Comparison of high-performance liquid chromatography and capillary zone electrophoresis in penciclovir biodegradation kinetic studies

High-performance liquid chromatography (HPLC) and capillary zone electrophoresis (CZE) were used in biodegradation kinetic studies. This paper describes a rapid penciclovir separation using CZE with detection limits comparable to HPLC. The ionic-strength mediated stacking technique was employed while good resolution was maintained. With a shorter analysis time, comparable detection limits and no organic solvent consumption, CZE is a better method for penciclovir biodegradation studies than conventional reversed-phase HPLC (RP-HPLC).     

A Capillary Electrophoresis-Based Assay for Protein Kinases and Protein Phosphatases Using Peptide Substrates

A novel procedure for detection and assay of protein kinase and phosphatase activities in complex biological mixtures was developed. By means of capillary zone electrophoresis (CZE) methodology, the phosphorylated and dephosphorylated forms of the peptide Kemptide, a 46-amino-acid fragment from protein phosphatase inhibitor-1 and a peptide fragment corresponding to the R_II subunit of cAMP-dependent protein kinase (PKA), were rapidly resolved. This facilitated nonradioactive detection of PKA and protein phosphatase-2B (calcineurin) in rabbit skeletal muscle extracts. In addition, the CZE procedure enabled a site-specific assay of a 14-amino-acid peptide from the glycogen-binding subunit of protein phosphatase-1 monophosphorylated on distinct sites by PKA and casein kinase-II. These results suggest that CZE may prove to be extremely useful for the analysis of peptides that are phosphorylated at multiple sites in vivo.     

Determination of amino acids in fodders and raw materials using capillary zone electrophoresis

Two schemes were offered for analysis of amino acid contents in fodders and raw materials for mixed fodders by capillary zone electrophoresis (CZE). The first variant provides express analysis of four technologically important amino acids (lysine, methionine, threonine, cystine) in borate buffer on characteristic absorption of aminogroup (190 nm), with limits of quantitation being on average 0.2%. The second scheme includes pre-capillary derivatization of amino acids using phenylisothiocyanate (PITC) and separation of phenylthiocarbamyl (PTC)-derivatives obtained by CZE with a detection on 254 nm, which allows to widen a list of detectable components up to 19 (without tryptophan) and significantly improve detection limits down to 0.01%. Acid hydrolysis was used for a sample preparation. The results of analysis of fodders were compared using such methods, as CZE, ion exchange chromatography (amino acid analyzer) and reversed-phase (RP)-HPLC (with gradient technique of elution).     

Evaluation of capillary electrophoresis with post-column derivatization and laser-induced fluorescence detection for the determination of substance P and its metabolites

A method for the detection of substance P and its metabolites using capillary electrophoresis with post-capillary derivatization and laser-induced fluorescence detection is described. The post-capillary chemical derivatization system employs naphthalene-2,3-dicarboxaldehyde and β-mercaptoethanol. Two reactor designs were evaluated for the determination of substance P and its metabolites. The fluorescent spectroscopic properties of the derivatives under optimal separation conditions were also examined. The final system was evaluated for the investigation of substance P metabolism in brain following perfusion of the striatum with substance P using microdialysis sampling.     

Comprehensive protein profiling by multiplexed capillary zone electrophoresis using cross-linked polyacrylamide coated capillaries

We have recently developed a new process to create cross-linked polyacrylamide (CPA) coatings on capillary walls to suppress protein-wall interactions. Here, we demonstrate CPA-coated capillaries for high-efficiency (>2 x 10(6) plates per meter) protein separations by capillary zone electrophoresis (CZE). Because CPA virtually eliminates electroosmotic flow, positive and negative proteins cannot be analyzed in a single run. A "one-sample-two-separation" approach is developed to achieve a comprehensive protein analysis. High throughput is achieved through a multiplexed CZE system.     

Determination of poorly separated monoclonal serum proteins by capillary zone electrophoresis

A capillary zone electrophoresis (CZE) technique was developed for the determination of poorly separated monoclonal serum proteins by agarose gel electrophoresis (AGE). A P/ACE 5500 capillary instrument (Beckman) was used under the following conditions: 57 cm x 50 microm I.D. fused-silica capillary, pH 9.6 borate buffer, and 214 nm on-line detection. Sixty patients (61 +/- 13 years) with a well isolated (n=24, group A) or poorly separated monoclonal band(s) by AGE (n=36, group B) were included in this study. Within- and between-run precision for CZE was below 4% for albumin and 7% for gamma-globulin. A 100% (group A) or 61% agreement (group B, more bands detected by CZE in 10 cases) was obtained between CZE and AGE for the number of monoclonal bands. In group B, quantification was possible in 92% of samples by CZE vs. 64% by AGE (P<0.05, chi-square). The proposed CZE method appears as an additional helpful technique for the determination of poorly separated monoclonal serum proteins by AGE.     

