In-line capillary electrophoretic evaluation of the enantioselective metabolism of verapamil by cytochrome P3A4

Asensi-Bernardi, Lucía; Martı́n-Biosca, Yolanda; Escuder-Gilabert, Laura; Sagrado, S.; Medina-Hernández, M.J.

doi:10.1016/j.chroma.2013.05.038

Cited by 22 publications

(34 citation statements)

References 34 publications

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“…Mixing by longitudinal diffusion through the interface between the plugs is also called "at-inlet reaction" due to the fact that enzyme and substrate plugs are placed at the inlet of the capillary. This simple approach has been applied successfully, for example, in the case of l-tyrosinase [76], protein kinase C [77], CYP enzymes [66,78,79] and α-glucosidase [80]. However, longitudinal diffusion as mixing mode is highly inefficient when relatively long plugs or more than two plugs have to be mixed because longitudinal diffusion is a rather slow process and, moreover, the interface between the plugs is relatively small.…”

Section: In-capillary Enzyme Assaysmentioning

confidence: 99%

“…This has been realized in a series of papers on the stereoselective CYP-mediated metabolism of the drugs fluoxetine [78], verapamil [79] and ketamine [86,92]. The partial filling mode was applied in all cases.…”

Section: In-capillary Enzyme Assaysmentioning

confidence: 99%

“…The background electrolyte contained either highly sulfated β-cyclodextrin (β-CD) or highly sulfated γ-CD as chiral selector. Mixing of enzyme and substrate/cofactor plugs was achieved either electrophoretically [92] or by longitudinal diffusion [78,79] and TDLFP [86], respectively. In the latter case, seven consecutive short plugs of enzyme and drug/cofactor solutions were efficiently mixed.…”

Section: In-capillary Enzyme Assaysmentioning

confidence: 99%

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Advances in Capillary Electrophoresis-Based Enzyme Assays

Scriba

Belal

2015

Chromatographia

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IntroductionEnzymes catalyze metabolic reactions in cells regulating homeostasis, growth and reproduction of living organisms. Enzymes are also often involved in human disease. The analysis of the human genome has revealed that within the so-called druggable genome a significant portion (in some analyses more than half) of the potential drug targets are enzymes [1][2][3]. Consequently, enzymes are important targets in the development of new drugs. Moreover, enzymes play a role in the clinical diagnosis of diseases. Therefore, effective methods for characterization of enzymes as well as for the determination of their activity are important, for example, for the understanding of enzyme-mediated metabolic reactions or the identification of substrates and inhibitors for the treatment of human diseases.Capillary electrophoresis (CE) has been applied to enzyme analysis for more than 20 years since the first report by Banke et al. on an assay of alkaline protease [4]. Since then, all aspects of enzyme-related analysis have been studied by CE methods including the evaluation of enzymatic activity, enzyme kinetics, identification and characterization of enzyme inhibitors and activators as well as metabolic pathways as, for example, summarized in [5][6][7][8][9][10][11][12].Generally, CE-based enzyme assays can be divided in three groups, pre-capillary (offline) assays, in-capillary (online) assays and post-capillary assays. In the pre-capillary assays, all required components including enzyme, substrate, co-factors, etc., are mixed in a vial and incubated for an intended period of time. Subsequently, the reaction is quenched and an aliquot is subjected to CE analysis for the determination of substrate(s) and/or co-substrate(s) and/ or product(s). Multiple sampling or repeated sampling in combination with sequential runs for the determination of Abstract Capillary electrophoresis (CE) has become a flexible and accurate, high-efficiency analytical separation technique in many areas requiring only minute amounts of sample and chemicals. Thus, CE has also been recognized as a suitable technique to study enzymatic reactions including the determination of Michaelis-Menten kinetic data or the identification and characterization of inhibitors. The most often applied CE-based enzyme assay modes can be divided into two categories: (1) pre-capillary assays where incubations are performed offline followed by CE analysis of substrate(s) and/or product(s) and (2) in-capillary assays in which the enzymatic reaction and analyte separation are performed in the same capillary. In case of the in-capillary assays, the enzyme may be immobilized or in solution. The latter is also referred to as electrophoretically mediated microanalysis (EMMA), while in the case of immobilized enzyme the term immobilized enzyme reactor (IMER) is used. The present review summarizes the literature on CEbased enzyme assays published between January 2010 and April 2015. Immobilized enzyme reactors as well as microfluidic devices applied to the study of enzymatic a...

