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At least 325 records · Page 18Linked to original sources
PubMed2006

On-capillary derivatization and analysis of amino acids in human plasma by capillary electrophoresis with laser-induced fluorescence detection: application to diagnosis of aminoacidopathies.

A capillary electrophoresis with laser-induced fluorescence detection method for the analysis of free amino acids (AA) in human plasma was developed. A mixture of 16 AA was on-capillary derivatized with 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ) and separated inside the capillary in less than 30 min using 70 mM borax-3.5 mM SDS pH 9.3 as running buffer. Four plasma samples from a healthy donor and patients suffering from phenylketonuria, propionic acidemia, and tyrosinemia type II were studied. Repeatabilities calculated as intra-day RSD (n = 3) values for the AA involved in these aminoacidopathies (glycine, phenylalanine, and tyrosine) were in the range of 0.3 to 1.2% for migration time and 3.7 to 8.2% for peak height. Reproducibilities calculated as inter-day RSD (n = 4) values for the same AA were between 0.7 and 1.4% for migration time and 4.7 and 9.1% for peak height. A fast qualitative analysis allowed the identification of the corresponding disease by comparing the electrophoretic profiles from the patient and the healthy donor and noting the increased level of the specific AA accompanying each individual disease. The results of the quantitative analysis for glycine, phenylalanine, and tyrosine in the plasma samples studied using the developed method showed a good agreement with those provided by the Center of Diagnosis of Molecular Diseases using a standard method for AA analysis.

Amino Acid Metabolism, Inborn Errors↗
PubMed2006

Capillary electrochromatography with zwitterionic stationary phase on the lysine-bonded poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolithic capillary column.

A polymer-based neutral monolithic capillary column was prepared by radical polymerization of glycidyl methacrylate and ethylene dimethacrylate in a 100 mum id fused-silica capillary, and the prepared monolithic column was subsequently modified based on a ring opening reaction of epoxide groups with 1 M lysine in solution (pH 8.0) at 75 degrees C for 10 h to produce a lysine chemically bonded stationary phases in capillary column. The ring opening reaction conditions were optimized so that the column could generate substantial EOF. Due to the zwitterionic functional groups of the lysine covalently bonded on the polymer monolithic rod, the prepared column can generate cathodic and anodic EOF by varying the pH values of running buffer during CEC separation. EOF reached the maximum of -2.0 x 10(-8) m2v(-1)s(-1) and 2.6 x 10(-8) m2v(-1)s(-1) with pH of the running buffer of 2.25 and 10, respectively. As a consequence, neutral compounds, ionic solutes such as phenols, aromatic acids, anilines, and basic pharmaceuticals were all successfully separated on the column by CEC. Hydrophobic interaction is responsible for separation of neutral analytes. In addition, the electrostatic and hydrophobic interaction and the electrophoretic migration play a significant role in separation of the ionic or ionizable analytes.

Electrophoresis, Capillary↗
PubMed2005

Development of a method for quantitative analysis of the major whey proteins by capillary electrophoresis with on-capillary derivatization and laser-induced fluorescence detection.

The main whey proteins have been derivatized on-capillary with 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ) and analyzed using a laboratory-made capillary electrophoresis apparatus provided with a laser-induced fluorescence detector. Several parameters controlling on-capillary derivatization of proteins, including pH, mixing time, reaction time, concentration of the reagents (potassium cyanide and FQ), and reaction temperature, were optimized. Coefficient variations were lower than 1% for migration time and 7% for peak height. Assay detection limits for the different proteins were in the range 5 nM to 10 nM. The method developed was applied to the separation of the major whey proteins in a laboratory-made cheese whey and in an infant food formulated with milk. In addition, the beta-LG content of these samples was quantitated. The results showed good agreement with those given by an RP-HPLC method and with those reported in the literature.

Electrophoresis, Capillary↗
PubMed2005

Ionene-dynamically coated capillary for analysis of urinary and recombinant human erythropoietin by capillary electrophoresis and online electrospray ionization mass spectrometry.