Simultaneous determination of phthalate di- and monoesters in poly(vinylchloride) products and human saliva by gas chromatography-mass spectrometry

This paper reports on the selectivity behaviour of tryptic peptides on a Cu(2+)-loaded immobilised metal ion affinity chromatography (IMAC) support. Ovalbumin was chosen as a model protein for investigation of the selection and separation of histidine-containing peptides by IMAC off-line coupled with capillary electrophoresis and matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF). Two of five histidine-containing peptides in addition to some non-histidine-containing peptides from a tryptic digest of ovalbumin were captured by IMAC. To separate and purify the selected peptides, the IMAC sample was analysed by capillary zone electrophoresis (CZE). The sample was not separated by capillary zone electrophoresis, therefore, micellar electrokinetic chromatography (MEKC) using 10-75 mM SDS was used. Analysis of IMAC sample by MEKC, using low concentrations of SDS (10 mM) was characterised by MALDI-TOF. When using SDS at 75 mM, the migration times of reversed-phase fractions of the IMAC sample, were used to identify the peaks. One of the two selected histidine-containing peptides with two histidine residues was identified, analysing the sample by CZE or MEKC.     

On-line combination of monolithic immobilized pH gradient-based capillary isoelectric focusing and capillary zone electrophoresis via a partially etched porous interface for protein analysis

An integrated platform consisting of monolithic immobilized pH gradient-based capillary isoelectric focusing (M-IPG CIEF) and capillary zone electrophoresis (CZE) coupled by a partially etched porous interface was established. Since carrier ampholytes (CAs) were immobilized on monolith in M-IPG CIEF to form a stable pH gradient, subsequent depletion of CAs at the interface to prevent the interference on CZE separation and detection were avoided. Moreover, a partially etched porous capillary column, which was facile for fabrication and durable for operation, was exploited as the interface to combine M-IPG CIEF and CZE. The RSD values in terms of the migration time for M-IPG CIEF separation, transfer protein from the first dimension to the second dimension, and CZE separation, were 2.4%, 3.9% and 2.3%, respectively. With a 6-protein mixture as the sample, two-dimensional capillary electrophoresis (2D-CE) separation was successfully completed within 116 min, yielding a peak capacity of ～200 even with minute sample amount down to 5.0 μg/mL. The limit of detection was 0.2 μg/mL. In addition, proteins extracted from milk were used to test the performance of such a 2D-CE separation platform. We expect that such a novel 2D-CE system would provide a promising tool for protein separation with high throughput and high peak capacity.     

Direct determination of diuretic drugs in urine by capillary zone electrophoresis using fluorescence detection

Four diuretic drugs banned in sport (amiloride, triamterene, bendroflumethiazide and bumetanide) have been separated by capillary zone electrophoresis (CZE) and detected using conventional fluorescence spectrometry. The effect of pH on electrophoretic parameters such as migration time, peak efficiency and peak height is discussed. Complete separation of the four drugs is achieved in less than 8 min at pH 8. No interference due to endogenous urine components is observed and thus direct urine analysis is feasible. Analytical figures of merit including precision and limits of detection are presented. Limits of detection range between 0.5 fmol for triamterene and 21.6 fmol for bumetanide.     

Methyl malondialdehyde as an internal standard for the determination of malondialdehyde

Methyl malondialdehyde (Me-MDA) is suggested as an internal standard for the determination of the lipid peroxidation product, malondialdehyde (MDA). A procedure for synthesising the Me-MDA sodium salt is described in detail. The purity and identity of the synthesised Me-MDA have been confirmed using nuclear magnetic resonance and UV spectroscopy, and by micellar electrokinetic chromatography. The applicability of Me-MDA as an internal standard has been demonstrated for rat brain homogenate samples. These samples were purified solely through ultrafiltration. The preferred analytical technique was capillary zone electrophoresis (CZE) with UV detection at 267 nm. The limits of detection (3 S/N) for the CZE separations of Me-MDA and MDA were 0.5 and 0.2 μM, respectively, and the total analysis time was approximately 10 min. Details of separations are also presented using high-performance liquid chromatography (HPLC) with UV detection at 245 nm, and gas chromatography, together with either electron capture or mass spectrometric detection. The GC separations require derivatisation of MDA and Me-MDA with pentafluorophenylhydrazine while the CZE and HPLC separations can be performed on the native molecules.     

Nonradioactive monitoring of organic and inorganic solute transport into single Xenopus oocytes by capillary zone electrophoresis.

