Capillary Electrophoresis Approach Research Articles

Applications of affinity interactions in capillary electrophoresis.

Capillary electrophoresis (CE) has proven useful for the study of reversible molecular interactions. This is because highly efficient and reproducible separations take place in an environment where molecular interactions may contribute to selectivity without being inhibited by adverse buffer conditions. Affinity CE may be used to estimate quantitative binding data (binding constants and in some cases binding stoichiometries and rate constants) for various molecular interactions. Specific binding interactions (e.g., based on antibodies or aptamers) may also be utilized to quantitatively measure specific analytes using CE. Applications within these areas are here reviewed with focus on the last three years and with emphasis on novel concepts as well as innovative methodology and technology. It is concluded that the affinity CE approach is of growing versatility and will continue to play an integral role in discovering, characterizing, and exploiting biomolecular interactions.

Applications of on-line weak affinity interactions in free solution capillary electrophoresis

The impressive selectivity offered by capillary electrophoresis can in some cases be further increased when ligands or additives that engage in weak affinity interactions with one or more of the separated analytes are added to the electrophoresis buffer. This on-line affinity capillary electrophoresis approach is feasible when the migration of complexed molecules is different from the migration of free molecules and when separation conditions are nondenaturing. In this review, we focus on applying weak interactions as tools to enhance the separation of closely related molecules, e.g., drug enantiomers and on using capillary electrophoresis to characterize such interactions quantitatively. We describe the equations for binding isotherms, illustrate how selectivity can be manipulated by varying the additive concentrations, and show how the methods may be used to estimate binding constants. On-line affinity capillary electrophoresis methods are especially valuable for enantiomeric separations and for functional characterization of the contents of biological samples that are only available in minute quantities.

Applications of on-line weak affinity interactions in free solution capillary electrophoresis

The impressive selectivity offered by capillary electrophoresis can in some cases be further increased when ligands or additives that engage in weak affinity interactions with one or more of the separated analytes are added to the electrophoresis buffer. This on-line affinity capillary electrophoresis approach is feasible when the migration of complexed molecules is different from the migration of free molecules and when separation conditions are nondenaturing. In this review, we focus on applying weak interactions as tools to enhance the separation of closely related molecules, e.g., drug enantiomers and on using capillary electrophoresis to characterize such interactions quantitatively. We describe the equations for binding isotherms, illustrate how selectivity can be manipulated by varying the additive concentrations, and show how the methods may be used to estimate binding constants. On-line affinity capillary electrophoresis methods are especially valuable for enantiomeric separations and for functional characterization of the contents of biological samples that are only available in minute quantities.

Methods for the determination of binding constants by capillary electrophoresis.

Apparent equilibrium constants for molecular association (e.g., association constants, binding constants, dissociation constants, partition coefficients) can be determined with a variety of different capillary electrophoresis (CE) approaches. In many cases, the investigated association behavior is between a smaller molecule or ion (i.e., the solute, drug, or analyte of interest) and a larger entity (e.g., proteins, micelles, polymers, chiral selectors such as cyclodextrins, etc.). Each experimental approach has advantages and disadvantages. Frequently, it is the nature of the system being evaluated that determines the optimal experimental approach. Six different CE-based techniques for evaluating binding constants are reviewed. Examples of each method, and recent references on its use are given.

Analysis of amphetamine and congeners in illicit samples by liquid chromatography and capillary electrophoresis.

Screening methods based on liquid chromatography (HPLC) and capillary electrophoresis (CE) have been developed for the identification and determination of amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine, and 3,4-methylenedioxyethylamphetamine in illicit tablets. Diethylpropione, (-)-ephedrine, and 3-amino-1-phenylbutane were also included in the study as amphetamino-related compounds. The HPLC-diode-array detection method involved on-line photochemical derivatization to enhance the selectivity of detection allowing amphetamines to be distinguished from related compounds such as diethylpropione (amfepramone). When the CE approach was adopted, two identification parameters (UV spectra and migration index) were used and the enantioresolution of the racemic amphetamines was achieved using hydroxypropyl-beta-cyclodextrin as chiral selector.