In this article, a series of ionene polymers were synthesized and used to coat fused-silica capillaries for the separation of recombinant and urinary human erythropoietin (rhEPO and uEPO) standards by CE. The influence of the charge density of coatings on the separation of rhEPO and uEPO glycoforms was investigated. Then, we further studied the method for fast separation and detection of rhEPO and uEPO standards by CE-ESI-MS. The influence of several CE and MS operating parameters, such as the concentration of CE running buffer, applied external pressure, and the composition and flow rate of sheath liquid on CE-ESI-MS was studied. The results demonstrated that when the capillary was permanently coated with 6,6-ionene and the pH value of acetic acid-ammonium acetate running buffer was 4.80 and 5.50, respectively, a significantly reproducible separation was achieved for rhEPO and uEPO glycoforms. In the online CE-ESI-MS experiments, we not only achieved the online MS signal of uEPO, but also obtained baseline separation of three major rhEPO glycoforms successfully and reproducibly on the 6,6-ionene-coated capillaries. Furthermore, the standard mixture of rhEPO and uEPO was separated, and two incompletely resolved peaks that were identified to be rhEPO and uEPO by the unique MS "fingerprint" were obtained. Additionally, the molecular weight of rhEPO and uEPO were verified and compared to the results by MALDI-TOF-MS. It can be concluded that, in contrast to other indirect methods, the online CE-ESI-MS technique with the combination of the advantages of both CE and MS shows great potential for the separation and detection of rhEPO doping directly in competitive sports.

Electrophoresis, Capillary↗
PubMed2006

Capillary isoelectric focusing of microorganisms in the pH range 2-5 in a dynamically modified FS capillary with UV detection.

The isoelectric points of many microbial cells lie within the pH range spanning from 1.5 to 4.5. In this work, we suggest a CIEF method for the separation of cells according to their isoelectric points in the pH range of 2-5. It includes the segmental injection of the sample pulse composed of the segment of the selected simple ampholytes, the segment of the bioanalytes and the segment of carrier ampholytes into fused silica capillaries dynamically modified by poly(ethylene glycole). This polymer dissolved in the catholyte, in the anolyte and in the injected sample pulse was used for a prevention of the bioanalyte adsorption on the capillary surface and for the reduction of the electroosmotic flow. Between each focusing run, the capillaries were washed with the mixture of acetone/ethanol to achieve the reproducible and efficient CIEF. In order to trace of pH gradients, low-molecular-mass pI markers were used. The mixed cultures of microorganisms, Escherichia coli CCM 3954, Candida albicans CCM 8180, Candida parapsilosis, Candida krusei, Candida glabrata, Candida tropicalis, CCM 8223, Proteus vulgaris, Klebsiela pneumoniae, Staphylococcus aureus CCM 3953, Streptococcus agalactiae CCM 6187, Enterococcus faecalis CCM 4224 and Staphylococcus epidermidis CCM 4418, were focused and separated by the CIEF method suggested here. This CIEF method enables the separation and detection of the microbes from the mixed cultures within several minutes. The minimum detectable number of microbial cells was less than 10(3).

Electrophoresis, Capillary↗
PubMed1996

Capillary isoelectric focusing and sodium dodecyl sulfate-capillary gel electrophoresis of recombinant humanized monoclonal antibody HER2.

