Applications of in-capillary reaction micellar electrokinetic chromatography in the food industry

This review describes the quantitative analysis of in-capillary reactions by using capillary electrophoresis (CE) in the food industry. An electrophoretic analysis of products of an enzyme reaction of a substrate by in-capillary reaction was useful for the activity measurement of glucoamylase in sake rice koji. p-Nitrophenyl-beta-D-maltoside was employed as a substrate and p-nitrophenyl-beta-D-glucopyranoside was the product of the enzyme reaction. The glucoamylase activity of sake rice koji samples gave a good linear relationship with the peak area observed in the in-capillary enzyme reaction method. Also, in-capillary micellar electrokinetic chromatography (MEKC) was used for analyzing the Monascus pigment-mediated degradation of mutagenic 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole. During the electrophoresis, the mutagen and the pigment, due to their different migration velocities, mix for a certain period of time to interact, and then they are separated and quantitated. The in-capillary reaction MEKC method can be applied to the routine quality control of enzyme activities in the food industry and the evaluation of mutagenic compounds in food materials.     

Study of enzyme kinetics of phenol sulfotransferase by electrophoretically mediated microanalysis

Electrophoretically mediated microanalysis (EMMA) was applied for the study of the kinetic parameters of the enzymatic reaction of phenol sulfotransferase SULT1A1 isoenzyme with 4-nitrophenol as a substrate. The SULT1A1 activity was determined by the quantitation of the product, 4-nitrophenyl sulfate, at 274 nm by using different injection and separation steps. This new approach solved the problem of the presence of the very strong inhibitor, adenosine 3',5'-bisphosphate (PAP), in the co-substrate solution (adenosine 3'-phosphate 5'-phosphosulfate, PAPS) which is unstable at room temperature. The inhibitor PAP was electrophoretically separated from the co-substrate PAPS before the injection of enzyme and substrate inside the capillary (and thus before their in-capillary encountering). With the developed in-capillary SULT1A1 activity assay an average Michaelis constant (Km) for 4-nitrophenol was calculated to be 0.84 microM, a value which is consistent with a previously reported value. Strong substrate inhibition (above a 4-nitrophenol concentration of 2.5 microM) was observed, and this is also in accordance with literature values.     

Kinetic study of angiotensin converting enzyme activity by capillary electrophoresis after in-line reaction at the capillary inlet

The in-capillary reaction of angiotensin converting enzyme (ACE) with the tripeptide substrate hippuryl-L-histidyl-L-leucine was studied. ACE activity was determined by the quantitation of the product, hippuric acid, at 230 nm. Reaction occurred at the capillary inlet during a predetermined waiting period, followed by the electrophoretic separation of the compounds. When the set-up was reversed, i.e. reaction at the opposite side after short-end injection of enzyme and substrate, separation was achieved in less than 5 min. Using the Lineweaver-Burk equation, an average Michaelis constant for ACE from rabbit lung was calculated to be 1.16 +/- 0.12 mM, a value consistent with previously reported data.     

Advances in Capillary Electrophoresis-Based Enzyme Assays

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 CE-based enzyme assays published between January 2010 and April 2015. Immobilized enzyme reactors as well as microfluidic devices applied to the study of enzymatic activity will also be briefly addressed.

     

Simultaneous determination of water-soluble vitamins in a vitamin-enriched drink by an in-capillary enzyme reaction method

