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. The cell medium of organoid models of Langerhans islets, regulating blood glucose levels by insulin secretion, can be analyzed by liquid chromatography-mass spectrometry (LC-MS). However, organoid medium complexity is a major challenge, as matrix interferences can reduce sensitivity and selectivity, even with optimized LC-MS conditions. By applying preparative agarose gel electrophoresis-electrodialysis (PGE-ED), we were able to decrease the cell medium background signal, allowing for reduced interferences affecting LC-MS analysis of human insulin.

Gene expression and metabolic activity of Streptococcus mutans during exposure to dietary carbohydrates glucose, sucrose, lactose, and xylitol.

Recent RNA sequencing studies have given us a deeper insight into the cariogenic impact of carbohydrate sources in the bacterium Streptococcus mutans, the principal microbial agent in dental caries etiopathogenesis. The process of dental caries development is facilitated by the ability of this bacterium to ferment some carbohydrates into organic acids contributing to a pH decrease in the oral cavity and the demineralization of the hard tissues of the tooth. Furthermore, in dental caries progression, biofilm formation, which starts and ends with free planktonic cells, plays an important role and has several unique properties called virulence factors. The most cariogenic carbohydrate is sucrose, an easily metabolizable source of energy that induces the acidification and synthesis of glucans, forming typical bacterial cell clumps. We used multifaceted methodological approaches to compare the transcriptomic and metabolomic profiles of S. mutans growing in planktonic culture on preferred and nonpreferred carbohydrates and in fasting conditions. Streptococcus mutans in a planktonic culture with lactose produced the same pH drop as glucose and sucrose. By contrast, xylitol and lactose showed high effectiveness in regulating intracellular polysaccharide metabolism, cell wall structure, and overall virulence involved in the initial phase of biofilm formation and structure but with an opposite pattern compared with sucrose and glucose. Our results confirmed the recent findings that xylitol and lactose play a vital role in biofilm structure. However, they do not reduce its formation, which is related to the creation of a cariogenic environment.

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. In this arrangement, a fused-silica capillary was used as both a separation column and a nanoESI emitter providing high ionization efficiency and sensitivity. The optimized procedure was found to have sufficient selectivity, linearity, accuracy, and precision. The established LOD and LOQ values were 0.3 and 1.0 ng/mL, respectively. The practical applicability of the method was tested by analyses of commercially available Valsacor® tablets. The results obtained prove that the developed procedure represents a promising alternative to currently available GC- and LC-based methods. Furthermore, after an adjustment of the separation conditions, the CE-nanoESI/MS system can be conceptually used for the determination of NDMA in other suspected pharmaceuticals.

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). Key parameters of the procedures are summarized in four concise tables. The presented applications cover analyses of active pharmaceutical ingredients and their related substances such as degradation products or enantiomeric impurities. The contribution of reported results to the current knowledge of separation science and general aspects of the practical applications of capillary electrophoretic methods are also discussed.

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. This review aims at providing a comprehensive overview of published capillary electrophoretic methods for the analysis of primary components present in the phloem and xylem saps of higher plants.

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. This chapter presents a method based on capillary electrophoresis coupled to mass spectrometry detection for kinetic and inhibition assays of the beta-secretase reaction with a decapeptide derived from an amyloid precursor protein.

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. Capillary electrophoresis is a promising technique for enantioselective drug metabolism studies due to highly effective separations, minuscule consumption of sample and reagents, compatibility with a variety of detection techniques, high-throughput via automation, and the implementation of online procedures. An online method comprised of the diffusion-based mixing of cytochrome P450 3A4 with racemic ketamine, incubation of the enzyme reaction, separation of the reaction products S- and R-norketamine, and their quantification is presented in this chapter. Since diffusion is an inherent property of all molecules, the method enables the addition of virtually any compound to the reaction mixture without the need for additional optimization of the mixing conditions, and thus can be favorably used for the rapid screening of putative cytochrome P450 3A4 inhibitors.

