulose (CMC). By adding ammonium sulphate to the sample mixture prior to the analysis, good quality mass spectra could be acquired. The usual time-consuming search for 'sweet-spots' at the crystalline rim of the MALDI target spot was also avoided. This quality improvement made it possible to investigate whether various positions on the target spot generated mass spectra in which the measured DS varied. The accuracy and reproducibility of the sample preparation procedure were tested by applying it on three commercial CMCs. The study shows that the DS values that were calculated from the spectra acquired from the centre region of the MALDI target spot were in better agreement with the DS provided by the supplier than were the values obtained from the large crystals at the target spot rim. This observation could be one reasonable explanation for the higher DS values reported in other publications. By applying our refined MALDI sample preparation procedure DS values that were in good agreement with the values provided by the manufacturer could be obtained. This indicates that MALDI-TOFMS of partially depolymerised CMCs can be used for an estimation of the DS as a complement to the more established methods, e.g. NMR, titrimetry, and chromatographic techniques.

Journal Article↗
PubMed1976

Transmission electron microscopy of biliary mixed lipid micelles.

One potential artifact problem in election microscopy of micellar systems is that structural changes and aggregation could be induced by high vacuum dehydration in sample preparation prior to viewing and photomicrography. This could account for an earlier report on "macroassemblies" of biliary lipid, a finding which has been discrepant with the predominance of other evidence on the size, configuration and behavior of biliary micelles. In the present work, this possibility was explored and no evidence for such macroassemblies demonstrated when freeze-drying sample preparation was used; whereas, with conventional technique, the "macroassemblies" were again observed. It is concluded that freeze-drying or freeze-etching sample preparation is required to avoid this form of artifact in electron microscopy of micellar systems. The present findings are compatible with a spherical-equivalent or oblate ellipsoid configuration for biliary mixed micelles.

Bile↗
PubMed2000

The limitations of MALDI-TOF mass spectrometry in the analysis of wide polydisperse polymers

Average molecular weight determination of polymers with polydispersities greater than 1.2 is an ongoing challenge in the field of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Mass discrimination effects observed in the analysis of these polymers have been attributed to sample preparation, desorption/ionization, and instrumental factors. In an effort to separate these factors, we studied poly(methyl methacrylate) (PMMA) standards using two different ion detection systems installed on the same time-of-flight mass analyzer. Equimass blends of narrow PMMA standards were used to simulate a polymer with a wide polydispersity. MALDI-MS analysis was also performed on a PMMA standard with a polydispersity of 1.7. All samples were analyzed by size exclusion chromatography for comparison. Although sample preparation and ionization/desorption factors were found to influence the spectral appearance of the MMA distributions, we demonstrate that, under similar sample preparation and instrument conditions, different ion detection systems produce different results for synthetic polymer blends. The differences in the detector responses for the blends and wide polydisperse standard arise from several factors related to the ion detection system: (1) detection mechanisms, (2) saturation effects, and (3) signal-to-noise limitations.

Journal Article↗
PubMed2006

A novel mutation detection approach of hMLH1 and hMSH2 genes for screening of colorectal cancer.

BACKGROUND: Recently, Denaturing High-Performance Liquid Chromatography (DHPLC) has been widely used for mutation detection hMLH1 and hMSH2 genes due to reduced cost of analysis, accuracy, and high sample throughput. Unfortunately, one major drawback in screening the hMLH1 and hMSH2 genes with any analysis technique involves sample preparation. Additionally, there are limitations to this technique which include: (1) amplicons for hMLH1 and hMSH2 exons cannot be generated under the same PCR thermal cycler condition due to differences in the annealing temperatures of the traditional primer sets which drastically increases sample preparation time; (2) due to minimal changes in the DHPLC chromatogram when compared to the corresponding wild-type amplicon, there is a possibility to not detect a homozygous mutation; and (3) lack of specialized mutation analysis software for automated screening of the hMLH1 and hMSH2 genes with the Transgenomic Wave system. METHODS: To overcome these limitations, the hMLH1 and hMSH2 condition-oriented-PCR primer-embedded-reactor (COPPER) plate was developed to reduce the sample preparation time and technological skill required for analysis, as well as standardize a mutation screening technique for hMLH1 and hMSH2 analysis using the Transgenomic Wave system for research and clinical genetics investigations. In this study, we validated the COPPER plate for simultaneous amplification of the exons of the hMLH1 and hMSH2 genes coupled to DHPLC detection for colorectal cancer (CRC) patients. RESULTS AND CONCLUSIONS: Our results suggest that the COPPER plate DHPLC approach is a simple, cost effective, accurate, universal, and reproducible technology for screening hMLH1 and hMSH2 genes which are associated with human CRC. We also believe that the COPPER plate DHPLC approach is amenable for characterization of other germline alterations in clinical genetics and pharmacogenetics.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Sampling uncertainties for the detection of chemical agents in complex food matrices.

