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At least 1,225 records · Page 68Linked to original sources
PubMed2001

Capillary electrophoretic bioanalysis of therapeutically active peptides with UV and mass spectrometric detection after on-capillary preconcentration.

An earlier developed capillary electrophoresis (CE) system with an on-capillary adsorptive phase is investigated for its suitability to quantitate low concentrations of angiotensin II and gonadorelin in plasma. An off-line solid-phase extraction is used for sample preparation. The on-line preconcentration CE system allows multiple capillary volumes of sample solution to be injected, increasing the concentration sensitivity of CE with 3-4 orders of magnitude. Furthermore, possible influence of matrix salts can be ruled out by employing a rinsing step after sample application. Using short-wavelength UV detection, reproducibility and linearity in the low nanomolar range were satisfactory. The capillary could be efficiently regenerated using a programmed between-run rinsing procedure, allowing 20-30 large injections of sample extracts. Coating of the capillary improved the robustness of the method. Mass spectrometric detection via a previously reported sheathless interface increased the selectivity and sensitivity substantially. Recommendations are provided for the sample preparation process, the most critical part of the system. Further purification of the sample is required to allow the loading of larger sample volumes and to optimize the system's robustness.

Angiotensin II↗
PubMed1985

Evidence for two isozymes of leukocyte alkaline phosphatase in leukemic leukocytes.

Leukocyte alkaline phosphatase (LAP) is a granulocyte enzyme whose level of expression is markedly altered in various disease states. We have characterized LAP from normal cells and leukemic cells with a high level of LAP activity in order to determine whether increased enzyme levels are caused by increased levels of the same enzyme or induction of a different alkaline phosphatase. Leukocyte alkaline phosphatase was purified from normal granulocytes and from leukemic cells of a patient with chronic granulocytic leukemia (CGL) in blast phase with an elevated LAP level. LAP was partially purified utilizing diethylaminoethyl (DEAE)-cellulose ion exchange chromatography, gel filtration, and preparative electrophoresis. The sample prepared from normal granulocytes contained a single protein with LAP activity having a molecular weight of 61,000 as determined by SDS gel electrophoresis. The sample from the CGL blast-phase cells, however, demonstrated two proteins with alkaline phosphatase activity, one with a molecular weight of 61,000 (LAPs) and one with a molecular weight of 45,000 (LAPf). Differential heat inactivation and distinct isoelectric points of the two isozymes suggest that they are different proteins. We interpret our data to suggest two closely related LAP alleles whose expression is controlled independently. This may represent either genetic heterogeneity or induction of "tumor marker" gene expression.

Alkaline Phosphatase↗
PubMedDate not supplied

Preparation of environmental samples for determination of sulfonylurea herbicides by solid-phase extraction using a polymeric sorbent.

An analytical procedure was developed for rapid determination of sulfonylurea herbicides in water or soil. Samples were prepared by solid-phase extraction using polymeric sorbents in cartridges and analyzed by liquid chromatography with photodiode array detection. Recoveries were consistent for all sample matrixes tested and were generally > 85%, except for tribenuron methyl (75%). Detection limits were < 100 ng/L for water samples.

Adsorption↗
PubMed2003

Enzyme-friendly, mass spectrometry-compatible surfactant for in-solution enzymatic digestion of proteins.

Improved in-solution tryptic digestion of proteins in terms of speed and peptide coverage was achieved with the aid of a novel acid-labile anionic surfactant (ALS). Unlike SDS, ALS solubilizes proteins without inhibiting trypsin or other common endopeptidases activity. Trypsin activity was evaluated in the presence of various denaturants; little or no decrease in proteolytic activity was observed in 0.1-1% ALS solutions (w/v). Sample preparation prior to mass spectrometry and liquid chromatography analysis consists of sample acidification. ALS degrades rapidly at low-pH conditions, which eliminates surfactant-caused interference with analysis. Described methodology combines the advantages of protein solubilization, rapid digestion, high peptide coverages, and easy sample preparation for mass spectrometry and liquid chromatography analyses.