Capillary electrophoresis integrated immobilized enzyme reactor for kinetic and inhibition assays of ß-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 ß-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 ß-secretase, optimization of a mutually compatible incubation protocol and separation method as well as the production of the capillary electrophoresis integrated immobilized enzyme reactor. The applicability of the capillary electrophoresis integrated immobilized enzyme reactor was demonstrated by kinetic assay with an unlabelled substrate and by inhibition assays using three structurally different reference inhibitors. The resulting kinetic and inhibition parameters clearly support the applicability of the herein presented method as well as document the fundamental phenomena which need to be taken in account when comparing the results to other methods.

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

Alzheimer's disease is the most common cause of dementia, afflicting over 34 million patients worldwide. Since ß-secretase is a rate-limiting enzyme of the production of neurotoxic ß-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 ß-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. The optimized procedures were thoroughly validated and applied for determining the enzyme's kinetic parameters and the inhibition characteristics of two potent probe inhibitors. The resulting values were found to be comparable to literature data obtained with other analytical techniques. The suitability of the employed methodologies for different experimental designs is discussed on the basis of a statistical evaluation of the experimental data. The presented methods constitute a miniaturized and fully automated tool, which should be suitable for kinetic and inhibition studies of ß-secretase as a target for Alzheimer's disease drug discovery in the early stages of the development of a new drug.

A capillary electrophoresis-mass spectrometry based method for the screening of ß-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 ß-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 12.5% acetic acid as a background electrolyte and detected by MS. The limits of detection and quantitation were estimated using the signal to noise ratio to be 5nM (S/N=3) and 15nM (S/N=10), respectively, both being well below the working range for kinetic and inhibition studies. Its applicability for the kinetic study of BACE1 was demonstrated using optimized enzyme assay conditions and the estimated kinetic parameter values were confirmed by classic CE-UV analyses. The method was finally used for the main purpose for which it was developed - to screen BACE1 inhibitors as potential AD therapeutics. The resulting kinetic and inhibition parameters values were compared to those published in the literature, which were almost exclusively obtained by FRET based assays. These comparisons brought up several issues that are further discussed below and favour the application of an unlabelled substrate. The proposed CE-MS based method offers a high-throughput capability for new drug development.

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. The simulator in the diffusion mode was used to predict transverse diffusional reactant mixing occurring during hydrodynamic plug injection of configurations featuring four and seven plugs. The same simulator in the electrophoretic mode was applied to study electrophoretic reactant mixing caused by voltage application in absence of buffer flow. Resulting conclusions were experimentally verified with enantioselective analysis of norketamine in a background electrolyte at low pH. Furthermore, simulations visualize buffer changes that occur upon power application between incubation buffer and background electrolyte and have an influence on the reaction mixture.

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. The resulting on-line system provides an integration of enzyme reaction mixing and incubation, reaction products separation, detection and quantification into a single fully automated procedure with the possibility of repetitive use of the enzyme and minuscule amounts of reactant consumption. The on-line kinetic and inhibition studies of CYP2C9's reaction with diclofenac as a model substrate and sulfaphenazole as a model inhibitor were conducted in order to demonstrate its practical applicability. Values of the apparent Michalis-Menten constant, apparent maximum reaction velocity, Hill coefficient, apparent inhibition constant and half-maximal inhibition concentration were determined on the basis of the calculation of the effective substrate and inhibitor concentrations inside the capillary IMER using a model described by the Hagen-Poisseulle law and a novel enhanced model that reflects the influence of the reactants' diffusion during the injection process.

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. All the estimated kinetic values--apparent Michaelis constants and apparent maximum reaction velocities were in agreement with literature data obtained using other techniques. In addition, the consumption of reactants was in the tens of nL per analysis. The method's usability was further demonstrated on tolbutamide, the other probe substrate of cytochrome P450 isoform 2C9. As a result, the method is conceptually applicable for the screening of any other cytochrome P450 isoform and its substrates and inhibitors after adapting the incubation and separation conditions.

