Miniaturized nanoelectrospray interface for coupling capillary electrophoresis with mass spectrometry detection.

A miniaturized electrospray interface consisting of a microfluidic nanosprayer and nanospray module is reported in the presented short communication. The nanosprayer was fabricated using silicon (Si) technology suitable for cost-efficient high-volume mass production. The nanospray module enabled the positioning of the nanosprayer in front of a mass spectrometry entrance and its coupling with capillary electrophoresis based on the liquid junction principle.

Preparative agarose gel electrophoresis for reducing matrix interferences of organoid cell medium prior to LC-MS analysis of insulin.

Organoids are 3D cell cultures with microanatomies mimicking aspects of real organs, useful for e.g. animal-free studies of development, disease, and drug discovery.

Current applications of capillary electrophoresis-mass spectrometry for the analysis of biologically important analytes in urine (2017 to mid-2021): A review.

Capillary electrophoresis coupled online with mass detection is a modern tool for analyzing wide ranges of compounds in complex samples, including urine. Capillary electrophoresis with mass spectrometry allows the separation and identification of various analytes spanning from small ions to high molecular weight protein complexes. Similarly to the much more common liquid chromatography-mass spectrometry techniques, the capillary electrophoresis separation reduces the complexity of the mixture of analytes entering the mass spectrometer resulting in reduced ion suppression and a more straightforward interpretation of the mass spectrometry data.

Application of capillary electrophoresis-nano-electrospray ionization-mass spectrometry for the determination of N-nitrosodimethylamine in pharmaceuticals.

After a presence of highly hepatotoxic and potentially carcinogenic N-nitrosodimethylamine was detected in certain lots of sartan, ranitidine, metformin, and other pharmaceuticals, local regulatory authorities issued recalls of suspected products, and concerns of the pharmacotherapy safety were widely discussed. Since then, testing of a representative sample of each produced lot of these pharmaceuticals is required as a part of quality control processes. Hence, an interface-free CE-nanoESI system coupled with MS detection was employed for the development of a simple and economical method for quantitative detection of this contaminant in the valsartan drug substances and finished formulations used as model matrices.

Capillary electrophoretic methods for quality control analyses of pharmaceuticals: A review.

Capillary electrophoresis represents a promising technique in the field of pharmaceutical analysis. The presented review provides a summary of capillary electrophoretic methods suitable for routine quality control analyses of small molecule drugs published since 2015. In total, more than 80 discussed methods are sorted into three main sections according to the applied electroseparation modes (capillary zone electrophoresis, electrokinetic chromatography, and micellar, microemulsion, and liposome-electrokinetic chromatography) and further subsections according to the applied detection techniques (UV, capacitively coupled contactless conductivity detection, and mass spectrometry).

Online Enantioselective Capillary Electrophoretic Method for Screening Cytochrome P450 3A4 Inhibitors.

The market share of single-enantiomer drugs is steadily increasing. The pharmacodynamics and pharmacokinetics of individual enantiomers can differ considerably. Thus, their characteristics have to be addressed as early as possible in the development process of new pharmaceuticals.

Screening of Beta-Secretase Inhibitors by Capillary Electrophoresis-Mass Spectrometry.

Alzheimer's disease is the most common cause of dementia, currently afflicting almost 40 million patients worldwide. According to the amyloid cascade hypothesis, the pathogenesis of the disease could be slowed down or even stopped by the inhibition of beta-secretase, making this aspartic acid protease a potentially important drug target site. Capillary electrophoresis is a promising technique for screening putative enzyme inhibitors due to highly effective separations, minuscule sample and other chemicals consumption, compatibility with a variety of detection techniques, and high throughput via automation.

Application of capillary electrophoretic methods for the analysis of plant phloem and xylem saps composition: A review.

Plant vascular tissue is essential for the exchange of water, nutrients, metabolic products, and signals among distant organs in cormophytes. The compositions of phloem and xylem saps are highly dependent on many internal and external factors, and thus their analysis provides a valuable insight into plant physiology, growth, and development as well as nutrition status or presence of biotic or abiotic stresses. Capillary electrophoresis characterized by highly efficient separations and minuscule sample requirements represents a suitable analytical technique for this purpose because the sap constitutes a complex mixture with generally minimal availability.

Capillary electrophoresis integrated immobilized enzyme reactor for kinetic and inhibition assays of β-secretase as the Alzheimer's disease drug target.

Capillary electrophoresis integrated immobilized enzyme reactors are becoming an increasingly popular alternative for enzyme kinetic and inhibition assays thanks to their unique set of features including cost effectiveness, repeated use of the enzyme, minuscule sample consumption, rapid analysis time and easy automation. In this work we present the development and application of a capillary electrophoresis integrated immobilized enzyme reactor based on magnetic particles for kinetic and inhibition studies of β-secretase, a key enzyme in the development of Alzheimer's disease and a promising drug target. We document the optimization of the immobilization procedure, characterization of immobilized β-secretase, optimization of a mutually compatible incubation protocol and separation method as well as the production of the capillary electrophoresis integrated immobilized enzyme reactor.

Development and comprehensive comparison of two on-line capillary electrophoretic methods for β-secretase inhibitor screening.

