•Classification of wine based on the content of carboxylic acids.•Capillary electrophoresis and biosensor analysis of carboxylic acids linked to chemometrics.•Principal component analysis on lactate ...and inhibition of sarcosine oxidase, vs. wine tasting.•Self-organized maps as a novel method linked to biosensor analysis.
Bio-electronic tongue was linked to artificial intelligence processing unit and used for classification of wines based on carboxylic acids levels, which were indirectly related to malolactic fermentation. The system employed amperometric biosensors with lactate oxidase, sarcosine oxidase, and fumarase/sarcosine oxidase in the three sensing channels. The results were processed using two statistical methods – principal component analysis (PCA) and self-organized maps (SOM) in order to classify 31 wine samples from the South Moravia region in the Czech Republic. Reference assays were carried out using the capillary electrophoresis (CE). The PCA patterns for both CE and biosensor data provided good correspondence in the clusters of samples. The SOM treatment provided a better resolution of the generated patterns of samples compared to PCA, the SOM derived clusters corresponded with the PCA classification only partially. The biosensor/SOM combination offers a novel procedure of wine classification.
The biology of hyaluronidase activity on age related turnover of the hyaluronic acid (HA) in skin dermis and epidermis has not been established. Elucidation of this phenomenon enables discovery of ...novel compounds for skin health.
As a simple and green technique, capillary electrophoresis (CE) was used for the first time for the determination of the kinetic constants (Km, Vmax and IC50) of the enzymatic degradation of HA. Reaction products were identified using CE/high-resolution mass spectrometry (HRMS) after appropriate optimization. Best results in terms of signal sensitivity were obtained using 10 mM ammonium acetate (pH 9.0) BGE, a sheath liquid composed of methanol–water (80:20, v/v) with 0.02% (v/v) formic acid at 10 μL min−1 and an ESI voltage at −4 kV. Km and Vmax were determined (n = 3) using CE/UV at 200 nm as 0.24 ± 0.02 mg mL−1 and 150.4 ± 0.1 nM s−1, respectively. They were also successfully obtained by CE/HRMS (n = 3) with Km of 0.49 ± 0.02 mg mL−1 and Vmax of 155.7 ± 0.2 nM s−1. IC50 of a standard natural inhibitor, epigallocatechin gallate, was also determined by CE-UV/HRMS. Kinetic constant values obtained by CE compared well with literature which validated the developed CE-based assay.
In addition, the activity of homemade tetrasaccharides of biotinylated chondroitin sulfate CS-A or CS-C (4- or 6- sulfated in a homogeneous or heterogeneous way) on the hydrolysis reaction of hyaluronidase was evaluated. Hyaluronidase was mostly dose-dependently inhibited by CS-A tetrasaccharides sulfated in a homogeneous way. Two trisaccharides from truncated linkage region of proteoglycans were also tested as inhibitors or activators. CE-based assay showed that even a small modification of one hydroxyl group changes the influence on hyaluronidase activity. CE-based assay can be used for the screening of natural and synthetic inhibitors of hyaluronidase activity for cosmetic and therapeutic applications.
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•Linearity and stability study of bovine testicular hyaluronidase activity by CE.•Km and Vmax for hyaluronic acid hydrolysis by hyaluronidase obtained by CE-UV/HRMS.•IC50 of epigallocatechin gallate toward hyaluronidase obtained by CE-UV/HRMS.•Effect of chondroitin sulfate (CS) oligosaccharides on hyaluronidase evaluated by CE.•Sulfate position and number in CS contribute to anti-hyaluronidase activity.
•Computer simulation provides insight into reactant mixing during plug injection.•Electrophoretic mixing of reactants and enzyme after plug injection is simulated.•Simulation visualizes buffer ...changes occurring in discontinuous buffer system.•Conclusions are experimentally verified by monitoring of norketamine enantiomers.
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.
CE is well known for its many advantages, including the speed of the analyses; most of which can be accomplished in less than 10 min. However many potential fields of application such as ...pharmaceutical research and industry and clinical diagnostics require analyses of large numbers of samples and so need an even shorter analysis time to be practical. There are generally three main options for enhancing the throughput of electromigration methods – using microchip or multi‐capillary systems or modifying the method preformed on single capillary instruments. The latter option will probably be the method of choice for most laboratories, since they are only equipped with classical commercial CE instrumentation. Although several approaches can be used – higher electric field, EOF modification, external pressure application during analysis, this article reviews one of the simplest and at the same time most used, the so‐called short‐end injection procedure.