Using uncertainty associated with detection of aflatoxin in shelled corn as a model, the uncertainty associated with detecting chemical agents intentionally added to food products was evaluated. Accuracy and precision are two types of uncertainties generally associated with sampling plans. Sources of variability that affect precision were the primary focus of this investigation. Test procedures used to detect chemical agents generally include sampling, sample preparation, and analytical steps. The uncertainty of each step contributes to the total uncertainty of the test procedure. Using variance as a statistical measure of uncertainty, the variance associated with each step of the test procedure used to detect aflatoxin in shelled corn was determined for both low and high levels of contamination. For example, when using a 1-kg sample, Romer mill, 50-g subsample, and high-performance liquid chromatography to test a lot of shelled corn contaminated with aflatoxin at 10 ng/g, the total variance associated with the test procedure was 149.2 (coefficient of variation of 122.1%). The sampling, sample preparation, and analytical steps accounted for 83.0, 15.6, and 1.4% of the total variance, respectively. A variance of 149.2 suggests that repeated test results will vary from 0 to 33.9 ng/g. Using the same test procedure to detect aflatoxin at 10,000 ng/g, the total variance was 264,719 (coefficient of variation of 5.1%). The sampling, sample preparation, and analytical steps accounted for 41, 57, and 2% of the total variance, respectively. A variance of 264,719 suggests that repeated test results will vary from 8,992 to 11,008 ng/g. Foods contaminated at low levels reflect a situation in which a small percentage of particles is contaminated and sampling becomes the largest source of uncertainty. Large samples are required to overcome the "needle-in-the-haystack" problem. Aflatoxin is easier to detect and identify in foods intentionally contaminated at high levels than in foods with low levels of contamination because the relative standard deviation (coefficient of variation) decreases and the percentage of contaminated kernels increases with an increase in concentration.

Aflatoxins↗
PubMed1996

Microchip-based Devices for Molecular Diagnosis of Genetic Diseases.

Microchips, constructed with a variety of microfabrication technologies (photolithography, micropatterning, microjet printing, light-directed chemical synthesis, laser stereochemical etching, and microcontact printing) are being applied to molecular biology. The new microchip-based analytical devices promise to solve the analytical problems faced by many molecular biologists (eg, contamination, low throughput, and high cost). They may revolutionize molecular biology and its application in clinical medicine, forensic science, and environmental monitoring. A typical biochemical analysis involves three main steps: (1) sample preparation, (2) biochemical reaction, and (3) detection (either separation or hybridization may be involved) accompanied by data acquisition and interpretation. The construction of a miniturized analyzer will therefore necessarily entail the miniaturization and integration of all three of these processes. The literature related to the miniaturization of these three processes indicates that the greatest emphasis so far is on the investigation and development of methods for the detection of nucleic acid, followed by the optimization of a biochemical reaction, such as the polymerase chain reaction. The first step involving sample preparation has received little attention. In this review the state of the art of, microchip-based, miniaturized analytical processes (eg, sample preparation, biochemical reaction, and detection of products) are outlined and the applications of microchip-based devices in the molecular diagnosis of genetic diseases are discussed.

Journal Article↗
PubMed2001

Determination of assay and impurities of gamma irradiated chloramphenicol in eye ointment.

A sample preparation method was developed to isolate chloramphenicol and its radiolytic products from an oily ointment base. The isolation method suspended the eye ointment in n-hexane at 45 degrees C, and isolated the target compounds as residue by centrifugation. It was found that the main element to ensure a satisfactory isolation was keeping the sample solution at 45 degrees C during sample preparation. Linearity, precision, accuracy and suitability of the method were confirmed valid for both assay and impurity tests. This isolation method was ideal for assay, unique for extraction of unexpected and complex radiolysis products, and had a number of advantages compared to the pretreatment methods described in The United Stares Pharmacopoeia and British Pharmacopoeia, in terms of accuracy, precision, and easy handling. The effect of gamma-irradiation on chloramphenicol eye ointment was studied by HPLC, after applying the developed sample preparation method. The present assay and impurity test methods with HPLC were confirmed to be suitable for irradiated chloramphenicol in eye ointment. Formation of radiolytic products induced by gamma-irradiation was evidenced by the impurity test. The assay test showed that active ingredient of chloramphenicol eye ointment decreased by 3.3% at an irradiation dose of 25 kGy and by 11.1% at 50 kGy.

Centrifugation↗