Capillary isoelectric focusing (cIEF) and IEF of recombinant humanized monoclonal antibody HER2 (rhuMAbHER2) show five charged isoforms with estimated pI values ranging from 8.6-9.1. The cIEF assay demonstrated good precision with relative standard deviations (R.S.D.) 0.7-3.7% and 0.4-4.2% for intra and interassay analysis, respectively. The method was linear for the area of the main peak over the concentration range 2-250 micrograms/ml with a Pearson correlation coefficient > 0.99. The limit of detection for the main peak was determined to be 2 ppm. With both sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) and SDS-polyacrylamide gel electrophoresis, the nonreduced rhuMAbHER2 migrated as a single major peak with minor peaks in the aggregate and clip regions. After reduction, the electropherogram and the slab gel showed the expected heavy chain and light chain fragments with minor peaks in the aggregate and clip regions. The SDS-CGE assay showed good precision with R.S.D. values of 0.1-7.8% and 0.1-8.1% for intra and interassay analysis, respectively. The Pearson correlation coefficient for the area of the main peak was > 0.99 demonstrating linearity for the concentration range 0.5-500 micrograms/ml. The limit of detection for intact rhuMAbHER2 was determined to be 0.5 ppm. The data presented demonstrates the feasibility of replacing the slab gel techniques with capillary electrophoresis in a quality control environment.

Antibodies, Monoclonal↗
PubMed1986

The shape of renal vasa recta capillaries and its effect on calculation of single capillary blood flow.

Previous studies of the renal papilla of the rat have suggested that the vasa recta capillaries can be well approximated by elliptical cylinders (C. Holliger, K. V. Lemley, S. L. Schmitt, F. C. Thomas, C. R. Robertson, and R. L. Jamison, 1983, Circ. Res., 53, 401-413). This hypothesis was validated in a morphological study employing several methods of specimen fixation and preparation. Papillas of young (body wt = 90 g) Wistar rats were fixed and subsequently examined by light and electron microscopy. Cross-sectional shapes and orientations were determined for 300 superficial vasa recta. The ratio, beta, of vessel cross-sectional major-axis-to-minor-axis lengths was 1.39 +/- 0.24 (SD). Values of beta greater than 1.0 (the value expected for circular vessels) could not be accounted for by either fixation artifact or the angle of histologic sectioning of the papillas. A quantitative estimate of the relationship between the apparent capillary diameter measured in vivo and the capillary cross-sectional area was made using a mathematical model which accounts for cross-sectional shapes and orientations of the vasa recta. This estimate implies that current methods of calculating single vas rectum blood flow using apparent diameters and blood velocities determined in vivo probably overestimate actual blood flow by about 25%.

Animals↗
PubMed2004

Assay of phenolic compounds in red wine by on-line combination of capillary isotachophoresis with capillary zone electrophoresis.

The on-line combination of capillary zone electrophoresis (CZE) with capillary isotachophoresis (ITP) increases significantly the separation capability and sensitivity of capillary electrophoresis. This technique was used for separation and quantification of fourteen selected natural constituents in red wine belonging to flavonoids and phenolic acids. The leading electrolyte (LE) in the ITP pre-separation step was 10 mM HCl of pH* 7.2 with Tris as counterion, the terminating electrolyte (TE) was 50 mM boric acid of pH* 8.2 (adjusted with barium hydroxide). The background electrolyte in the electrophoretic step contained 25 mM beta-hydroxy-4-morpholinopropanesulfonic acid (MOPSO), 50 mM Tris, 15 mM boric acid and 5 mM beta-cyclodextrin of pH* 8.5. The content of methanol in all electrolytes was 20% (v/v). For exact timing of the transfer of isotachophoretically stacked analyte zones into the CZE column and for the control of the residual amount of leading and terminating ITP electrolytes picric acid was used as coloured marker. The R.S.D. values (n = 6) ranged between approximately 0.1% (for 0.25 microg ml(-1) rutin) and approximately 11% (for 0.25 microg ml(-1) of quercitrin). Detection limits were 30 ng mi(-1) for phenolic acids, quercitrin and rutin, 100 ng ml(-1) for quercetin, kaempferol and epicatechin and 250 ng ml(-1) for catechin. A single analysis took 45 min.

Electrophoresis↗
PubMed2004

In-capillary derivatization approach applied to the analysis of insulin by capillary electrophoresis with laser-induced fluorescence detection.