The in-capillary enzyme reaction method was used to determine riboflavin phosphate in a vitamin-enriched drink based on its conversion to riboflavin (vitamin B2) with alkaline phosphatase. Simultaneously, three water-soluble vitamins [thiamine nitrate (vitamin B2 mononitrate), pyridoxine hydrochloride (vitamin B6 hydrochloride) and nicotinamide (vitamin PP)] and anhydrous caffeine in the drink were subjected to quantitative analysis. In the system, electrophoretic migration was used to mix zones containing the substrate (riboflavin phosphate) and the enzyme (alkaline phosphatase). The reaction was then allowed to proceed in the presence of a weak electric field and, finally, the product (riboflavin) of enzyme reaction and other water-soluble vitamins migrated under the influence of an applied electric field to the detector. All the active ingredients and the formulation excipients were successfully separated by micellar electrokinetic chromatography with 135 mM sodium dodecyl sulfate. To prevent inhibition of enzyme reaction by the addition of sodium dodecyl sulfate to the reaction zone, sandwich mode injection, in which plugs of sandwich solution without sodium dodecyl sulfate were introduced into the capillary on both sides of the reaction zone, was utilized as a barrier to protect the enzyme reaction from the inhibitor. The relationship between the peak area of the product and the concentration of the substrate was calculated in the in-capillary enzyme reaction method. Excellent linearity was obtained, with correlation coefficients of 0.9999. The established method was validated and demonstrated to be applicable to the determination of the five active ingredients, including riboflavin phosphate, in a commercial vitamin-enriched drink. No interference from the formulation excipients was observed. Good linearities were obtained, with correlation coefficients above 0.999. Recoveries and precisions ranged from 99.3 to 101.8%, and from 0.1 to 2.5% RSD, respectively. Good agreement was obtained between the established method and traditional high-performance liquid chromatographic methods. These results suggest that the in-capillary enzyme reaction method can be used for the simultaneous determination of riboflavin phosphate and other water-soluble vitamins in pharmaceuticals.     

A rapid and sensitive CE method with field-enhanced sample injection and in-capillary derivatization for selenomethionine metabolism catalyzed by flavin-containing monooxygenases

A rapid and sensitive electrophoretically mediated microanalysis method with field-enhanced sample injection (FESI) for in-capillary derivatization was developed to determine selenomethionine (SeMet) and selenomethionine selenoxide (SeOMet). Phthalic anhydride (PA) was selected as the derivatization reagent due to the fast reaction at room temperature and the stability of derivatives. The in-capillary derivatization was accomplished by electrophoretically mixing PA and sample plugs. PA reagent was introduced hydrodynamically into the capillary, whereas the sample solution was injected electrokinetically, thus allowing a selective preconcentration of the analytes by FESI. For FESI, the optimum sample solvent was 2 mM borate solution. The borate buffer was suitable for both in-capillary derivatization and separation of the derivatives. The combination of electrophoretically mediated microanalysis with FESI for in-capillary derivatization was successfully achieved with about 800-fold concentration sensitivity enhancement compared to direct CE-UV detection in the same setup. The present method is miniaturized and fully automated, which ensures the on-line derivatization, stacking, separation and detection in 10 min. Finally, the developed method was successfully applied to measure enzyme activities by analyzing the reaction mixtures of SeMet with human flavin-containing monooxygenases (FMO). The results showed that both FMO1 and FMO3, but not FMO5 could catalyze the Se-oxygenation of SeMet.     

Analysis of calcineurin activity by capillary electrophoresis with laser-induced fluorescence detection using peptide substrate

Reversible protein phosphorylation and dephosphorylation are very important activities in understanding cellular signaling networks. In this paper we described a CE-LIF-based assay method of calcineurin (CN), a protein phosphatase important in cardiac hypertrophy, in which a fluorescence-labeled 19-amino acid phosphopeptide was used as a substrate. The substrate was converted to a dephosphorylated product by CN and both the substrate and product were detected by the LIF detector. This assay method was tested for various separation parameters as well as reaction parameters. It was found that 100 mM of a boric acid buffer with a pH of 9.00 produced optimum separation at 10 kV of applied voltage using a 47 cm capillary. After obtaining the suitable reaction conditions the method was used to detect and quantify the CN activity in HL-1 cell extracts where the picogram level of CN activity was obtained per microgram total protein. It was also observed that immunosuppressive drugs like okadaic acid and cyclosporine A inhibit in vitro CN activity.     