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Section: In-capillary Enzyme Assaysmentioning

confidence: 99%

Section: In-capillary Enzyme Assaysmentioning

confidence: 99%

Section: In-capillary Enzyme Assaysmentioning

confidence: 99%

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Advances in Capillary Electrophoresis-Based Enzyme Assays

Scriba

Belal

2015

Chromatographia

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“…Chiral compounds have been resolved with cyclodextrin [28], highly sulfated cyclodextrins [29–33], or micelles [34]. In these techniques, analytes are separated based on differences in their interaction with the secondary selector either due to the hydrophobicity or the electrostatic charge.…”

Section: Adapting the Separation To Determine Km Valuesmentioning

confidence: 99%

“…Most of the on-line incubations described in the paper either use the same pH [13,18,23,24,34,43,56–62], or a pH close to enzyme reaction [26,32,33,41,55]. In some instances a wide difference of pH existed between the enzyme incubation buffer and the separation buffer [31,63].…”

Section: Adapting the Separation To Determine Km Valuesmentioning

confidence: 99%

Advances in enzyme substrate analysis with capillary electrophoresis

Gattu

Crihfield

et al. 2018

Methods

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Capillary electrophoresis provides a rapid, cost-effective platform for enzyme and substrate characterization. The high resolution achievable by capillary electrophoresis enables the analysis of substrates and products that are indistinguishable by spectroscopic techniques alone, while the small volume requirement enables analysis of enzymes or substrates in limited supply. Furthermore, the compatibility of capillary electrophoresis with various detectors makes it suitable for KM determinations ranging from nanomolar to millimolar concentrations. Capillary electrophoresis fundamentals are discussed with an emphasis on the separation mechanisms relevant to evaluate sets of substrate and product that are charged, neutral, and even chiral. The basic principles of Michaelis-Menten determinations are reviewed and the process of translating capillary electrophoresis electropherograms into a Michaelis-Menten curve is outlined. The conditions that must be optimized in order to couple off-line and on-line enzyme reactions with capillary electrophoresis separations, such as incubation time, buffer pH and ionic strength, and temperature, are examined to provide insight into how the techniques can be best utilized. The application of capillary electrophoresis to quantify enzyme inhibition, in the form of KI or IC50 is detailed. The concept and implementation of the immobilized enzyme reactor is described as a means to increase enzyme stability and reusability, as well as a powerful tool for screening enzyme substrates and inhibitors. Emerging techniques focused on applying capillary electrophoresis as a rapid assay to obtain structural identification or sequence information about a substrate and in-line digestions of peptides and proteins coupled to mass spectrometry analyses are highlighted.

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Chiral Separations by Capillary Electrophoresis

Sánchez-López

Castro-Puyana

Marina

et al. 2015

Analytical Separation Science

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The enantiomeric separation of chiral compounds is nowadays a promising topic in analytical sciences. CE is considered as a well‐established tool to perform chiral separations of a wide range of analytes in different research fields such as pharmaceutical, food and chemical industries, and clinical and environmental laboratories. Due to its numerous advantages such as high efficiency, simplicity, and low consumption of reagents, chiral selectors, and samples, CE plays an important role in the separation of enantiomers. Advantages and limitations of the different CE modes are displayed in the present chapter, discussing their possibilities and the actual trends. Hyphenation with MS detection is also discussed, highlighting the potential of this coupling as well as its weaknesses and, the most commonly used strategies to minimize its drawbacks. Most recent and relevant applications of chiral CE are summarized and illustrated with tables including the analysis of pharmaceutical, biological, and environmental samples, food and beverages, and agrochemicals. Finally, concluding remarks and future trends are also included describing the actual state of the art and challenges of chiral CE techniques.

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In-line capillary electrophoretic evaluation of the enantioselective metabolism of verapamil by cytochrome P3A4

Cited by 22 publications

References 34 publications

Advances in Capillary Electrophoresis-Based Enzyme Assays

Advances in Capillary Electrophoresis-Based Enzyme Assays

Advances in enzyme substrate analysis with capillary electrophoresis

Chiral Separations by Capillary Electrophoresis

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