Alzheimer's disease is the most common cause of dementia, afflicting over 34 million patients worldwide. Since β-secretase is a rate-limiting enzyme of the production of neurotoxic β-amyloid peptide oligomers abnormally accumulated in the affected brain tissue, its specific inhibition appears to be a promising approach to slowing down or even stopping the progression of the disease. Hence two on-line capillary electrophoretic methods for studies of β-secretase activity based on the principles of transverse diffusion of laminar flow profiles and electrophoretically mediated microanalysis were developed, both using a simple unlabeled peptide substrate and UV detection.

A capillary electrophoresis-mass spectrometry based method for the screening of β-secretase inhibitors as potential Alzheimer's disease therapeutics.

In this work a novel capillary electrophoresis-mass spectrometry (CE-MS) based method was developed and validated for the assay of β-secretase (BACE1) activity as a potential target for Alzheimer's disease (AD) treatment. In contrast with the typically used Förster resonance energy transfer (FRET) assays, an unlabelled decapeptide derived from the amyloid precursor protein BACE1 site with the "Swedish mutation" was used as the substrate. The CE usage enabled the enzymatic reaction to be carried out in as small a volume as 100μL in 60min with sufficient yields of proteolytic product, which was subsequently separated in a bare fused silica capillary using 12.

Simulation and experimental study of enzyme and reactant mixing in capillary electrophoresis based on-line methods.

The establishment of an efficient reaction mixture represents a crucial part of capillary electrophoresis based on-line enzymatic assays. For ketamine N-demethylation to norketamine mediated by the cytochrome P450 3A4 enzyme, mixing of enzyme and reactants in the incubation buffer at physiological pH was studied by computer simulation. A dynamic electrophoretic simulator that encompasses Taylor-Aris diffusivity which accounts for dispersion due to the parabolic flow profile associated with pressure driven flow was utilized.

On-line coupling of immobilized cytochrome P450 microreactor and capillary electrophoresis: A promising tool for drug development.

In this work, the combination of an immobilized enzyme microreactor (IMER) based on the clinically important isoform cytochrome P450 2C9 (CYP2C9) with capillary electrophoresis (CE) is presented. The CYP2C9 was attached to magnetic SiMAG-carboxyl microparticles using the carbodiimide method. The formation of an IMER in the inlet part of the separation capillary was ensured by two permanent magnets fixed in a cassette from the CE apparatus in the repulsive arrangement.

Combination of on-line CE assay with MS detection for the study of drug metabolism by cytochromes P450.

A new CE-MS method with enzymatic reaction inside the capillary was developed for the study of drug metabolism by cytochromes P450. This automated method, based on the transverse diffusion of laminar flow profiles methodology, is comprised of the injection of substrates and enzyme, their mixing, incubation, and separation of the reaction products, all performed by CE, and their detection, identification, and quantification by MS. The developed and validated method was finally used to conduct a kinetic study of cytochrome P450 isoform 2C9 or human liver microsomes with diclofenac in order to demonstrate its practical functionality.

Optimized on-line enantioselective capillary electrophoretic method for kinetic and inhibition studies of drug metabolism mediated by cytochrome P450 enzymes.

Pharmacokinetic and pharmacodynamic properties of a chiral drug can significantly differ between application of the racemate and single enantiomers. During drug development, the characteristics of candidate compounds have to be assessed prior to clinical testing. Since biotransformation significantly influences drug actions in an organism, metabolism studies represent a crucial part of such tests.

New capillary electrophoretic method for on-line screenings of drug metabolism mediated by cytochrome P450 enzymes.

A new method for the determination of kinetic and inhibition parameters of cytochromes P450 reactions by means of on-line CE was developed. It is based on transverse diffusion of laminar flow profiles methodology introduced by Krylov et al. that injection procedure was modified.

On-line drug metabolites generation and their subsequent target analysis by capillary zone electrophoresis with UV-absorption detection.

This study presents the in-capillary enzymatic biotransformation of dextromethorphan, an antitusive drug and opioid receptor antagonist, and subsequent electrophoretic separation of its products. The study includes the optimization of separation parameters to fulfill the requirements of an online microreaction. The analyses were performed in a bare fused-silica capillary using 100 mM sodium tetraborate (pH 10.

Study of atypical kinetic behaviour of cytochrome P450 2C9 isoform with diclofenac at low substrate concentrations by sweeping-MEKC combination.

In view of the fact that several studies have shown that diclofenac hydroxylation by cytochrome P450 2C9 deviated from Michaelis-Menten kinetics at low substrate concentrations, sweeping combined with MEKC was applied for the kinetic study of this pharmacologically important reaction. A 50  μm fused silica capillary (56  cm effective length) was used to carry out all separations. 70  mM SDS in 20  mM phosphate 20  mM tetraborate buffer, pH 8.

Application of micellar electrokinetic capillary chromatography for evaluation of inhibitory effects on cytochrome P450 reaction.

The major aim of this work is to demonstrate the applicability of micellar electrokinetic capillary chromatography with SDS based pseudostationary phase for the screening of cytochrome P450 inhibitors. In contrast with the other capillary electrophoresis modes the cytochrome P450 reaction mixture thus could be used for the analysis without any pre-treatment. Cytochrome P450 2C9, one of the most important isoforms in human liver, was chosen as a model example for this study in combination with diclofenac as a probe substrate.