Micellar electrokinetic chromatography and laser induced fluorescence detection of botulinum neurotoxin type A activity using a dual-labelled substrate

The activity of Botulinum neurotoxin type A (BoNT A) can be measured by monitoring the toxin's endopeptidase reaction with its peptide substrate. In this report, a sensitive and simple capillary electrophoresis (CE) method for analysing BoNT A activity was developed using a peptide substrate labelled with Fluorescein isothiocynate (FITC) at the N-terminal and biotin at the C-terminal. This dual labelling enables not only highly sensitive laser induced fluorescence (LIF) detection of the reaction product, but also good analytical separation of the product from the peptide substrate by Micellar Electrokinetic Chromatography (MEKC). The separation between the product peak and the substrate peak was approximately 5 min using the dual-labelled substrate, while just about 1 min using the FITC-labelled substrate without biotinylation. Using the current assay method, BoNT A with concentration as low as 0.1 ng ml^?1 (3.6 U mL^?1 in mouse LD₅₀) in water was detected with a S:N ratio of 3 (RSD <19%) and a linear range of four orders of magnitude. With CE's advantages of very small sample volume needed, this method may find particular applications as in assays of BoNT A activity in water samples and kinetic analyses of toxin activity.     

In-capillary derivatization and laser-induced fluorescence detection for the analysis of organophosphorus pesticides by micellar electrokinetic chromatography

We developed a rapid and sensitive method using in-capillary derivatization and laser-induced fluorescence (LIF) detection for the fully automated analysis of organophosphorus pesticides (OPPs), including glufosinate, aminomethylphosphonic acid (AMPA) and glyphosate by micellar electrokinetic chromatography (MEKC). The potential of 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) as in-capillary derivatization reagent is described for the first time. The unique feature of this MEKC method is the capillary being used as a small reaction chamber. In in-capillary derivatization, the sample and reagent solutions were injected directly into the capillary by tandem mode, followed by an electrokinetic step to enhance the mixing efficiency of analytes and reagent plugs in accordance with their different electrophoretic mobilities. Standing a specified time for reaction, the derivatives were then immediately separated and determined. Careful optimization of the derivatization and separation conditions allowed the determination of glufosinate, AMPA and glyphosate with detection limits of 2.8, 3.6 and 32.2 ng/mL, respectively. These detection limits were comparable to those of 1.4, 1.9 and 23.8 ng/mL obtained from conventional pre-capillary derivatization. Furthermore, repeatability better than 0.40% for migration time and 3.4% for peak area, as well as shorter migration time, was obtained. The method was successfully applied to the analysis of spiked river water sample with satisfactory results.     

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.     

RNA separation by in-capillary denaturing polymer electrophoresis with 1,2,5-thiadiazole as an additive

1,2,5-Thiadiazole improved RNA separation with in-capillary denaturing polymer electrophoresis. 1,2,5-Thiadiazole was synthesized as an extraction solvent substituted for a halogenated solvent. While 1,2,5-thiadiazole was an excellent extraction solvent and an environmentally friendly solvent, we found that 1,2,5-thiadiazole was a strong hydrophobic compound for RNA and the RNA separation performance by in-capillary denaturing polymer electrophoresis was dramatically improved. We suggest "in-capillary denaturing polymer electrophoresis" as an RNA separation that realizes the denaturing and separation simultaneously. RNA separation by the method required a strong denaturant, acetic acid, to cleave the intramolecular hydrogen. The running buffer containing acetic acid was of high conductivity and low pH, in which the condition introduced Joule heating and low sensitivity. While conventional denaturants, formaldehyde and urea, maintained small electric conductivity and neutral pH, these denaturants were too weak to achieve the RNA separation by in-capillary denaturing polymer electrophoresis. 1,2,5-Thiadiazole being a neutral molecule, both conductivity and buffer pH were able to be adjusted to a desirable strength for RNA separation. In this paper, we report that RNA separation by in-capillary denaturing polymer electrophoresis in neutral pH was achieved and the sensitivity for RNA separation was higher than that for RNA separation by in-capillary denaturing polymer electrophoresis with acetic acid.     