Alkanesulfonates↗
PubMed1981

Ovalbumin subfractionation and individual difference in ovalbumin microheterogeneity.

Purified ovalbumin from hen egg white was fractionated by concanavalin A (Con A)/Sepharose chromatography. Four major fractions were separated, one unadsorbed fraction OA, followed by a minor fraction OA', and three adsorbed fractions OB, OC, and OD. The recovery was over 90%. Fractional ratios (OA:OB:OC:OD) were 16:6:37:41. The amino acid composition of the four fractions were quite similar. Galactose was only detected in OA, and the mannose content in OD was high as compared to that of the other fractions. The glycopeptide resulting from pepsin digestion of ovalbumin was also fractionated by Con A/Sepharose. Four fractions were obtained, and they corresponded to fractions OA, OB, OC, and OD. The amino acid compositions of the peptides were identical, indicating that they were derived from the same region in ovalbumin. In order to study individual differences in ovalbumin microheterogeneity, ovalbumin samples prepared from individual eggs were subfractionated on Con A/Sepharose. Four ovalbumin samples prepared from the eggs of a single hen revealed the same elution profile and the same fractional ratios. Fractional ratios of 17 individual preparations varied from 7:2:19:72 to 27:5:40:28 (OA:OB:OC:OD). A constant relation was found among the fractional ratios: the OB, OC and OD contents were dependent on the OA content, and the proportion of OD was in inverse relation to those of OA and OC. The OB content was low and did not change in all ranges of OA content. The present observations indicate that a single hen produces ovalbumin with qualitatively and quantitatively similar carbohydrate chains by unknown mechanisms; moreover, individual differences exist in ovalbumin microheterogeneity which follow constant patterns.

Amino Acids↗
PubMed1997

Immunoassay analysis of lysergic acid diethylamide.

Screening large numbers of urine samples for drugs of abuse is typically accomplished using immunoassays that allow for processing large numbers of samples without the requirement of sample preparation before analysis. Until fairly recently, screening of lysergic acid diethylamide (LSD) in urine samples could only be accomplished by the use of radioimmunoassays (RIA). Recently, new nonisotopic immunoassays have been developed for the screening of samples for LSD. These assays lend themselves to rapid, high-volume, automated analysis compared with RIA procedures. In order to evaluate the current commercially available assays, samples prepared at known concentrations were tested by each of the assays. In addition, samples from known use of LSD were tested and the performance of each of the assays compared. The assays examined in this study included RIA assays from Roche Diagnostics (Abuscreen) and Diagnostic Products (coat-a-count) and nonisotopic assays from Roche (OnLine), Behring (EMIT), Boehringer Mannheim (CEDIA), and STC (Microplate EIA). Assays that could readily be carried out in a semiquantitative mode (determining concentration based on a calibration curve) were evaluated as to their relative response to the samples tested. All of the assays evaluated identified all of the samples which confirmed positive by gas chromatography-mass spectrometry (GC-MS). Likewise, each of the assays identified some samples which did not confirm as positive by GC-MS.

Animals↗
PubMed2001

Application of a high-performance liquid chromatographic assay for the neuromuscular blocker gallamine to analysis of rat plasma, muscle and microdialysate samples.

A reliable high-performance liquid chromatographic method has been validated for determination of gallamine in rat plasma, muscle tissue and microdialysate samples. A C18 reversed-phase column with mobile phase of methanol and water containing 12.5 mM tetrabutyl ammonium (TBA) hydrogen sulphate (22:78, v/v) was used. The flow-rate was 1 ml/min with UV detection at 229 nm. Sample preparation involved protein precipitation with acetonitrile for plasma and muscle tissue homogenate samples. Microdialysate samples were injected into the HPLC system without any sample preparation. Intra-day and inter-day accuracy and precision of the assay were <13%. The limit of quantification was 1 microg/ml for plasma, 1.6 microg/g for muscle tissue and 0.5 microg/ml for microdialysate samples. The assay was applied successfully to analysis of samples obtained from a pharmacokinetic study in rats using the microdialysis technique.

