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At least 649 records · Page 36Linked to original sources
PubMed1996

Microparticle-enhanced nephelometric immunoassay of human plasminogen.

A microparticle-enhanced nephelometric immunoassay was developed for plasminogen quantitation in human plasma. It is based on the nephelometric measurement of the light scattered by microparticle clusters formed during a sandwich reaction between plasminogen, microparticle--anti-plasminogen conjugate, and the free antibodies of anti-plasminogen rabbit antiserum. This immunoassay was sensitive (detection limit in reaction mixture, 34 micrograms/L) and could be performed in 500-fold diluted human plasma, excluding any interference or sample pretreatment. It allowed the quantitation of plasminogen on a large range of concentrations (17-550 mg/L), with a security in antigen excess reaching 1,100 mg/L, with accuracy (linear recovery in dilution-overloading assay and correlation with conventional immunonephelometry), and precision (within- and between-run coefficients of variation lower than 8%). A normal reference range from 54 to 148 mg/L (mean +/- 2 SD) was calculated from plasminogen concentration in plasma from 130 adults. Easy to perform (no washing or phase separation) and rapid (two steps of 30 minutes then 1 hour of incubation at room temperature), this microparticle-enhanced nephelometric immunoassay could be an interesting alternative method for human plasminogen quantitation.

Adult↗
PubMed1999

Bilirubin-human serum albumin interaction monitored by capillary zone electrophoresis.

Capillary zone electrophoresis was used to monitor the interaction between bilirubin and human serum albumin. Cord blood serum samples were injected directly into an uncoated fused-silica capillary (30 cm x 50 microns i.d.) and separation was accomplished within 4 min without extensive sample pretreatment. The most suitable running buffer to separate free bilirubin from albumin bound bilirubin was found to contain 1.0 mmol/L EDTA, 5% acetonitrile and 15 mmol/L phosphate with pH adjusted to 8.4. Approximately two bilirubin dianions could be bound per human serum albumin molecule in the cord blood serum. The binding constant was estimated to be 1.1 x 10(5) (L/mol) at 25 degrees C and pH 8.4. The peak area ratio of free bilirubin to total bilirubin can be used to determine the bilirubin binding capacity of cord blood serum for the concentration range of total bilirubin from 204 to 340 mumol/L using 1:5 diluted cord blood seras.

Bilirubin↗
PubMed1999

Determination of aspartic acid enantiomers in bio-samples by capillary electrophoresis.

Enantiomeric separation and detection of D,L-aspartic acid (Asp) derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) by capillary electrophoresis (CE) using modified cyclodextrins as chiral selectors was studied. Heptakis(2,3, 6-tri-O-methyl)-beta-cyclodextrin(TM-beta-CD) was most effective for enantiomeric separation of NBD-D,L-Asp with optimum conditions of 30 mM TM-beta-CD in 50 mM phosphate buffer (pH 4.0) and the limit of detection (LOD) attained was 100 nM for each enantiomer. The method proposed in the present study was convenient for both D- and L-Asp determination since the other amino compounds migrated differently and D-Asp in bio-samples such as rat pineal gland and foods was determined with a simple sample pretreatment and a short analysis run time.

Animal Feed↗
PubMed1999

Direct HPLC analysis of ketoprofen in horse plasma applying an ADS-restricted access-phase.

Making up part of the unique family of restricted access materials (RAM) the Lichrospher ADS (alkyl-diol silica) sorbents have been developed as special packing materials for precolumns used for LC-integrated sample processing of biofluids. The advantage of such phases consists of direct injection of untreated biological fluids without sample clean-up and elimination of the protein matrix together with an on-column enrichment. The plasma samples, with internal standard phenacetin added (not essential), were brought onto the precolumn (C-18 ADS, 25 micron, 25 x 4 mm i.d.) using a phosphate buffer, 0.1 M, pH 7.0. After washing with the buffer, the ADS column was backflushed with the mobile phase phosphate buffer 0. 05 M pH 7.0: acetonitrile (80:20), thus transporting the analytes onto a reversed-phase column Ecocart 125-3 HPLC cartridge with a LiChrocart 4-4 guard column, both packed with LiChrospher 5 micron 100 RP-18; after separation detection was performed in UV at 260 nm. Essential features of the method include the novel precolumn packing, the absence of sample pretreatment, a quantitave recovery, good precision and accuracy, as well as a considerable reduction of analysis time compared to conventional manual methods applied in bioavailability studies.