An in-capillary derivatization procedure of insulin for its subsequent capillary electrophoretic analysis (with laser-induced fluorescence detection) was developed. The in-capillary derivatization performed using the 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) in a borate buffer pH 8.9, was achieved by successive introduction of plugs of sample and AQC reagent followed by application of a voltage (30 kV). Derivatization reaction results from the differential transport velocities that permit the distinct zones to penetrate each other under the applied field. Reagent/sample molar ratio (Rm) and plug lengths ratio were shown to have an influence on the efficiency of the derivatization reaction. A single peak could be obtained with a high reagent/sample molar ratio (Rm > or = 68). The tagged derivative peak intensity and efficiency were improved when reagent solution time injection was at least twice higher than that of insulin sample. The validation of the method showed a good linearity between the corrected area of the derivative peak and insulin concentrations. The relative standard deviations of the migration times and the corrected areas obtained for the tagged derivative were 2.3 and 4.6%, respectively. An efficient derivatization and separation of a mixture of insulin and two glycated forms of insulin was obtained using the technique.

Electrophoresis, Capillary↗
PubMed2005

Amino acids determination using capillary electrophoresis with on-capillary derivatization and laser-induced fluorescence detection.

Free amino acids have been derivatized on-capillary with 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ) and analyzed using a laboratory-made capillary electrophoresis apparatus with laser-induced fluorescence detection. Several parameters that control on-capillary derivatization of amino acids, including pH, mixing time, reaction time, concentration of the derivatization reagents (potassium cyanide and FQ) and solvent of FQ, as well as the temperature of mixing and reaction were optimized. Repeatabilities better than 1.8% for migration time and 7.8% for peak height were obtained. Assay detection limits for the different amino acids ranged from 23 nM for glycine to 50 nM for lysine and glutamic acid. The methods developed were applied to the analysis of several amino acids in pharmaceutical preparations and plasma samples. Results showed a good agreement with those obtained using an amino acid autoanalyzer for the same samples.

Amino Acids↗
PubMed2006

Direct comparison of capillary electrophoresis and capillary liquid chromatography hyphenated to collision-cell inductively coupled plasma mass spectrometry for the investigation of Cd-, Cu- and Zn-containing metalloproteins.

Capillary liquid chromatography (cLC) and capillary electrophoresis (CE) have been critically compared for the separation of metalloproteins when using collision-cell inductively coupled plasma mass spectrometry (ICP-CC-MS) as detection system. For cLC separation, the selected column was a C8 (0.3 mm I.D.) and the separation conditions involved a gradient up to 80% methanol in 10mM ammonium acetate buffer (pH 7.4). The low flow rate used (3 microL min(-1)) permitted the utilization of a high methanol content maintaining the sensitivity along the whole chromatographic run. For this purpose, a new low-flow interface has been developed based on a total consumption nebulizer. Similarly, CE has been studied as separation technique using a 75 microm I.D. fused silica capillary and a running buffer of 20 mM Tris-HNO3 (pH 7.4) and working at 30 kV. Metallothionein (mixture of MT-I and -II) and superoxide dismutase (SOD) have been used as protein models in order to evaluate the separation/detection capabilities using the same injection volumes in both systems (20 nL). For both hybrid systems, separation parameters such as retention factor, numbers of theoretical plates, tailing factor and resolution have been critically compared. Also, the analytical performance characteristics of both hybrid systems have been evaluated and tested by analyzing the Cu-, Zn-species present in red blood cell extracts in order to explore more adequate separation methodology for the analysis of metalloproteins in complex matrices.

Animals↗
PubMed2005

Enantiomeric purity determination of tamsulosin by capillary electrophoresis using cyclodextrins and a polyacrylamide-coated capillary.