Animals↗
PubMed2001

Proton binding by hydrous ferric oxide and aluminum oxide surfaces interpreted using fully optimized continuous pKa spectra.

A modified regularized least squares pKa spectrum approach is proposed to determine proton stability constants and concentrations for binding sites on hydrous ferric oxide (HFO) and aluminum oxide surfaces. Acid-base titration data are fit to a continuous binding site model for the system represented as a pKa spectrum. The modified parameter fitting method optimizes simultaneously for both smoothness of the pKa spectrum and goodness-of-fit, whereas other methods optimize for goodness-of-fit given a fixed smoothness factor. The modified method is tested with aluminum oxide and recovers values consistent with theoretical values. The regularized pKa spectrum method optimized for smoothness is applied to prepared samples of two types of HFO. The prepared HFO samples differ only in the total iron concentration of the parent solution. The resultant pKa distributions are compared to proton binding constants from MUSIC model results for crystalline iron oxides. The types of binding sites in the HFO sample are consistent with theoretical binding site stability constants for crystalline iron oxides. Overall, the prepared HFO samples have binding constants most consistent with values for lepidocrocite and goethite.

Aluminum Oxide↗
PubMed1999

Electron diffraction provides new information on human stratum corneum lipid organization studied in relation to depth and temperature.

The outermost layer of mammalian skin, the stratum corneum, provides the body with a barrier against transepidermal water loss and penetration of agents from outside. The lipid-rich extracellular matrix surrounding the corneocytes in the stratum corneum is mainly responsible for this barrier function. In this study (cryo-) electron diffraction was applied to obtain information about the local lateral lipid organization in the extracellular matrix in relation to depth in human stratum corneum. For this purpose, stratum corneum grid-strips were prepared from native skin in vivo and ex vivo. It was found that the lipid packing in samples prepared at room temperature is predominantly orthorhombic. In samples prepared at 32 degrees C the presence of a hexagonal packing is more pronounced in the outer layers of the stratum corneum. Gradually increasing the specimen temperature from 30 to 40 degrees C induced a further transition from an orthorhombic to a hexagonal sublattice. At 90 degrees C all lipids were present in a fluid phase. These results are in good agreement with previously reported wide angle X-ray diffraction and Fourier transformed infrared spectroscopy studies. We conclude that the lipids in human stratum corneum are highly ordered throughout the stratum corneum and that electron diffraction allows monitoring of the local lipid organization, which contributes to the understanding of stratum corneum barrier function.

Cryoultramicrotomy↗
PubMed2006

Sample treatments prior to capillary electrophoresis-mass spectrometry.

Sample preparation is a crucial part of chemical analysis and in most cases can become the bottleneck of the whole analytical process. Its adequacy is a key factor in determining the success of the analysis and, therefore, careful selection and optimization of the parameters controlling sample treatment should be carried out. This work revises the different strategies that have been developed for sample preparation prior to capillary electrophoresis-mass spectrometry (CE-MS). Namely the present work presents an exhaustive and critical revision of the different samples treatments used together with on-line CE-MS including works published from January 2000 to July 2006.

Analytic Sample Preparation Methods↗
PubMed2003

Determination of amoxycillin in human plasma by direct injection and coupled-column high-performance liquid chromatography.