Animals↗
PubMed1988

Serum neuron-specific/nonneuronal enolase ratio in the diagnosis of neuroblastomas.

Pretreatment samples from 24 children with neuroectodermal tumors (two ganglioneuromas, 22 neuroblastomas) and from 106 others with various tumors were submitted to the enzymatic determination of the serum neuron-specific enolase (NSE). The enzymatic procedure employed in this study allows the systematic determination of the NSE and of the nonneuronal enolase (NNE), thus permitting the calculation of the ratio of the two enolase components. Like results obtained with other procedures, enzymatic determined serum NSE results were raised in a high proportion of Stage IV neuroblastoma (100%) but elevated values also were found in a considerable number of the other tumors (29.2%) like Wilms' tumor, lymphomas, and soft tissue sarcomas. The use of the NSE/NNE ratio which characterizes NSE elevations originating from relative poor or rich sources of NSE, represents an additional index for improving the specificity of the NSE results in the diagnosis of neuroblastomas. With a cutoff value fixed at 7.5%, the specificity of the test is 85.9%. When this limit is fixed at 15%, the specificity reaches 95.3% whereas 81.8% of the results of Stage IV neuroblastomas are still above this value.

Child↗
PubMed2001

Determination of free amino acids in infant food by capillary zone electrophoresis with mass spectrometric detection.

A method for the quantitative determination of free amino acids in infant food samples using capillary electrophoresis (CE) with electrospray-mass spectrometric (ESI-MS) detection is presented. According to the zwitterionic nature of the analytes, two different modes of separation as well as detection were tested; highly acidic carrier electrolytes combined with MS detection in the positive ion mode proved to be the optimum solution. Sensitivity as well as linearity of the method were sufficient to allow the analysis of all solutes of interest in a single run. In this way, free amino acids could be analyzed in a variety of infant food preparations without any sample pretreatment or derivatization step.

Amino Acids↗
PubMed2001

Application of capillary zone electrophoresis with off-line solid-phase extraction to in vitro metabolism studies of antifungals.

A simple and robust solid-phase extraction (SPE) procedure for the cleanup and sample preconcentration of antifungals (ketoconazole, clotrimazole, itraconazole, fluconazole, and voriconazole) and their metabolites after incubation with human liver microsomes, as well as a simplified capillary zone electrophoresis (CZE) method for their rapid analysis, have been developed to determine the stability of these compounds in in vitro samples. Three different sample pretreatment procedures using SPE with reversed-phase sorbents (100 mg C8, 100 mg C18, and 30 mg Oasis-HLB) were studied. The highest and most reproducible recoveries were obtained using a 30 mg Oasis-HLB sorbent and methanol containing 2% acetic acid as eluent. Enrichment by a factor of about four times was achieved by reconstituting the final SPE eluates to a small volume. For the CZE separation, good separations without interfering peaks due to the in vitro matrix were obtained with a simple running electrolyte using a fused-silica capillary. The best separation for all components originated by each tested drug after incubation with human liver microsomes (unmetabolized parent drug and its metabolites) was obtained using a 0.05 M phosphate running buffer (pH 2.2) without additives. The effect of the injection volume was also investigated in order to obtain the best sensitivity. Performance levels in terms of precision, linearity, limits of detection, and robustness were determined.

Acetic Acid↗
PubMed2001

Capillary electrophoresis of proteins 1999-2001.

This review article with 223 references describes recent developments in capillary electrophoresis (CE) of proteins and covers papers published during last two years, from the previous review (V. Dolnik, Electrophoresis 1999, 20, 3106-3115) through Spring 2001. It describes the topics related to CE of proteins including modeling of the electrophoretic properties of proteins, sample pretreatment, wall coatings, improving selectivity, detection, special electrophoretic techniques, and applications.

Electrophoresis, Capillary↗
PubMed2001

Maximization of injection volumes for DNA analysis in capillary electrophoresis.

We report concentration and separation of DNA in the presence of electroosmotic flow (EOF) using poly(ethylene oxide) (PEO) solution. DNA fragments migrating against EOF stacked between the sample zone and PEO solution. To maximize the injection volume, several factors, such as concentrations of Tris-borate (TB) buffer and PEO solution, capillary size, and matrix, were carefully evaluated. The use of 25 mM TB buffers, pH 10.0, containing suitable amounts (less than 10 mM) of salts, such as sodium chloride, sodium phosphate, and sodium acetate, to prepare DNA is essential for the concentration of large-volume samples. In the presence of salts, the peaks also became sharper and the fluorescence intensity of DNA complexes increased. Using 2.5% PEO and a 150 microm capillary filled with 400 mM TB buffer, pH 10.0, up to 5 microL DNA samples (phiX 174 RF DNA-HaeIII digest or the mixture of pBR 322/HaeIII, pBR 328/Bg/I, and pBR 328/HinfI digests) have been analyzed, resulting in more than 400-fold improvements in the sensitivity compared to that by conventional injections (ca. 36 nL). Moreover, this method allows the analysis of 3.5 microL PCR products amplified after 17 cycles without any sample pretreatment.