In-capillary derivatization and analysis of ephedrine and pseudoephedrine by micellar electrokinetic chromatography with laser-induced fluorescence detection

This paper describes an automatic rapid approach for in-capillary derivatization of ephedrine (E) and pseudoephedrine (PE) and subsequent sensitive determination of the derivatives by micellar electrokinetic chromatography (MEKC) with laser-induced fluorescence (LIF) detection using 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) as fluorescent reagent. The unique feature of this method is the capillary being used as a small reaction chamber, in which the sample, derivatization buffer and reagent solutions were injected directly into the capillary by tandem mode, followed by an electrokinetic step (5 kV, 15s) to enhance the mixing efficiency of analytes and reagent plugs. Standing a specified time of 1 min for reaction, the derivatives were then immediately separated and determined. Several parameters for in-capillary derivatization and subsequent MEKC separation were systematically investigated. Under these optimized conditions, a baseline separation of the two analytes was achieved within 10 min and the derivatization concentration limits of detection were found to be 4.8 ng mL(-1) for E and 1.6 ng mL(-1) for PE, respectively. The method was validated in terms of precision, linearity, accuracy and successfully applied for the determination of the two alkaloids in ephedra herb and its preparations.     

Evaluation of an in-capillary approach for performing quantitative cytochrome P450 activity studies

An automated in-capillary assay requiring very small quantities of reagents was developed for performing in vitro cytochrome P450 (CYP450) drug metabolism studies. The approach is based on the following: (i) hydrodynamic introduction of nanoliter volumes of substrate and enzyme solutions in the sandwich mode, within a capillary; (ii) mixing the reagents by diffusion across the interfaces between the injected solutions; (iii) collection of the capillary content at the end of the in-capillary assay; and (iv) off-line analysis of the incubation mixture by ultrahigh pressure liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS). After optimizing the injection sequence of the reagents, the in-capillary approach was applied to the quantitative determination of the kinetics of drug metabolism reactions catalyzed by three CYP450 isozymes involved in human drug metabolism: CYP1A2, CYP2D6, and CYP3A4. It was demonstrated that this in-capillary method was able to provide similar kinetic parameters for CYP450 activity (e.g., Michaelis constants and turnover values) as the classical in vitro method, with a drastic reduction of reagent consumption.     

Electrophoretically mediated microanalysis for in-capillaryelectrical cell lysis and fast enzyme quantification by capillary electrophoresis

In this study, a novel capillary electrophoresis (CE)-based enzymatic assay was developed to evaluate enzymatic activity in whole cells. β-Galactosidase expression was used as an example, as it is a biomarker for assessing replicative senescence in mammalian cells. It catalyzes the hydrolysis of para-nitrophenyl-β-d-galactopyranoside (PNPG) into para-nitrophenol (PNP). The CE-based assay consisted of four main steps: (1) hydrodynamic injection of whole intact cells into the capillary, (2) in-capillary lysis of these cells by using pulses of electric field (electroporation), (3) in-capillary hydrolysis of PNPG by the β-galactosidase—released from the lysed cells—by the electrophoretically mediated microanalysis (EMMA) approach, and (4) on-line detection and quantification of the PNP formed. The developed method was applied to Escherichia coli as well as to human keratinocyte cells at different replicative stages. Results obtained by CE were in excellent agreement with those obtained from off-line cell lysates which proves the efficiency of the in-capillary approach developed. This work shows for the first time that cell membranes can be disrupted in-capillary by electroporation and that the released enzyme can be subsequently quantified in the same capillary. Enzyme quantification in cells after their in-capillary lysis has never been conducted by CE. The developed CE approach is automated, economic, eco-friendly, and simple to conduct. It has attractive applications in bacteria or human cells for early disease diagnostics or insights for development in biology.

gamma-Glutamyl transpeptidase acylation with peptidic substrates: free energy relationships measured by an HPLC kinetic assay

gamma-Glutamyl transpeptidase (GGT, EC 2.3.2.2) is a highly glycosylated heterodimeric enzyme linked to the external cellular membrane that catalyzes the hydrolysis of glutathione as well as the transfer of its gamma-glutamyl group to amino acids and dipeptides in a transpeptidation reaction. The measurement of both the hydrolytic and transpeptidation activity of this important enzyme is a challenge, since its native substrates are not highly chromogenic. We have developed an HPLC-based method for the quantitative photometric detection of numerous enzyme substrates and products, after their pre-column derivation with dabsyl chloride. The broad applicability of this method was demonstrated in the kinetic investigation of transpeptidation reactions of rat kidney GGT with glutathione, its native substrate, as well as a series of pertinent glutathione analogues. The pH-rate profile constructed for glutathione confirmed the dependence on the ionisation state of at least two residues. Analysis of the free-energy relationships in the series of synthetic peptidic substrate analogues revealed the importance of enzyme-substrate interactions unrelated to amine leaving group basicity during the acylation step. These results are further interpreted in the context of the recently published structure for a similar GGT.     

