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PubMed1994

Detection of point mutations by solid-phase methods.

Several techniques exist that permit the efficient distinction among characterized DNA sequence variants. In this review we discuss a number of such analytic procedures. These techniques all take advantage of a variety solid supports to prepare and analyze reaction products. The described diagnostic principles are now being applied for the development of miniaturized assay formats, suitable for automated detection of large sets of sequences in clinical samples.

Animals↗
PubMed1991

Determination of tin in human blood serum by radiochemical neutron activation analysis.

A method was developed for the determination of tin in human serum by radiochemical neutron activation analysis, using the long-lived radioisotope Sn(T1/2 = 115.09 days). This radioisotope decays to a daughter isotope 113mIn, the most suitable nuclide for counting (T1/2 = 1.658 h, gamma-ray of 391.7 keV). Experience showed that, with the exception of the serum samples with the lowest tin levels, in the experimental conditions of the present study tin could mostly also be determined by using its radioisotope 117mSn(T1/2 = 13.61 days, gamma-ray of 158.5 keV). Samples were collected and prepared by using the procedure elaborated by the authors, which proved its effectiveness in preventing significant sample contamination on several occasions. Because samples had to be irradiated at 10(14) n.cm-2.s-1, dry ashing was necessary. After irradiation, tin was separated by solvent extraction of tin(IV) iodide from a sulfuric acid-ammonium iodide solution with toluene. The dry ashing and solvent extraction steps were exhaustively tested by means of radioactive tracer experiments whereas the accuracy and precision of the analytical method were thoroughly checked by analyzing biological reference materials (Bowen's kale powder, the NBS' bovine liver, the NBS' nonfat milk powder, and the "second-generation" biological reference material--freeze-dried human serum--for trace element determinations, developed by the authors).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMedDate not supplied

[Validation of triiodothyronine sodium analysis method].

OBJECTIVE: To validate the ultraviolet spectrophotometry analytical method for triiodothyronine sodium in order to use this method in the quality control process of a diluted powder of said active principle for the manufacturing of 25 mcg capsules. MATERIAL AND METHODS: An ultraviolet spectrophotometry analysis is performed on triiodothyronine sodium to obtain its spectrum. Subsequently, a mother solution of triiodothyronine sodium is made, from which 5 different-concentration dilutions are prepared in order to obtain a calibration line allowing the study of the method's linearity. Then 3 of these 5 dilutions are selected for repeatability and reproducibility studies. Two pattern solutions with differing concentrations are prepared for the accuracy study. Three different-concentration dilutions are prepared from each of them. The absorbencies of controls for both pattern solutions are measured to compare results obtained. RESULTS: A scan is performed between 350 and 200 nm, and an experimental peak of 319 nm is obtained. The method proves linear for the considered range of concentrations (r(2) = 0.9985). Variation coefficients are around 2%, thus indicating good repeatability and reproducibility. The mean percentage of recovery in known-concentration samples does not significantly differ from the theoretical 100%. CONCLUSION: The studied analytical method is adequately reliable to result in a foreseen result within defined intervals.

Capsules↗
PubMed2026

On-filter fractionation by empFASP improves identification of membrane peptides in proteomic experiments.