Buffers↗
PubMed2002

Microfluidic chips for clinical and forensic analysis.

This review gives an overview of developments in the field of microchip analysis for clinical diagnostic and forensic applications. The approach chosen to review the literature is different from that in most microchip reviews to date, in that the information is presented in terms of analytes tested rather than microchip method. Analyte categories for which examples are presented include (i) drugs (quality control, seizures) and explosives residues, (ii) drugs and endogenous small molecules and ions in biofluids, (iii) proteins and peptides, and (iv) analysis of nucleic acids and oligonucleotides. Few cases of microchip analysis of physiological samples or other "real-world" matrices were found. However, many of the examples presented have potential application for these samples, especially with ongoing parallel developments involving integration of sample pretreatment onto chips and the use of fluid propulsion mechanisms other than electrokinetic pumping.

Antibodies↗
PubMed2002

Fast capillary electrochromatographic analysis of parabens and 4-hydroxybenzoic acid in drugs and cosmetics.

A fast capillary electrochromatographic method was developed for the analysis of paraben preservatives in drugs and cosmetics in the presence of their main metabolite and/or impurity, 4-hydroxybenzoic acid. The separation was optimized in a 75 num ID capillary, fully packed with 5 num C18 stationary phase, studying the effects of mobile phase pH and composition (buffer type and organic solvent content). The mobile phase 5 mM ammonium formate, pH 3.0, containing 65% acetonitrile allowed us to obtain the baseline separation of methyl-, ethyl-, propyl-, butyl-, and benzylparabens from a mixture in less than 2.5 min with repeatability and linearity using the short-end injection method (8 cm separation capillary effective length). Under the optimum experimental conditions, the method provided high separation efficiency for parabens, in the range of 129 312-140 325 number of theoretical plates per meter, and analyte quantitation limits (LOQs) in the range of 1.25-2.50 nug/mL. The method was successfully applied to the quantitative analysis of paraben preservatives in pharmaceutical and cosmetic industrial samples with direct injection or after reduced sample pretreatment.

Chromatography, High Pressure Liquid↗
PubMedDate not supplied

The use of sequential extraction procedures for the characterization and management of contaminated soils.

Metal mobility and availability in solid samples, such as soils, sediments and sludges, is frequently studied with sequential extraction procedures, which allow to partition the total metal content into classes of different reactivity. From the experimental point of view, each extraction step requires many practical operations, which can represent sources of error if they are not properly executed. The first part of the paper gives indication on how to perform sequential extractions, from sample pretreatment to extract analysis, in order to reduce experimental errors. The second part describes the main applications of sequential extraction schemes for the characterization and management of contaminated soils: the assessment of the risks associated to metal pollution, the evaluation of the feasibility and efficiency of remediation techniques, the study of soil properties and of metal behaviour and plant availability.

Agriculture↗
PubMed2005

Amyloid-beta oligomers are inefficiently measured by enzyme-linked immunosorbent assay.

Amyloid-beta (Abeta) peptide levels are widely measured by enzyme-linked immunosorbent assay (ELISA) in Alzheimer's disease research. Here, we show that oligomerization of Abeta results in underestimated Abeta ELISA levels. The implications are that comprehensive analysis of soluble Abeta requires either sample pretreatment at denaturing conditions or novel conformation-dependent immunoassays. Our findings might be of relevance for many neurodegenerative disorders in which soluble protein aggregates are the main neurotoxic species.

Alzheimer Disease↗
PubMed2006

Simultaneous determination of essential basic amino acids in pharmaceuticals by capillary isotachophoresis.