The chiral separation of racemic tamsulosin hydrochloride (TH) was carried out using cyclodextrin (CD)-mediated capillary electrophoresis (CE) with DAD at 200 nm. The best separation of enantiomers of the studied compound was achieved at 20 kV with 30 cm x 50 microm I.D. polyacrylamide (PAA)-coated fused-silica capillary (effective length 20 cm) and running buffer with sulfated-beta-CD (S-beta-CD) as chiral selector. Other selected native or derivatized CDs were also tested: beta-CD (5, 15 mmol l(-1)), carboxymethyl-beta-CD (5, 30 mmol l(-1)), dimethyl-beta-CD (15 mmol l(-1)) and hydroxypropyl-beta-CD (5, 30 mmol l(-1)). Several parameters such as capillary pretreatment, buffer type and concentration, pH of background electrolyte, methanol content, separation temperature and voltage, were optimized. The excellent baseline separation of chiral TH was successfully achieved within 12 min using 100 mmol l(-1) phosphate buffer with pH 2.5 containing 1.7 mmol l(-1) S-beta-CD. Rectilinear calibration range was 50.0-500.0 mumol l(-1) of each enantiomer (r = 0.9993-0.9996). The method was applied to the assay of R-TH in Omnic, capsules (nominal content 0.4 mg per capsule) with R.S.D. 2.75% (n = 6), recovery 99.3-101.7% and it was suitable for the chiral purity control of the active enantiomer in the pharmaceutical.

Acrylic Resins↗
PubMed2005

On-line combination of capillary isoelectric focusing and capillary non-gel sieving electrophoresis using a hollow-fiber membrane interface: a novel two-dimensional separation system for proteins.

A novel two-dimensional (2D) separation system for proteins was reported. In the system, a piece of dialysis hollow-fiber membrane was employed as the interface for on-line combination of capillary isoelectric focusing (CIEF) and capillary non-gel sieving electrophoresis (CNGSE). The system is similar equivalent to two-dimensional polyacrylamide gel electrophoresis (2D PAGE), by transferring the principal of 2D PAGE separation to the capillary format. Proteins were focused and separated in first dimension CIEF based on their differences in isoelectric points (pIs). Focused protein zones was transferred to the dialysis hollow-fiber interface, where proteins hydrophobically complexed with sodium dodecyl sulfate (SDS). The negatively charged proteins were electromigrated and further resolved by their differences in size in the second dimension CNGSE, in which dextran solution, a replaceable sieving matrix instead of cross-linked polyacrylamide gel was employed for size-dependent separation of proteins. The combination of the two techniques was attributed to high efficiency of the dialysis membrane interface. The feasibility and the orthogonality of the combined CIEF-CNGSE separation technique, an important factor for maximizing peak capacity or resolution elements, were demonstrated by examining each technique independently for the separation of hemoglobin and protein mixtures excreting from lung cancer cells of rat. The 2D separation strategy was found to greatly increase the resolving power and overall peak capacity over those obtained for either dimension alone.

Animals↗
PubMed2001

Capillary electrophoretic determination of the constituents of Artemisiae Capillaris Herba.

Two capillary electrophoretic methods, a micellar electrokinetic electrophoretic (MEKC) one and a capillary zone electrophoretic (CZE) one, were developed for the separation of 12 constituents in Artemisiae Capillaris Herba. Detection at 254 nm with 20 mM sodium dodecyl sulfate and 20 mM sodium borate buffer (pH 9.82) in MEKC or with 25 mM sodium borate and 6.75 mg/ml 2,3,6-tri-O-methyl-beta-cyclodextrin buffer in CZE was found to be the most suitable approach for this analysis. Within 42 min, the MEKC method could successfully separate 12 authentic constituents, whereof chlorogenic acid, however, appeared as a broad and split peak, and capillarisin and chlorogenic acid overlapped partially with other coexisting substances in crude extract of the herb. The CZE method could completely overcome these problems and was used to determine the amounts of capillarisin, chlorogenic acid, scopoletin and caffeic acid in the extract. The effect of buffers on the constituent separation and the validation of the two methods were discussed.

Asteraceae↗
PubMed2001

New configuration in capillary isotachophoresis-capillary zone electrophoresis coupling.