Transport of organic and inorganic solutes into and out of cells requires specialized transport proteins. Given a sufficiently sensitive analytical method for measuring cellular solute concentrations, it should be possible to monitor solute transport across the plasma membrane at the level of single cells. We report a capillary zone electrophoresis approach that is generally applicable to monitor solute transport into Xenopus laevis oocytes, requires only nanoliters of sample, and involves no radioactive materials. The sensitivity of capillary electrophoresis with UV detection is typically on the order of 10(-5)-10(-6) M, resulting in the mass detection limits in the low femtomole range. We show that capillary zone electrophoresis serves as a simple technique to measure solute transport into oocytes. Studies of the mammalian oligopeptide transporter PepT1 and the Na(+)- and K(+)-coupled epithelial and neuronal glutamate transporter EAAC1 expressed in oocytes demonstrate that transport of the dipeptide Trp-Gly via PepT1 and transport of Na+ and K+ via EAAC1 across the oocyte plasma membrane can be monitored by measuring intracellular tryptophan absorption and by indirect UV detection of inorganic ions, respectively. The CZE method allowed the simultaneous detection of changes of intracellular Na+ and K+ concentrations in response to EAAC1-mediated Na+ cotransport and K+ countertransport. This is the first report of a capillary zone electrophoresis-based quantitative analysis of intracellular components of a single cell in response to transport activity.     

Interaction study between double-stranded DNA and berberine using capillary zone electrophoresis

Two non-self-complementary 17-mer double-stranded DNA (dsDNA) with four different central base pairs were designed to systematically investigate the binding affinity and sequence specificity of berberine with dsDNA by capillary zone electrophoresis (CZE). The data analysis with the Kenndler model proved only low affinity between dsDNA and berberine and suggested some weak binding preference of berberine for AATT-containing to GGCC-containing dsDNA. The binding constant, Ka, between berberine and dsDNA(AB) was about (1.0 +/- 0.7) x 10(3) M(-1). In addition, the separation of single-stranded DNA (ssDNA) from dsDNA under simple electrophoretic conditions enabled CZE to be a potentially alternative tool to check the extent of DNA annealing, which is usually done by the time-consuming and labor-intensive slab electrophoresis.     

Heart-cut two-dimensional separation method via hyphenation of micellar electrokinetic capillary chromatography and capillary zone electrophoresis using analyte focusing by micelle collapse

A novel two-dimensional (2D) separation method, which hyphenated micellar electrokinetic capillary chromatography (MEKC) and capillary zone electrophoresis (CZE), was developed for analysis of flavonoids in Leonurus cardiaca. The Leonurus cardiaca sample was separated and purified in first dimension by MEKC. Then only a selected portion of the first dimension separation was transferred into the second dimension by pressure. Finally, the zone of flavonoids was separated by CZE. As the key to successful hyphenation of MEKC and CZE, an analyte focusing by micelle collapse (AFMC) concentration method was employed between the two dimensions to release analytes from the micelle interior to a liquid zone and to overcome the sample zone diffusion caused by mobilization pressure. The whole heart-cut 2D separation process can be performed in a conventional CE analyzer. The relative standard deviation of peak height, peak area and migration time were in the range of 2.3-4.2%, 1.5-3.8% and 3.6-5.5%, respectively, and detection limits (S/N=3) were 15-55 ng/mL. The new methodology was applied with success for the flavonoids separation of Leonurus cardiaca.     

Capillary electrophoresis determination of the binding affinity of bioactive sulfated polysaccharides to proteins: study of the binding properties of fucoidan to antithrombin

The interaction of proteins with polysaccharides represents a major and challenging topic in glycobiology, since such complexes mediate fundamental biological mechanisms. An affinity capillary electrophoresis method has been developed to evidence the complex formation and to determine the binding properties between an anticoagulant polysaccharide of marine origin, fucoidan, and a potential target protein, antithrombin. This method is a variant of zonal electrophoresis in the mobility shift format. A fixed amount of protein was injected into a capillary filled with a background electrolyte containing the polysaccharide in varying concentrations. The effective mobility data of the protein were processed according to classical linearization treatments to obtain the binding constant for the polysaccharide/antithrombin complex. The results indicate that fucoidan binds to antithrombin in a 1:1 stoichiometry and with an affinity depending on the molecular weight of the polysaccharide. For heparin, the binding constant obtained similarly is in accordance with the literature. This is the first report showing the implementation of a capillary electrophoresis method contributing to the mechanistic understanding of the biological activities of fucoidan and providing evidence for the complex formation between fucoidan and the protein inhibitor of the coagulation antithrombin.     

Determination of salicylic acid in human serum with capillary zone electrophoresis

The determination of salicylic acid (SA), a metabolite of aspirin, in human serum was developed using capillary zone electrophoresis (CZE) with diode array detection. The reproducibility of separation and quantification with CZE analysis of the extract of SA from human serum was appropriate for the intra- and inter-day assay coefficients. A high correlation was revealed between the serum SA levels in volunteers determined by CZE and those determined by a fluorescence polarization immunoassay (r=0.973, n=12), although the former values were slightly higher than the latter. There were no peaks interfering with the assay of SA by internal standard method. This CZE method could provide a simple and efficient method for monitoring SA in patients.     