Capillary isotachophoresis (ITP) in cationic regime of the separation with conductometric detection has been used for the separation and determination of basic amino acids (arginine, histidine, and lysine) in pharmaceutical preparations. Several electrolyte systems of different compositions and pH were examined. The optimized ITP electrolyte system consisted of 10 mmol/L potassium acetate adjusted to pH 4.0 with acetic acid as the leading electrolyte with the electroosmotic-flow (EOF) suppressing additive, 0.2% (w/v) methylhydroxyethylcellulose (m-HEC), and 10 mmol/L beta-alanine as the terminating electrolyte. The proposed electrophoretic method was successfully validated. It was convenient for the sensitive, simple, rapid and highly reproducible assay of these amino acids. Good quantitation was obtained in short analysis times (a single analysis took about 10 min). The minimal sample pretreatment and low running costs make the proposed ITP method a good alternative to commonly used analytical methods. The obtained results suggest that the proposed method is suitable for routine assay of basic amino acids in pharmaceuticals.

Amino Acids, Essential↗
PubMed1989

A chemiluminescent immunoassay for the direct measurement of urinary 5 alpha-androstane-3 alpha, 17 beta-diol-glucuronide in urine.

We described a chemiluminescent immunoassay (CIA) for 5 alpha-androstane-3 alpha, 17 beta-diol-glucuronide (3 alpha-diol-G) in human diluted urine. This method allowed the direct measurement in 1 microliter of urine avoiding the hydrolysis and extraction steps for sample pretreatment commonly used in routine methods. The hapten 3 alpha-diol-G was synthesized by a Koenigs-Knorr reaction. The immunogenic complex, 3 alpha-diol-G conjugated to bovine serum albumin (BSA), was employed to induce the formation of specific antibodies in New Zealand rabbits. In addition, the required chemiluminescent (CL) tracer was prepared. The characteristics of the antibody was determined as regard to specificity and sensitivity and the precision of the assay methods established. In 22 hirsute women affected by polycystic ovarian syndrome we found 3 alpha-diol-G values significantly (p less than 0.01) higher (146.28 +/- 73.77 micrograms/g of creatinine; mean +/- SD) than those observed in normal women (72.1 +/- 32.58 micrograms/g of creatinine; mean +/- SD).

Adult↗
PubMed1990

A new method for the quantitation of aflatoxin M1 in urine by high performance liquid chromatography and its application to the etiologic study of hepatoma.

A high performance liquid chromatographic (HPLC) method for the analysis of aflatoxin M1 (AFM1) in urine is described. Urine samples were treated with saturated lead acetate and AFM1 was extracted with chloroform. After washing with water to remove impurities the compound was derivatized with trifluoroacetic acid and the AFM1 derivative was analyzed quantitatively by HPLC. The sample pretreatment is simple and more selective. A good line correlation between AFM1 peak height and its concentration was obtained when AFM1 content was in the range of 50-400 pg. The ratio of recovery was 87.42%. Sensitivity is 0.01 ppb. The method is applicable to trace analysis. Results in urine of residents who live in the high/low liver cancer incidence area in Fushui county were the same as that of previous epidemiological investigation.

Aflatoxin B1↗
PubMed1991

Mobile phase versus stationary phase approaches to the direct injection of biological fluids in liquid chromatography.

The liquid chromatographic analysis of drugs in urine through direct injection without any sample pretreatment was extended to micellar chromatography with nonionic surfactants, the Pinkerton ISRP column and the shielded hydrophobic phase (Hisep) column. The feasibility of using each was demonstrated through the determination of the diuretic, hydrochlorothiazide, in urine. Good separation, recovery, precision and linearity, and adequate limits of detection were obtained for this analysis with all three techniques. The advantages and limitations of the mobile phase approach of micellar chromatography and the two stationary phase approaches are discussed for the direct injection of urine as well as other biological fluids.

Chromatography, High Pressure Liquid↗
PubMed2004

High-performance liquid chromatographic determination of Levetiracetam in human plasma: comparison of different sample clean-up procedures.

An accurate and precise high-performance liquid chromatographic method using diode array detection for the determination of the novel antiepileptic, Levetiracetam, has been developed. Three clean-up procedures for the analysis of Levetiracetam in human plasma were implemented and evaluated, namely solid-phase extraction, deproteinization by addition of organic solvents and formation of insoluble salts. Adenosine was used as the internal standard for all three sample pretreatment procedures. Among the several cartridges used for solid-phase extraction, the hydrophilic-lypophilic balance (Oasis) HLB) phase provides the best extraction yield of Levetiracetam, together with high precision. With the two other clean-up procedures involving plasma deproteinization by addition of methanol or zinc sulphate, lower sensitivity and precision of the assays were obtained. However, they are cheaper and faster when compared with the solid-phase extraction procedure.

Anticonvulsants↗