Enzymes play an essential role in many aspects of pharmaceutical research as drug targets, drug metabolizers, enzyme drugs and more. In this specific field, enzyme assays are required to meet a ...number of specific requirements, such as low cost, easy automation, and high reliability. The integration of an immobilized‐enzyme reactor to capillary electrophoresis represents a unique approach to fulfilling these criteria by combining the benefits of enzyme immobilization, that is, increased stability and repeated use, as well as the minute sample consumption, short analysis time, and efficient analysis provided by capillary electrophoresis. In this review, we summarize, analyze, and discuss published works where pharmaceutically relevant enzymes were used to prepare capillary electrophoresis‐integrated immobilized‐enzyme reactors in an online manner. The presented assays are divided into three distinct groups based on the drug–enzyme relationship. The first, more extensively studied group employs enzymes that are considered to be therapeutic targets, the second group of assays present tools to assess drug metabolism and the third group assesses enzyme drugs. Furthermore, we examine various methods of enzyme immobilization and their implications for assay properties.
Evaluating the physiological state of an organism is of clinical importance. In assisted reproduction, knowledge of the embryo's physiology is crucial for selecting the embryo with the highest ...developmental capacity to ensure high pregnancy rates. Amino acids (AAs) are involved in many biochemical processes during embryo development, which means that the determination of AA fluctuations in the embryo's surroundings can determine the embryo's physiological state. Since current embryo selection methods are mainly based on visual assessment, which lacks proper accuracy, a novel method for the analysis of AAs in the embryo's surroundings was developed. AAs were analyzed by means of MEKC‐LIF after on‐capillary derivatization by naphthalene‐2,3‐dicarboxaldehyde. The reactants were injected under the three zone arrangement and mixed using the transverse diffusion of laminar flow profiles methodology. The resulting derivatives of all the standard AAs were baseline resolved in the BGE comprised of 35 mM sodium tetraborate, 55 mM SDS, 2.7 M urea, 1 mM BIS‐TRIS propane, and 23 mM NaOH within 50 min. The method was applied on an analysis of spent culture media used in assisted reproduction to culture embryos after in vitro fertilization.
Amino acids are essential compounds for living organisms, and their determination in biological fluids is crucial for the clinical analysis and diagnosis of many diseases. However, the detection of ...most amino acids is hindered by the lack of a strong chromophore/fluorophore or electrochemically active group in their chemical structures. The highly sensitive determination of amino acids often requires derivatization. Capillary electrophoresis is a separation technique with excellent characteristics for the analysis of amino acids in biological fluids. Moreover, it offers the possibility of precapillary, on‐capillary, or postcapillary derivatization. Each derivatization approach has specific demands in terms of the chemistry involved in the derivatization, which is discussed in this review. The family of homocyclic o‐dicarboxaldehyde compounds, namely o‐phthalaldehyde, naphthalene‐2,3‐dicarboxaldehyde, and anthracene‐2,3‐dicarboxaldehyde, are powerful derivatization reagents for the determination of amino acids and related compounds. In the presence of suitable nucleophiles they react with the primary amino group to form both fluorescent and electroactive derivatives. Moreover, the reaction rate enables all of the derivatization approaches mentioned above. This review focuses on articles that deal with using these reagents for the derivatization of amino acids and related compounds for ultraviolet‐visible spectrometry, fluorescence, or electrochemical detection. Applications in capillary and microchip electrophoresis are summarized and discussed.
Separation technologies play an important role in revealing biological processes at various omic levels, in pharmacological and clinical research. In this context, CE is a strong candidate for ...analyses of samples with rapidly increasing complexity. Even though CE is well known for its many advantages in this regard, the sensitivity of CE analyses is insufficient for many applications. Accordingly, there are generally three main options for enhancing the sensitivity of CE analyses – using special detection techniques, using sample pre‐concentration and derivatisation. Derivatisation is often the method of choice for many laboratories, since it is simple and provides several advantages such as small sample volume demand and the possibility of automation. Although it can be performed in different ways depending on where the reaction takes place, this article reviews one of the simplest and at the same time most useful approaches on‐capillary derivatisation. Even if in many cases the use of on‐capillary derivatisation alone is enough to improve the detection sensitivity, on other occasions it needs to be employed in combination with the other above‐mentioned strategies. After a simple discussion of derivatisation in general, special attention is focused on the on‐capillary approach and methodologies available for on‐capillary reactant mixing. Its applications in various fields are also described.
Recommendations are given concerning the terminology of methods of bioanalytical chemistry. With respect to dynamic development particularly in the analysis and investigation of biomacromolecules, ...terms related to bioanalytical samples, enzymatic methods, immunoanalytical methods, methods used in genomics and nucleic acid analysis, proteomics, metabolomics, glycomics, lipidomics, and biomolecules interaction studies are introduced.
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.
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