Capillary electrophoresis for pharmacokinetic studies

Different analytical techniques involving capillary electrophoresis for the determination of drugs and metabolites in biological fluids are described. Pharmacokinetic studies carried out using capillary electrophoresis are presented, as well as the in vitro metabolism investigations. The advantages and the limitations of capillary electrophoresis for pharmacokinetic studies are discussed.     

Use of nanomaterials in capillary and microchip electrophoresis

This review gives an overview of different separation strategies with nanomaterials and their use in capillary electrophoresis (CE) and capillary electrochromatography, as well as in microchip electrophoresis, including metal and metal oxide nanoparticles, carbon nanotubes, fullerene and polymer nanoparticles, as well as silica nanoparticles. The paper highlights the new developments and innovative applications of nanoparticles as pseudostationary phases or immobilized on the capillary surface for CE separation. The separation and characterization of target nanoparticles with different sizes by CE are reviewed likewise.     

Use of nanomaterials in capillary and microchip electrophoresis

This review gives an overview of different separation strategies with nanomaterials and their use in capillary electrophoresis (CE) and capillary electrochromatography, as well as in microchip electrophoresis, including metal and metal oxide nanoparticles, carbon nanotubes, fullerene and polymer nanoparticles, as well as silica nanoparticles. The paper highlights the new developments and innovative applications of nanoparticles as pseudostationary phases or immobilized on the capillary surface for CE separation. The separation and characterization of target nanoparticles with different sizes by CE are reviewed likewise.     

Synergism of capillary isotachophoresis and capillary zone electrophoresis

The combination of capillary isotachophoresis and capillary zone electrophoresis may enhance greatly the performance of analytical capillary electrophoresis with respect to both separation power and the concentration sensitivity. The concentrating effects and the separation power of isotachophoresis allow the analysis of diluted samples and the elimination of interferences due to bulk components. The separation process of zone electrophoresis enables one to resolve the stack of trace analytes and detect the resulting individual zones with high sensitivity. The transition of isotachophoresis into zone electrophoresis plays the key role in the overall performance of this hyphenated technique. This article describes the dynamics of the conversion of isotachophoresis into zone electrophoretic mode and shows that the key role is played by the segments of the leading and terminating zones from the isotachophoretic stage. The magnitude of these segments directly effects the detection time as well as the separation width of the peaks of analytes. It is shown that these effects are also important in the analyses by capillary zone electrophoresis where isotachophoresis is induced by the sample itself. Finally, the paper presents a list of recommended, user-friendly, electrolyte systems which enable one to simply predict the performance of the combination isotachophoresis-zone electrophoresis.     

Clinical applications of capillary electrophoresis

Capillary electrophoresis (CE) is an extremely sensitive technique, which has been used in the clinical laboratory for almost 10 yr. The components of CE instrumentation are described, as are injection modes, buffers, and effects of electroosmotic flow. The modes of separation used in CE, namely, capillary zone electrophoresis, capillary isoelectric focusing, capillary isotachophoresis, and micellar electrokinetic capillary chromatography, are explained. References for 26 different clinical applications of CE are included, among them assays that are used routinely as well as niche assays for specialized applications of CE. Verification of CE assays, current instrumentation, and future development of CE in the clinical laboratory are addressed.     

Miniaturized separation techniques in glycomic investigations

High-sensitivity glycomic analyses are becoming of a great interest in modern biomedical and clinical research, as well as in the development of recombinant protein products. The evolution of separation techniques for glycomic analysis at high sensitivity is highlighted in this review. These methodologies include capillary liquid chromatography, capillary electrophoresis (CE) and capillary electrochromatography (CEC). The potential of such methodologies in glycomic analysis is demonstrated for model glycoproteins as well as total glycomes derived from biological samples.     

Separation of bovine serum albumin and its monoclonal antibody from their immunocomplexes by sodium dodecyl sulfate–capillary gel electrophoresis and its application in capillary electrophoresis-based immunoassay

A non-competitive immunoassay was performed by sodium dodecyl sulfate–capillary gel electrophoresis with UV detection using bovine serum albumin (BSA) and monoclonal anti-BSA. BSA, anti-BSA and their immunocomplexes were well resolved under non-denaturing conditions. A linear calibration curve was obtained and can be used for the quantification of anti-BSA. The limit of detection of anti-BSA was 0.1 μM under the present conditions. Compared with capillary zone electrophoresis, we believed that this method has the potential to be used as a more general format for performing capillary electrophoresis-based immunoassay of medium- and large-sized analytes.     