Membrane proteins remain among the most analytically challenging targets in bottom-up proteomics due to their limited solubility and low abundance of protease-accessible sites within transmembrane domains. In addition, hydrophobic peptides are frequently lost during detergent removal and the on-filter processing steps. Here, we present empFASP, a straightforward on-filter-fractionation-based modification of the enhanced filter-aided sample preparation (eFASP) workflow that enhances recovery of membrane-embedded peptides otherwise lost during digestion and cleanup. The method combines controlled on-filter inversion with sequential ethyl acetate extraction at defined pH values, enabling recovery of peptide material retained on the filter and redistributed into detergent micelles. Compared with SP3 and SP4 in HEK293T lysates, empFASP increased unique hydrophobic peptide identifications by up to 48% and increased the proportion of detected transmembrane peptides. Application to mouse mitochondrial membranes and phosphatidylethanolamine-deficient and PE-containing Escherichia coli membranes showed that the additional fractions of empFASP contribute complementary recovery of hydrophobic and membrane-associated peptides, with the strongest gains observed at the peptide level. Because empFASP requires no specialized reagents or instrumentation, it can be readily implemented in standard proteomics workflows to improve coverage of membrane-embedded regions. SIGNIFICANCE: The empFASP (enhanced membrane peptide) workflow offers a practical solution to one of the persistent limitations in membrane proteomics-the underrepresentation of hydrophobic and transmembrane peptides in standard digests. By integrating simple pH-controlled extractions into an on-filter format, empFASP recovers peptides otherwise lost through adsorption or detergent micelle retention, substantially improving coverage of the membrane proteome. This method expands the analytical reach of bottom-up proteomics without requiring specialized instrumentation, making it immediately applicable for studies of membrane topology, protein-lipid interactions, and the structural consequences of altered membrane composition.

Proteomics↗
PubMedDate not supplied

Preparative method for isolating alpha-zearalenol and zearalenone using extracting disk.

A liquid chromatographic method is described for the determination of zearalenol and zearalenone in corn. Zearalenol and zearalenone are extracted from corn with methanol-water (1 + 1) and cleaned up using a solid-phase extraction (SPE) disk, separated on a reversed-phase analytical column, and detected with a fluorescence detector. The SPE disk concentrated and cleanly separated zearalenol and zearalenone from sample interferences. Standard calibration curves for zearalenol and zearalenone for the concentration range 25-500 ng/mL were linear. The small extract disk had a column capacity equivalent to 1 g extracted corn. Zearalenol and zearalenone were added at levels ranging from 10 to 2000 ng/g to a control sample that contained no detectable levels of zearalenol and zearalenone. Both toxins were recovered from spiked samples at 106.3 and 103.8%, with coefficients of variation of 7.6 and 13.0%, respectively. The method has an estimated reliable limit of detection and limit of quantitation around 10 and 40 ng/g for each toxin, respectively.

Chromatography, Liquid↗
PubMed1999

New developments in the standardization of total prostate-specific antigen.

OBJECTIVE: Analytical evaluation of the calibration of three recently launched assays for the measurement of total prostate-specific antigen, i.e., IMx Total PSA (Abbott), Elecsys PSA (Roche), and IMMULITE 3rd Generation PSA (DPC). DESIGN AND METHODS: For accuracy assessment two reference materials were applied namely, Stanford 90:10 PSA Calibrator and Certified Reference Material 613 Prostate-Specific Antigen. Dilutions of these preparations were analyzed with all assays. In addition, clinical specimens from known prostate cancer or benign prostate hyperplasia patients and samples taken from an ongoing prostate cancer screening study were used for comparison. RESULTS: Application of the Stanford Calibrator revealed results well within 10% of the calculated values for all assays. Regarding the CRM Calibrator only the IMx Total PSA proved to approach the line of identity. The IMMULITE results differed about 40% and the Elecsys about 18% from the calculated values. The comparison with clinical specimens showed statistically different results for the combination IMMULITE-IMx and for IMMULITE-Elecsys. The regression lines for both collections were: y(IMx) = 0.86x(IMMULITE) +0.12 (n = 104, r = 0.970, Sy/x = 0.883 microg/L) and y(Elecsys) = 0.98x(IMMULITE) +0.38 (n = 97, r = 0.976, Sy/x = 0.733 microg/L). In the lower measuring range (PSA <5.0 microg/L) as measured with the screening samples (n = 43), these differences were less pronounced. CONCLUSION: In analytical sense a difference was found for both reference preparations in the assays studied. Clinically, despite improvements in methodology, results for total prostate-specific antigen are still not interchangeable. The possible consequences need to be elaborated.

Calibration↗
PubMedDate not supplied

Sampling and preparation of sample for chemical examination.