This work reports the use of multidimensional HPLC by coupling a restricted access medium (RAM) bovine serum albumin (BSA) octadecyl column (100 x 4.6 mm I.D., 10 microm particle size and 120 A pore size) to an octadecyl Hypersil column (150 x 4.6 mm I.D., 5 microm particle size and 120 A pore size) to the analysis of amoxycillin in human plasma by direct injection. Ion pairing was necessary to extract amoxycillin with good recovery from the plasma proteins. To prepare the spiked samples, aliquots (60 microl) of the appropriated standard solutions were added to each culture tube containing an 180 microl of plasma and a solution of 0.30 mM tetrabuthylammonium phosphate (60 microl). They were vortexed for 15 s and then 290 microl were transferred to autosampler vials. Aliquots (250 microl) of the spiked plasma samples were injected to a column-switching HPLC system. An analysis time of 25 min with no time spent on sample preparation was achieved. The developed method showed good selectivity, sensitivity, accuracy and precision for direct analysis of this polar low wavelength ultraviolet absorption antibiotic using only 180 microl of human plasma. The validated method proved to be reliable and sensitive for the determination of amoxycillin in plasma samples of five healthy volunteers to whom test and reference formulations were administered as an oral dose (500 mg).

Amoxicillin↗
PubMed1987

Glutamine and glutamate: automated quantification and isotopic enrichments by gas chromatography/mass spectrometry.

A method is described for simultaneous quantification of glutamine and glutamate plasma levels and isotopic enrichments in these compounds. Glutamine and glutamate are analyzed intact as their tertiary-butyldimethylsilyl derivatives. Deuterated glutamine and glutamate are used as internal standards for quantification by reverse isotope dilution. Preparation of plasma samples is accomplished by adding ammonium formate as an ion-pairing agent followed by extraction of the amino acids into 4.3:1 methanol:water. Negligible amounts of glutamine to glutamate conversion are observed during the sample preparation and GC/MS analysis. Since glutamine is analyzed intact, both single and double [15N]glutamine labels can be quantified. [15N]Glutamine at 0.2 to 11 mol% excess was measured in plasma with an average relative standard error of 3.8%, and [15N]glutamate over a range of 0.4 to 9 mol% excess was measured with a mean relative standard error of 12%. At glutamate levels above 1 mol% excess 15N, the mean relative standard error was 6%. Finally, automated sample injection into the GC/MS and automated data reduction are used for the analysis of samples by GC/MS.

Animals↗
PubMed2004

Chiral analysis of butaclamol enantiomers in human plasma by HPLC using a macrocyclic antibiotic (vancomycin) chiral stationary phase and solid phase extraction.

An enantioseparation of the antipsychotic drug butaclamol in human plasma by high-performance liquid chromatography (HPLC) with solid phase extraction is presented. The separation was achieved on the vancomycin macrocyclic antibiotic chiral stationary phase (CSP) Chirobiotic V with a polar ionic mobile phase (PIM) consisting of methanol : glacial acetic acid : triethylamine (100:0.2:0.05, v/v/v) at a flow rate of 0.5 ml/min. The detection wavelength was 262 nm. Bond Elut C18 solid phase extraction cartridges were used in the sample preparation of butaclamol samples from plasma. The method was validated over the range of 100-3,000 ng/ml for each enantiomer concentration (R(2) > 0.999). Recoveries for (+)- and (-)-butaclamol were in the range of 94-104% at the 300-2,500 ng/ml level. The method proved to be precise (within-run precision ranged from 1.1-2.6% and between-run precision ranged from 1.9-3.2%) and accurate (within-run accuracies ranged from 1.5-5.8% and between-run accuracies ranged from 2.7-7.7%). The limit of quantitation (LOQ) and limit of detection (LOD) for each enantiomer in human plasma were 100 ng/ml and 50 ng/ml, respectively.

Anti-Bacterial Agents↗
PubMed2005

Design and characterization of a compact dual channel virus counter.

BACKGROUND: Although there is a growing need in the field of biotechnology to rapidly and accurately quantify viruses, time-consuming techniques such as the plaque titer method remain the "gold standard." Flow cytometric methods for virus quantification offer the advantages of rapid analysis and statistical treatment. The technique presented in this work represents the first demonstration of a flow cytometric determination of a viral count that is directly related to the count obtained by plaque titer. METHODS: A flow cytometric instrument for rapid quantification of virus particles was designed, constructed, and thoroughly characterized. A two-color method, which involved staining the viral genome and the protein coat for baculoviruses, was developed in addition to an algorithm to identify simultaneous events on the DNA and protein channels. RESULTS: The instrument was fully characterized, which included analysis of the data acquisition rate, sampling time, flow rate, detection efficiency, linear dynamic range, channel cross-talk, and the limit of detection. Baculovirus samples were analyzed and the results were compared with concentrations obtained by a one-channel flow cytometer and plaque assay. CONCLUSIONS: The dual channel virus counter yields a representative value for the concentration of active viruses in an unpurified sample when compared with plaque assay and a one-channel flow cytometer. The technique is rapid (within minutes), requires only minimal sample preparation and minimum sample size (approximately 100 microl).