Determination of PyPuPu (PyPuPy) intermolecular triple-stranded DNA by capillary electrophoresis

The PyPuPu and PyPuPy intermolecular triple-stranded DNA (tsDNA) can be determined more easily by capillary electrophoresis (CE) than by traditional methods. The tsDNA and its component compounds can be well separated by using a sieving matrix of 1.0% hydroxypropylmethylcellulose (HPMC) containing 2.5 mM magnesium ions. Such factors as buffer pH, the concentration of triplex-forming oligonucleotide (TFO), temperature, and the concentration of magnesium cation in the formation and stabilization of triple-stranded helices have been studied with capillary electrophoresis. The triplex cannot be formed when the buffer pH is lower than 4.0. When the concentration of TFO is four times higher than that of dsDNA, all of the dsDNA molecules can be associated. The limit of capillary electrophoresis detection with good reproducibility is 0.5-1 nM (S/N = 3). The CE analysis of short tsDNA takes only 40 min, whereas gel electrophoresis needs at least 5 h.     

Comparison of three microsatellite analysis methods for detecting genetic diversity in <Emphasis Type="Italic">Phytophthora sojae</Emphasis> (Stramenopila: Oomycete)

Analysis of an organism’s genetic diversity requires a method that gives reliable, reproducible results. Microsatellites are robust markers, however, detection of allele sizes can be difficult with some systems as well as consistency among laboratories. In this study, our two laboratories used 219 isolates of Phytophthora sojae to compare three microsatellite methods. Two capillary electrophoresis methods, the Applied Biosystems 3730 Genetic Analyzer and the CEQ 8000 Genetic Analysis system, detected an average of 2.4-fold more alleles compared to gel electrophoresis with a mean of 8.8 and 3.6 alleles per locus using capillary and gel methods, respectively. The two capillary methods were comparable, although allele sizes differed consistently by an average of 3.2 bp across isolates. Differences between capillary methods could be overcome if reference standard DNA genotypes are shared between collaborating laboratories.     

Evaluation of ADA Gene Expression and Transduction Efficiency in ADA/SCID Patients Undergoing Gene Therapy

A capillary electrophoresis (CE) method was developed for ADA/SCID diagnosis and monitoring of enzyme replacement therapy, as well as for exploring the transfection efficiency for different retroviral vectors in gene therapy.     

Evaluation of ADA gene expression and transduction efficiency in ADA/SCID patients undergoing gene therapy

A capillary electrophoresis (CE) method was developed for ADA/SCID diagnosis and monitoring of enzyme replacement therapy, as well as for exploring the transfection efficiency for different retroviral vectors in gene therapy.     

Capillary electrophoresis procedures for serum protein analysis: comparison with established techniques

Methods using automated capillary electrophoresis (CE) instrumentation are available for serum protein electrophoresis with monoclonal band quantitation, isoelectric focusing and sodium dodecyl sulphate-polyacrylamide gel electrophoresis separations. The advantages of CE over previous gel methods relate to the time and labour saved by the automated instrumentation. High pI monoclonal bands and cryoglobulin specimens can be successfully analysed by CE. However, if the CE application uses a standard company supplied kit, then the cost savings are often negated by the high cost of the kit. Improvements such as the inclusion of both a UV-Vis as well as a fluorescence detector as standard within the one commercial instrument, the production of coated IEF capillaries with a useful life of at least 100 samples, and the introduction of a capillary array into all commercial instrumentation would ensure greater use of CE within both the clinical and other protein laboratories.     

Affinity capillary electrophoresis: important application areas and some recent developments

Affinity capillary electrophoresis (ACE) is a broad term referring to the separation by capillary electrophoresis of substances that participate in specific or non-specific affinity interactions during electrophoresis. The interacting molecules can be found free in solution or can be immobilized to a solid support. Every ACE mode has advantages and disadvantages. Each can be used for a wide variety of applications. This paper focuses on applications that include purification and concentration of analytes present in diluted solutions or complex matrices, quantitation of analytes based on calibration curves, and estimation of binding constants from direct and derived binding curves based on quantitation of analytes or on analyte migration shifts. A more recent chemicoaffinity strategy in capillary electrophoresis/capillary electrochromatography (CE/CEC) termed molecular imprinting (`plastic antibodies') is discussed as well. Although most ACE studies are aimed at characterizing small-molecular mass analytes such as drugs, hormones, and peptides, some efforts have been pursued to characterize larger biopolymers including proteins, such as immunoglobulins. Examples of affinity interactions that have been studied are antigen–antibody, hapten–antibody, lectin–sugar, drug–protein, and enzyme–substrate complexes using ultraviolet, laser-induced fluorescence, and mass spectrometer detectors. This paper also addresses the critical issue of background electrolyte selection and quantitation of analytes. Specific examples of bioaffinity applications are presented, and the future of ACE in the biomedical field is discussed.     