Sampling and methods for reducing a laboratory sample to a test sample are discussed, with particular emphasis on sampling peanuts for aflatoxin analysis as a practical example. The only way to control the total error in the analysis of this heterogeneous product is to take and to analyze many and large samples.

Aflatoxins↗
PubMed1990

Effects of analytical method and lyophilized sera on measurements of apolipoproteins A-I and B: an international survey.

In 1987 a collaborative study was initiated with 140 laboratories worldwide to evaluate the effects of analytical method and lyophilization on the measurement of different concentrations of apolipoproteins (apo) A-I and B in four lyophilized serum pool samples. This survey confirmed that the lyophilized apo Reference Material of the International Union of Immunological Societies (IUIS) is useful for apo A-I assays as an international serum-based reference material, because among-method variation is negligible. The apo A-I concentration value of 1.24 g/L is now assigned to the IUIS Reference Material (CDC 1883) by a Centers for Disease Control RIA in-house reference method. Use of lyophilized serum preparation as a reference material for some modes of apo B measurement is questionable because of lyophilization and matrix effects. Both radial immunodiffusion and liquid immunoprecipitin methods demonstrated bias in measured apo B concentrations, compared with overall method-weighted means values on the IUIS Reference Material. Because of the uncertainty associated with LDL primary standard, protein analysis, and concentration differences among analytical methods, assigning a single apo B concentration value to the IUIS Reference Material appears inadvisable at present.

Analysis of Variance↗
PubMed1994

Confirmatory analysis for spiramycin residue in bovine muscle by liquid chromatography/particle beam mass spectrometry.

To ensure that residues of veterinary drugs, above their respective maximum residue limits, do not reach the human food supply, European Community regulations specify requirements for detection, quantification and confirmation analytical methods and control procedures. The European Community member states base meat controls on these protocols. A liquid chromatographic/mass spectrometric analysis of spiramycin in calf muscle is presented as a confirmatory method for this compound. A particle beam interface was used, with negative ion chemical ionization mass spectrometry, using methane as the reagent gas. Samples (2 g muscle were prepared by liquid/liquid extraction followed by solid-phase extraction clean-up. On-line liquid chromatography/mass spectrometry of extracts was carried out on a C-18 bonded silica column. The specificity required for a regulatory confirmation procedure was achieved by monitoring five fragment ions with m/z 304, 330, 475, 683 and 684. Variation of the relative ion abundances was less than 20% at the maximum limit of residue, 50 micrograms kg-1. The method specificity was tested for three related compounds: neospiramycin, erythromycin and tylosin. The detection limit based on ion chromatogram peaks areas obtained with control samples was determined to be 20 micrograms kg-1.

Animals↗
PubMed2001

Sampling and processing of biopsy samples for speciation studies of cytosolic metalloproteins.

Studies to specify various metalloproteins in cell cytosol of tissues using chromatographic separation methods and plasma mass spectrometry for element detection require a careful step by step sample preparation. These steps involve (i) bioptic sample removal from tissue, (ii) extraction and preparation of the tissue supernatant, (iii) the chromatographic separation of the proteins, and finally (iv) the on-line transfer of the column eluate into the plasma mass spectrometer. Each of the analytical steps has to be carefully monitored to avoid undesired changes in the sample composition which could be caused by enzymatic and/or oxidative processes as well as by external element contamination. This presentation introduces a concept to ensure that environmental element contamination does not occur to bias the analytical results.

Animals↗
PubMed1988

Densitometric assay of nanogram quantities of proteoglycans precipitated on nitrocellulose membrane with Safranin O.