The paper surveys possible configurations of a coupling capillary column operating in various electromigration modes. Special attention is given to capillary isotachophoresis-capillary zone electrophoresis (cITP-CZE) coupling and its description from the theoretical point of view. Computer simulations of separation are presented and compared with experiments. Further, we propose a new configuration of electrolyte systems in cITP-CZE coupling, which offers a possibility to perform complex analyses of micro- and macro-constituents in one run. The electrolyte system is verified by practical experiments for both anionic and cationic modes of analysis. The advantages and disadvantages of the new combination are discussed.

Beverages↗
PubMed2001

Post-capillary reaction detection in capillary electrophoresis based on the streptavidin-biotin interaction. Optimization and application to single cell analysis.

A class-selective post-capillary reaction detection method for capillary electrophoresis is described in which a streptavidin-fluorescein isothiocyanate (streptavidin-FITC) conjugate is used to detect biotin moieties. The selective binding of biotin moieties to the streptavidin-FITC conjugate causes an enhancement of fluorescence proportional to the concentration of biotin present. After capillary electrophoresis the separated analytes react with streptavidin-FITC in a coaxial reactor and are then detected either by a benchtop spectrofluorometer (2.5 microM detection limit) or by an epi-fluorescence microscope (1 x 10(-7) M detection limit). The method is used to examine biotinylated species in a crude mammalian cell lysate which was found to contain 83+/-3 fmol in 3600 cell volumes. In addition, it is used to examine the uptake of biotin by individual sea urchin oocytes. The results indicate that, in the oocytes, biocytin is the prevalent form of biotin and its concentration varies widely between cells (mean=2+/-2 microM).

Animals↗
PubMed2001

Capillary electrochromatographic enantioseparations using a packed capillary with a 3 microm OD-type chiral packing.

Enantiomer separations by capillary electrochromatography (CEC) using a packed capillary were investigated. As a chiral stationary phase, an OD-type packing material of 3 microm particle diameter, which is a silica-gel based material coated with cellulose tris(3,5-dimethylphenylcarbamate), was employed. The chiral packing was packed into a 100 microm I.D. fused-silica capillary by a pressurized method. Several racemic enantiomers were successfully resolved with acidic or basic buffers containing acetonitrile as mobile phases. The separation efficiencies obtained in this chiral CEC system were superior to those in the previously reported chiral CEC system using 5 microm particles. The typical plate heights obtained for several enantiomers were 4.6-6.6 microm or reduced plate heights of 1.5-2.2. Good repeatabilities in the migration time, peak height, and corrected peak area were observed as well as for the plate number. As an application study of this CEC system, an optical purity test was carried out by using benzoin enantiomers. A 1% content of one enantiomer in the other enantiomer was successfully separated and detected.

Chromatography, Micellar Electrokinetic Capillary↗
PubMed2001

Capillary electrophoretic determination of sulfite using the zone-passing technique of in-capillary derivatization.

A new capillary electrophoretic (CE) method was developed for the simple and selective determination of sulfite. The proposed method is based on the in-capillary derivatization of sulfite with iodine using the zone-passing technique and direct UV detection of iodide formed. The optimal conditions for the separation and derivatization reaction were established by varying concentration of iodine, electrolyte pH and applied voltage. The optimised separations were carried out in 20 mmol l(-1) Tris-HCl electrolyte (pH 8.5) using direct UV detection at 214 nm. Experimental results showed that the injection of the iodine zone from anodic end of the capillary gives significantly better precision. Common UV absorbing anions such as Br-, l-, S2O3(2-), NO3-, NO2-, SCN- did not give any interferences. Valid calibration (r2=0.998) is demonstrated in the range 1 x 10(-5) - 8 x 10(-4) mol l(-1) of sulfite. The detection limit (SIN=3) was 2 x 10(-6) mol l(-1). The proposed system was applied to the determination of free sulfite in wines. The recovery tests established for wine samples were within the range 92-103%. The CE results were compared with those obtained by iodometric titration technique.

Electrophoresis, Capillary↗