A simple method for the preparation of single-stranded polydeoxynucleotides of desired length.

A method for the preparation of homogeneous, single-stranded polydeoxynucleotides of desired length up to 800 bases is described. The procedure entails 1) generation of double-stranded DNA of desired length by PCR using a pair of primers of which one is biotinylated and the other is either unlabeled or fluorescently labeled, 2) isolation of PCR products by agarose slab gel electrophoresis, 3) recovery of desired product by electroelution, 4) binding of the product to streptavidin-coated magnetic beads and is followed by 5) duplex denaturation and removal of the unbound single strand that is either unlabeled or fluorescently labeled. Final product characteristics were determined by capillary gel electrophoresis with fluorescence detection. Up to microgram quantities of homogeneous single-stranded DNA of a desired length were obtained. These can be used as single-stranded size standards in capillary gel electrophoresis experiments as well as in other techniques requiring such standards.     

Ups and downs of protein crystallization: studies of protein crystals by high-performance capillary electrophoresis

High-performance capillary electrophoresis is a high-technology micro-separation method. Short run time, full automation and minute amounts of sample make it a very attractive technique. In this report we describe studies of protein crystals by capillary electrophoresis. We show how high-performance capillary electrophoresis can be used effectively for rapid evaluation and examination of the protein solution used for crystallization, the protein crystals (solubilized) and surrounding mother liquor. With coated capillaries, the runs were reproducible and disturbing effects, such as electroendosmosis and interaction of the proteins with the capillary wall, were suppressed efficiently. We recommend this new technique as a powerful and routine companion to protein crystallography.     

Polymer based micro-reactors

In this paper, we describe the fabrication technologies necessary for the production of polymer-based micro-fluidic devices. These technologies include hot embossing as a micro-structuring method as well as so-called back-end processes to complete the micro-devices. Applications such as capillary electrophoresis, micro-mixers and nanowell plates are presented.     

An assay for angiotensin-converting enzyme using capillary zone electrophoresis

A sensitive and rapid method was developed for angiotensin-converting enzyme (ACE) activity determination by capillary zone electrophoresis. Hippuryl-l-histidyl-l-leucine, a synthetic tripeptide, was used as the ACE-specific substrate. Capillary zone electrophoresis was employed to separate the products of the enzymatic reaction and the ACE activity was determined by quantification of hippuric acid, a result of the enzymatic reaction on the tripeptide. The capillary electrophoresis was performed in a 27 cm x 75 micrometer i.d. fused-silica capillary using 200 mM boric acid-borate buffer (pH 9.0) as a run buffer with an applied voltage of 8.1 kV at a capillary temperature of 23 degrees C. The electrophoresis was monitored at 228 nm. Each electrophoretic run requires only a nanoliter of the enzymatic reactant solution, at only 6 min, rendering a powerful tool for the ACE assay.     

Isoelectric focusing by free solution capillary electrophoresis.

A reproducible, quantitative isoelectric focusing method using capillary electrophoresis that exhibits high resolution and linearity over a wide pH gradient was developed. RNase T1 and RNase ba are two proteins that have isoelectric points (pI's) at the two extremes of a pH 3-10 gradient. Site-directed mutants of the former were separated from the wild-type form and pI's determined in the same experiment. The pI's of RNase T1 wild-type, its three mutants, and RNase ba were determined for the first time as 2.9, 3.1, 3.1, 3.3, and 9.0, respectively. The paper describes the protocol for isoelectric focusing by capillary electrophoresis, as well as presenting data describing the linearity, resolution, limits of mass loading, and reproducibility of the method.     

The use of capillary electrophoresis in a micropreparative mode: methods and applications.

The ability to collect sufficient quantities of analytes from capillary electrophoresis for subsequent analyses is demonstrated. Fractions collected have been analyzed using the following techniques: capillary electrophoresis, mass spectrometry, and protein sequencing. Fractions can be collected directly into small volumes of buffer or directly onto membrane surfaces. Relevant parameters such as capillary diameter, mass loading, and separation parameters are addressed.     

Rapid CE microbial assays for consumer products that contain active bacteria

Recent advances in high efficiency separation methods of bacteria allow their rapid identification and quantitation in some cases. A specific capillary electrophoresis (CE) technique is used to identify and quantitate Lactobacillus acidophilus in both pill and syrup health products as well as Bifidobacterium infantis in a powdered formula supplement. Cell viability can be evaluated as well. In some cases, both the living and dead bacterial cells as well as the molecular excipients can be evaluated in a single run.