Capillary zone electrophoresis method development for the analysis of Hippeastrum hybrid agglutinin samples

A capillary zone electrophoresis (CZE) method was developed aiming the analysis of Hippeastrum hybrid agglutinin (HHA) samples. HHA is presently being tested as a vaginal microbicide to prevent HIV transmission. It acts by direct binding to mannose residues that are abundantly present on the HIV gp120 envelope and so interrupts the virus entry process. The final CZE method employs 50 mM sodium tetraborate (pH 9.9) as background electrolyte. In this condition, a cluster of about 30 isoform peaks is obtained, with very repeatable patterns. RSDs in the order of 0.2% for the migration time and detection sensitivity in the order of 70 μg ml^?1 were achieved.     

Carrier ampholytes as potential buffers in electrophoresis: physico-chemical study

Joule heating is a limiting factor when separating proteins in capillary zone electrophoresis (CZE). Low conductivity buffers, are required for high-speed separations. We investigated the use of carrier ampholytes (CA) as background electrolytes (BGE) in CZE. We prepared 25 "narrow pH cuts" of wide pH range (3-10) CA mixture in order to know if these fractions were suitable to be used as BGE in CZE. Each fraction was characterised by CZE analysis, giving an idea of its heterogeneity (number and relative abundance of molecular ampholytes). Conductivities and buffering capacities of each fraction have been also measured. Our conclusion is that "narrow pH cuts" of CA might be well suited buffers for electrophoretic separations.     

Determination of heterocyclic amines by capillary electrophoresis with UV-DAD detection using on-line preconcentration

This paper proposed a method with capillary zone electrophoresis (CZE) for analysis of eight heterocyclic amines in a commercial meat matrix. The influence of composition, pH and concentration of buffer, and applied voltage were investigated. A 5 mmol/L formic acid-ammonium formate solution at pH 2.20 was chosen as the running electrolyte. Also several solid-phase extraction actions were performed for the clean-up of the samples. With 3s hydrodynamic injection, detection limits ranging from 0.554 to 1.783 microg/g was obtained. To improve sensitivity, field-amplified sample injection (FASI) was used with the conditions of 3 s hydrodynamic injection of a water plug and 25 s electrokinetic injection of the sample. And methanol-water (1:1 in volume) was applied as the sample solvent. Under above conditions, detection limits ranged from 1.329 to 19.39 ng/g, which were at least 50 times lower than those with hydrodynamic injection.     

Small molecular ion adsorption on proteins and DNAs revealed by separation techniques

Ion binding is a term that assumes that the ion is included in the solvation sphere characterising the biomolecule. The binding forces are not clearly stated except for electrostatic attraction; weak forces (hydrogen bonds and Van der Waals forces) are likely involved. Many publications have dealt with ion binding to proteins and the consequences over the past 10 years, but only a few studies were performed using high-performance liquid chromatography (HPLC: ion exchange, reversed phase without the well-identified immobilised metal affinity chromatography) and capillary zone electrophoresis (CZE). This review focuses on the binding of proteins and DNAs mainly to the oxyanions (phosphate, borate, citrate) and amines used as buffers for both the HPLC eluent and the background electrolyte of CZE. Such specific ion adsorption on biomolecules is evidenced by physico-chemical characteristics such as the mobility or retention volume, closely associated with the net charge, which differ from the expected or experimental data obtained under the conditions of an indifferent electrolyte. It is shown that ion binding to proteins is a key parameter in the electrostatic repulsion between the free protein and a fouled membrane in the ultrafiltration separation of a protein mixture.     

Separation and detection of vitellogenin in fish plasma by capillary zone electrophoresis

A method for coupling capillary zone electrophoresis (CZE) with rapid membrane chromatography purification (RMCP) was established for the analysis of vitellogenin (VTG) in male fish plasma induced with 17ss-estrodiol. CZE analyses of purified VTG were performed in a buffer containing 25 mM sodium borate (pH 8.4). A 50 microm i.d. fused-silica capillary was used for separation and the detection was carried out by UV-diode array at 214 nm. Inter- and intra-assay variabilities of the proposed method were less than 10.06 and 1.95%, respectively. The method has good linear relationship over the scope of 15-2250 microg/ml with a correlation coefficient of R2 = 0.9965 and a detection limit of 7.0 microg/ml. The established CZE method was also applied to directly separate and identify VTG from fish plasma. The results indicated this method could minimize interferences from plasma proteins, allowing the detection of at least 62.5 microg/ml of VTG proteins in total proteins. This is a rapid and easy method to determine the quantity and purity of VTG compared to Bradford method and SDS-PAGE.