Sugar–lectin interactions investigated through affinity capillary electrophoresis

The affinity interactions of Concanavalin A (Con A) with various saccharide oligomers (dextrins, dextrans, and selected N-linked glycans from various glycoproteins) have been investigated through a capillary electrophoresis approach. Con A has shown a notable binding discrimination between the α-1,6-linked dextran and α-1,4-linked dextrin oligomers. Both the binding capacity and binding discrimination appear to decrease with an increase in sugar chainlength. While the core structure of N-linked glycans is deemed to be responsible for the overall binding of various glycans to Con A, the presence of mannose units at the non-reducing ends was found to be very beneficial to the affinity interaction with Con A. Finally, a connection between the glycan–lectin interaction and glycoprotein–lectin interaction has also been suggested.

Analysis of ACE-inhibitors by CE using alkylsulfonic additives

Capillary electrophoresis (CE) was applied to the determination of angiotensin-converting enzyme (ACE) inhibitors in pharmaceuticals (tablets). Since a free solution CE system failed to reach a complete separation of closely related compounds (lisinopril, ramipril, benazepril, quinapril), alkylsulfonic additives (sodium heptansulfonate and (+)-10-camphorsulphonic acid) were added to the running buffer: improved separations were obtained suggesting a favourable effect of ion-pairing interactions between analytes and additives. The separations were carried out in acidic medium and a systematic investigation of electrophoretic parameters was made to evaluate the performance of the selected additives. Under the optimized conditions, ramipril and benazepril in their commercial dosage forms were determined confirming the applicability of the developed CE approach to the analysis of pharmaceutical samples; the results were also compared with those obtained applying a previously described and validated HPLC method.

Measuring conformational stability of proteins using an optimized temperature-controlled capillary electrophoresis approach

The thermal denaturation process of a model protein, bovine β-lactoglobulin, was analyzed using capillary zone electrophoresis (CZE). For this purpose, a commercial CE apparatus was improved, allowing efficient control and accurate measurement of the temperature up to 95°C. Under various pH conditions, transition temperature ( T m), enthalpy change (Δ H) and entropy change (Δ S) associated with the thermal denaturation were determined. Moreover, the technique is unique in its ability to estimate the heat capacity change (Δ C p). This work shows that CZE, performed even when electroosmotic flow occurs, is an innovative approach for determining the stability curves of proteins. Accordingly, CZE is a powerful tool to study protein unfolding/folding quickly and with minimal sample requirements.

Evaluation of β-lactoglobulin as a stationary phase in high-performance liquid chromatography and as a buffer additive in capillary electrophoresis: observation of a surprising lack of stereoselectivity

Previous studies have reported that α 1-acid glycoprotein is quite similar in amino acid sequence and disulfide bond arrangements to members of a group of proteins which include β-lactoglobulin (BLG). Since generally homologous proteins retain some similarity in function at the molecular level, we decided to evaluate the enantioselective properties of BLG as an high-performance liquid chromatographic chiral stationary phase (HPLC-CSP), and as an additive in capillary electrophoresis (CE). Two columns with differences in internal diameter and method of immobilisation on epoxide silica were prepared. Chiral acidic, basic and uncharged drugs were chromatographed and mobile phase parameters, namely pH and type of organic modifier, were varied in order to test the column performance. The CE approach has some advantages in that there is no need for immobilisation and only a small amount of protein is required. BLG was therefore tested as a CE buffer additive, using the same analytes as in the HPLC study. Although one would expect that a protein would display some enantioselectivity, BLG did not show any enantioselectivity whatsoever in either system; the protein has fairly weak interaction with the majority of the test solutes, as indicated by both techniques.

A heparin-binding peptide from human serum amyloid P component characterized by affinity capillary electrophoresis.