Proteoglycan (PG) and glycosaminoglycan (GAG) samples corresponding to a minimum of 10 ng of uronic acid were reliably quantified as precipitates with the cationic dye Safranin O, collected by vacuum-aided filtration onto a cellulose acetate/nitrate membrane in a standard 96-well dot assay apparatus. The reflectances of the precipitation dots were measured by automatic densitometric scanning of the membrane sheets. Standard GAGs produced reflectance values which were related to the number of anionic groups per unit disaccharide; hyaluronate and keratan sulfate gave lower values while heparin yielded values higher than those of chondroitin sulfates. The presence of 8 M urea, 1% Triton X-100, 30% sucrose, 0.02% NaN3, or mixtures of proteinase inhibitors and various buffers did not markedly influence the reflectances, while 4 M guanidinium chloride and 3 M CsCl reduced the sensitivity of the assay to 30-50 ng. Samples containing sodium dodecyl sulfate (SDS) were not applicable because SDS precipitated with Safranin O. Proteins showed virtually no response, while nucleic acids gave significant although smaller reflectances than GAGs. Owing to its marked sensitivity and convenience the method is particularly suitable for the detection of PGs during their preparative purification and fractionation as well as in various analytical assays.

Chondroitin Sulfates↗
PubMed2006

Low-level quantitation of oxycodone and its oxidative metabolites, noroxycodone, and oxymorphone, in rat plasma by high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry.

A method was developed for quantification of oxycodone, noroxycodone, and oxymorphone in small volumes (50 microl) of rat plasma by high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry using turbo ion-spray. Deuterated (d3) opioid analogues acted as internal standards. Sample preparation involved protein precipitation with acetonitrile, centrifugal evaporation, and reconstitution in mobile phase; analyte separation was performed on a C18 (5 microm, 2.1 mm x 50 mm) column using a linear gradient program. Lower limits of quantitation (ng/ml) and their between-day accuracy and precision were-oxycodone, 0.9 (-0.2 and 7.8%); noroxycodone, 1.0 (0.6 and 6.2%); oxymorphone 1.0 (-1.8 and 9.5%).

Analgesics, Opioid↗
PubMed1989

Preparation and certification of standard reference material 1507: 11-nor-delta 9-tetrahydrocannabinol-9-carboxylic acid in freeze-dried urine.

The National Institute of Standards and Technology (NIST) has prepared and certified SRM 1507, a freeze-dried urine fortified with 11-nor-delta 9-tetrahydrocannabinol-9-carboxylic acid (THC-9-COOH), the major urinary metabolite of marijuana. The certified concentration of 20 +/- 1 ng/mL for the analyte was obtained from the concordant results of analyses of the material by gas chromatography/mass spectrometry (GC/MS) and high-performance liquid chromatography with electrochemical detection (HPLC-EC). Solid-phase extraction was used to prepare the sample for GC/MS analyses, and liquid-liquid extraction was used for the HPLC-EC analyses. The multistep HPLC method was developed at NIST to circumvent interferences from urinary constituents. The results of a round robin test on this material among five Department of Defense laboratories involved in drug testing are reported.

Chromatography, High Pressure Liquid↗
PubMed2003

Direct determination of benzamides in serum by column-switching high-performance liquid chromatography.

A column-switching high-performance liquid chromatographic method with fluorescence detection was developed for the simultaneous determination of four benzamide-type anti-psychotic drugs: sulpiride, tiapride, sultopride and metoclopramide in human serum. In this method, a TSKgel Super-ODS column was used as an analytical column, and a TSKgel G 2000SW was prepared as a pretreatment column. Under the optimized analytical conditions, four benzamide-type anti-psychotic drugs were eluted within 18 min. The detection limits (S/N = 3) for sulpiride, tiapride, sultopride and metoclopramide are 1 ng/ml, 4 ng/ml, 2 ng/ml and 0.5 ng/ml, respectively. Finally, the method was applied to the determination of sulpiride in human serum samples obtained after a single oral dose of sulpiride.

Benzamides↗
PubMed2001

Analysis of ubidecarenone (CoQ10) aqueous samples using reversed phase liquid chromatography.