Baculoviridae↗
PubMed2005

Chip-based microfluidic devices coupled with electrospray ionization-mass spectrometry.

We present the current status of the development of microfluidic devices fabricated on different substrates for coupling with electrospray ionization-mass spectrometry (ESI-MS). Until now, much success has been gained in fabricating the ESI chips, which show better performances due to miniaturization when compared with traditional methods. Integration of multiple steps for sample preparation and ESI sample introduction, however, remains a great challenge. This review covers the main technical development of electrospray device that were published from 1997 to 2004. This article does not attempt to be exclusive. Instead, it focuses on the publications that illustrated the breath of the development and applications of microchip devices for MS-based analysis.

Electrophoresis, Microchip↗
PubMed2006

Pharmacokinetic applications of capillary electrophoresis: a review on recent progress.

This article covers recent publications from 2003 to 2005 on the subject of pharmacokinetic applications of CE. Many analytical methods were validated and more importantly, they were shown to have sufficient sensitivities to access pharmacokinetic data on different models. Because of unique advantages, such as simplified sample preparation methods, small sample amount required, high separation power, and speedy analysis, CE-based assays were found to gain popularity not only as a second method but also as a major method for many pharmacokinetic studies.

Animals↗
PubMed2006

A novel tool for high-resolution transmission electron microscopy of intact interfaces between bone and metallic implants.

A key feature in the understanding of the mechanisms of integration versus rejection of implanted materials is a deepened understanding of the elemental and molecular compositions of the interface zone between the surface of the synthetic man-made material and the biological components of tissue. Intact interfaces between metallic implants and tissues have not been able to image and analyse on the ultrastructural level with the common transmission electron microscopy (TEM) sample preparation techniques. By using focused ion beam microscopy for site-specific preparation of TEM samples, intact interfaces between metal implants and calcified tissue were imaged for the first time. The interface's elemental and crystallographic compositions were determined using energy dispersive X-ray mapping and electron diffraction. The developed technique fulfills a long-sought-for demand to correlate the surface properties of implanted metal prostheses with the fine structure and composition of preserved interfaces with tissues.

Animals↗
PubMed2006

Proteomic analysis and comparison of the biopsy and autopsy specimen of human brain temporal lobe.

The proteomic study on human temporal lobe can help us to understand the physiological function of CNS in normal as well as in pathological state. Proteomic tools are potent for the assessment of protein stability post mortem. In this pilot study, the human temporal lobe biopsy specimen with chronic pharmacoresistant temporal lobe epilepsy (TLE) and autopsy specimen in control were separated by 2-DE. Using MALDI-TOF-MS and MS/MS, 375 protein spots were identified which were the products of 267 genes. Six down-regulated and 23 up-regulated protein spots in the autopsy specimen were ascertained after the gel image analysis with the ImageMaster software. A number of proteins that include neurotransmitter metabolic and glycolytic enzymes, cytoprotective proteins and cytoskeleton were found decreased while the precursor of apolipoprotein A-I increased in the TLE brain. We tried several methods to prepare the protein samples and found that DNase and RNase treatment, ultracentrifugation and Amersham clean-up kit purification can improve gel separation quality. This work optimized the sample preparation method and constructed a primary protein database of human temporal lobe and found some proteins with remarkable level change probably involved in the post-mortem process and chronic pharmacoresistant TLE pathogenesis.

Autopsy↗