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. Hence, an optimized and economical capillary electrophoretic method for on-line studies of the enantioselective drug metabolism mediated by cytochrome P450 enzymes was developed. It comprises a diffusion-based procedure, which enables mixing of the enzyme with virtually any compound inside the nanoliter-scale capillary reactor and without the need of additional optimization of mixing conditions. For CYP3A4, ketamine as probe substrate and highly sulfated γ-cyclodextrin as chiral selector, improved separation conditions for ketamine and norketamine enantiomers compared to a previously published electrophoretically mediated microanalysis method were elucidated. The new approach was thoroughly validated for the CYP3A4-mediated N-demethylation pathway of ketamine and applied to the determination of its kinetic parameters and the inhibition characteristics in presence of ketoconazole and dexmedetomidine. The determined parameters were found to be comparable to literature data obtained with different techniques. The presented method constitutes a miniaturized and cost-effective tool, which should be suitable for the assessment of the stereoselective aspects of kinetic and inhibition studies of cytochrome P450-mediated metabolic steps within early stages of the development of a new drug.

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. The solutions of an enzyme and its substrates are injected by hydrodynamic pressure as a series of repeated consecutive plugs. Proposed injection of three plugs of enzyme surrounded with plugs of substrates represents a certain trade-off to obtain the reaction mixture with the satisfying homogeneity by the short-injection procedure as possible. Mathematical modeling confirmed the assumption of a consistent distribution of reactants in the final reaction mixture. Kinetic and inhibition studies of cytochrome P450 isoform 2C9's reaction with diclofenac as a probe substrate and sulfaphenazole as a probe inhibitor were conducted in order to prove the practical applicability of the proposed method for on-line screenings of drug metabolism mediated by cytochrome P450 enzymes. As a result, an apparent Michaelis constant of 2.66 ± 0.18 µM, apparent maximum reaction velocity of 7.91 ± 0.22 nmol min(-1) nmol(-1) , Hill coefficient of 1.59 ± 0.16, half maximal inhibitory concentration of 0.94 ± 0.04 µM and apparent inhibition constant of 0.39 ± 0.07 µM were determined. All these values are in agreement with literature data obtained using different techniques. In addition, less than 30 nL of cytochrome P450 2C9 solution was consumed per analysis in the kinetic and inhibition studies using this method.

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 8.6, was used as the BGE. Injection was accomplished by the application of 50 mbar (5 kPa) pressure to the sample vial for 52 s. Separation was performed at 22 kV (positive polarity), with a capillary temperature of 25°C and detection at 200 nm. The higher sensitivity of the sweeping-MEKC combination compared with the simple MEKC method enabled this reaction to be fitted to a Hill kinetic model and confirmed the findings of other authors. A Michaelis constant of 2.91±0.10 µM, maximum reaction velocity of 9.16±0.16 nmol/min/nmol and Hill coefficient of 1.66±0.08 were determined. This value of Hill coefficient confirms the presence of a positive cooperativity at low diclofenac concentrations and supports the hypothesis of two substrates binding at or near the active site.

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 10.0) mixed with linear polyacrylamide (20%, v/v) and 2-propanol (10%, v/v). This BGE was suitable for monitoring both off-line and in-capillary incubations. The partial filling technique enabled the enzymatic reaction to be carried out in its optimal environment (20 mM sodium phosphate, pH 7.4). Finally, in-capillary microreaction in the presence of cytochrome P450 3A4 gave satisfactory outcomes.

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. The inhibitory effect on the given cytochrome P450 reaction was evaluated for two inhibitors with different inhibition potential - strong inhibitor sulfaphenazole and moderate inhibitor ketoconazole. As a result 50% inhibitory concentrations IC(50) and inhibition constants K(i) were evaluated; their values for both inhibitors were in a good agreement with the literature data determined by different methods.