Affinity capillary electrophoresis (CE) was used for a detailed characterization of the binding between heparin and a peptide isolated from the heparin-binding serum protein amyloid P component (SAP). The peptide corresponds to a tryptic fragment (T3) comprising amino acids 14-38 of SAP. By including ligands in the electrophoresis buffer various glycosaminoglycans could be screened for binding of T3 using one sample aliquot. The binding was found to be highly specific for heparin and heparin fragments down to tetramers and appeared strongest at a slightly alkaline pH while no binding could be demonstrated with heparan sulfate, chondroitin sulfate, desulfated heparin, mannose 6-phosphate and phosphotyrosine. The T3-heparin complexes were sufficiently stable to perform quantitative measurements of the binding using preequilibration of samples prior to a CE-mediated separation of bound and free T3-peptide. Plots based on quantitation of analyte peaks corresponding to free and complexed T3 yielded a dissociation constant of 1.5 microM for the interaction with heparin. The results indicate that a specific subfraction of the heparin molecules is active in binding interactions with the peptide. The affinity CE approach proved to be useful for these studies because of its sensitivity to complex formation involving charged ligands and the possibility of achieving separations under native conditions. Also advantageous is the low sample consumption and the ability to analyze unlabeled reactants in solution.

Analysis of amino acid enantiomers derived from antitumor antibiotics using chiral capillary electrophoresis

The chiral separation of enantiomeric forms of derivatized amino acids have been achieved based on a metal–chelate chiral capillary electrophoretic method and a cyclodextrin mediated host–guest interaction approach in micellar electrokinetic chromatography (MEKC) mode with laser-induced fluorescence detection. This approach has been applied to the determination of enantiomeric forms of amino acids derived from novel depsipeptide antitumor antibiotics, BMY-45012 and its analogs. Amino acids were analyzed by complete hydrolysis and the hydrolysate was derivatized with either dansyl chloride for UV absorbance detection or fluorescein isothiocyanate for laser based fluorescence detection. The presence of several amino acids, serine and β-hydroxyl- N-methy-valine in the proposed structure have been confirmed as d-serine and l- β-hydroxyl- N-methy-valine enantiomeric forms by both chiral capillary electrophoresis (chiral CE) and MEKC approaches. A non-chiral amino acid, sarcosine, was also confirmed. These methodologies provide a quick and sensitive approach for the determination of amino acids racemization of pharmaceutical natural products and have proven to be useful for structural elucidation refinement.

Methods for the estimation of binding constants by capillary electrophoresis.

Capillary electrophoresis (CE) has developed into a particularly effective means to determine apparent equilibrium constants for molecular association in solution (e.g., to micelles, cyclodextrins, antibiotics, proteins, RNA, DNA, etc.). The various experimental, graphical and mathematical approaches for determining association constants are reviewed. In CE, association constants can be calculated because there is a relationship between substrate concentration and the measured electrophoretic mobility of the solute. Most of the approaches for obtaining association constants by CE are conceptually and mathematically related to one another. Likewise, they are analogous to many spectroscopic techniques that are used for obtaining association constants. The advantages, limitations and proper use of the various CE approaches are examined.

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.

Automated Capillary Electrophoresis Applied to Therapeutic Drug Monitoring

Chromatographic separations in conjunction with physical chemical detection methods can provide extremely accurate and highly precise measurements of drugs in biological matrices. Immunochemical methods for drug analyses, on the other hand, although usually fully automated, often lack accuracy, may be subject to matrix interference effects, and frequently exhibit nonlinearities that contribute to suboptimal analytical results. Automated capillary electrophoresis (CE) promises the high accuracy and precision of physical methods with no operator intervention once the sample is loaded into the sample cup. A clean-up step is necessary for the analysis of drugs in biological matrices, but that could also be fully automated. Reagent costs per test with CE approach zero, because of the use of extremely small volumes of buffers, standards, and controls. We present data supporting CE as an alternative separation strategy for 28 commonly used therapeutic drugs. These can be resolved in less than 16 min with an optimized micelle-based buffer system.