A novel method was developed for the quantitative determination of ubidecarenone (CoQ10) in aqueous media using non aqueous reversed phase liquid chromatography. Standards were prepared by melting the compound in Cremophor EL followed by dilution with distilled water. Samples were then analyzed by a Reverse Phase HPLC method using a Waters Novapak C18, 3.9 x 150 mm column. The mobile phase used was methanol: n-hexane (9:1) at a flow rate of 1.5 ml/min. Response of the detector to the analyte was linear (r: 0.999) over the range of 2.5-100 micrograms ml-1, with a limit of detection of 0.17 microgram ml-1. Acetone used to solubilize CoQ10 in the surfactant and Cremophor EL did not interfere with sample analysis. This method provided a convenient and alternative approach to the existing methods that require organic solvent extractions prior to analysis. Besides, this method enabled the separation of major photolytic decomposition products of CoQ10.

Chromatography, High Pressure Liquid↗
PubMed1976

Routine titration of foot and mouth disease virus suspensions by analytical ultracentrifugation. 1: Sedimentation method.

Infectivity and Complement Fixation (CF) tests are commonly used for the routine titration of Foot and Mouth Disease (FMD) virus suspensions. Only recently were techniques published for the routine determination of the virus concentration by the physical properties of the virions (Fayet et al., 1971; Barteling et al., 1974). These techniques are based on the separation of the virions from the culture fluid by sedimentation through a sucrose gradient, in a preparative ultracentrifuge. The ultraviolet absorption pattern of the tube content is recorded by a flow colorimeter. The virus concentration is estimated using either standard curves or direct caculation by the specific extinction coefficient (Bachrach et al., 1964). In our own attempts to develop a preparative ultracentrifugation technique for the routine titration of FMD virus suspensions, we had to deal with some problems such as remixing of the virus band at the end of the run. We therefore turned over to analytical ultracentrifugation methods. The manipulations are less complicated and the virus band is traced and measured while the rotor is spinning. Four samples are analyzed simultaneously and the scans are repeated to follow the move of the virus band. The sedimentation rate of the virus band, calculated from the repeated scans, helps to detect artifacts. The present paper describes the technique we developed for the routine titration of FMD virus suspensions, by the band sedimentation method, using an ultraviolet scanning analytical ultracentrifuge.

Aphthovirus↗
PubMed1999

Chromatographic separation of carotenoids.

The carotenoids are extremely reactive and consequently unstable due to their long system of conjugated double bonds. Several precautions, such as protection against light and oxygen, use of low temperature and antioxidants, analysis in the shortest possible time, should be taken during isolation and chromatography. The food samples, preferably fresh, are homogenized and immediately extracted with a suitable organic solvent. Saponification has been employed in order to hydrolyze the carotenoid esters, remove fatty material and destroy chlorophyll. This optional step facilitates subsequent carotenoid separation, identification and quantification. The separation of carotenoids is usually carried out by column chromatography, thin layer chromatography and high performance liquid chromatography, in analytical or preparative scale, on many stationary phases such as silica-gel, alumina, MgO, Ca(OH)2 and reversed-phase material (C18 and C30). The choice of the most suitable chromatographic method depends on the amount of sample, carotenoid composition, resolution, speed and purity required. Examples of carotenoid separation in different stationary phases will be shown and discussed.

Carotenoids↗
PubMed2005

Fluorimetric liquid chromatographic analysis of amantadine in urine and pharmaceutical formulation.

A simple and sensitive liquid chromatographic method is described for the analysis of amantadine and memantine. The method is based on the derivatization of amantadine and memantine extracted from alkalified samples with (2-naphthoxy)acetyl chloride at mild conditions. The resulting derivatives were analyzed by isocratic HPLC with a fluorimetric detector (lambdaex, 227 nm; lambdaem, 348 nm). The linear range for the determination of amantadine or memantine spiked in urine (1.0 ml) was 1.0-10.0 nmol with a detection limit of about 0.2 nmol (S/N = 3; injected sample 20 microl). Only amantadine preparations are available on our local market, and application of the method to the analysis of amantadine in formulation and in the urine of a dosed subject was demonstrated and proved feasible. Quantitation of AT in tablets or capsules is capable in the linear range of 2.0-50.0 microM. Toluene was used as the solvent for extracting amantadine or memantine in samples and the resulting toluene extract was directly subjected to subsequent derivatization without solvent replacement leading to a simpler analytical procedure.

Amantadine↗