Measuring the activity of farnesyltransferase by capillary electrophoresis with laser-induced fluorescence detection

Enzymatic farnesylation of oncogenic forms of Ras proteins is the initial step in a series of posttranslational modifications essential for Ras activity. The modification is catalyzed by the enzyme, protein farnesyltransferase (PFTase), which transfers a farnesyl moiety from farnesyl diphosphate to the protein. We employed capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection to develop a rapid and sensitive method for the determination of PFTase activity in vitro. The limited substrate specificity of PFTase allowed us to use a fluorescently labeled pentapeptide instead of a Ras protein as a substrate for the enzyme; the product of the enzymatic reaction was the farnesylated pentapeptide. The product was separated from the substrate by CE and quantified with LIF detection. Under optimal conditions, the separation was achieved within 10 min with a resolution of 86. The mass and concentration limits of detection for the farnesylated product were 10(-19) mol and 0.28 nM, respectively. By measuring the rate of accumulation of the farnesylated product, we were able to determine the kinetic parameters of the enzymatic reaction. For yeast PFTase as an enzyme and difluorocarboxyfluorescein-labeled GCVIA peptide as a substrate, the values of k(cat) and K(M) were found to be (3.1 +/- 0.3)x10(-3) s(-1) and (12.0 +/- 1.2) nuM, respectively. Our results suggest that CE-LIF can be efficiently used for the determination of enzymatic activity of PFTase in vitro. After minor modifications, the developed method can be also applied to other reactions of enzymatic prenylation of proteins.     

Michaelis-Menten analysis of bovine plasma amine oxidase by capillary electrophoresis using electrophoretically mediated microanalysis in a partially filled capillary

A method for determining bovine plasma amine oxidase (PAO; EC 1.4.3.6) activity with benzylamine (Bz) as substrate is described. Electrophoretically mediated microanalysis (EMMA) combined with micellar electrokinetic capillary chromatography (MEKC) was used to perform an on-capillary enzymatic reaction and to separate the generated benzaldehyde from the other reaction products. The capillary was only partially filled with the separation solution, since the enzyme was unstable in the presence of the applied surfactant. The initial reaction velocity of the enzyme-catalyzed reaction was estimated from the peak area of the enzyme product, benzaldehyde. An amplification step was introduced by means of an on-capillary incubation of 15 min, in order to accumulate enough reaction product to detect spectrophotometrically at 254 nm. This set-up resulted in a fully automated assay, which can be carried out in less then 35 min. Using the Lineweaver-Burk equation, an average Michaelis constant (K(M)) for PAO was calculated to be 0.74 mM +/- 0.05 mM, which is consistent with previously reported values.     

High-performance liquid chromatographic assay for acid and alkaline phosphatase in serum.

High-performance liquid chromatography was used to assay serum acid and alkaline phosphatase. Samples were incubated with adenosine-5'-monophosphoric acid (AMP) in a buffer of required pH, 5'-nucleotidase was inhibited with Ni2+ ions, and the phosphatase activity was determined by measuring the concentration of the reaction product, adenosine. The analysis time, after the incubation is terminated, is short (7 min), and the assay is quantitative and reproducible. Complete separation of the reaction product from the substrate and the naturally occurring serum constituents and the high sensitivity of the ultraviolet detection system eliminate some of the problems commonly encountered in spectrophotometric assays.     

Screening neuraminidase inhibitors from glycosaminoglycan and natural extract by capillary electrophoresis

An electrophoretically mediated microanalysis (EMMA) method for screening neuraminidase inhibitors in depolymerized glycosaminoglycan and natural extracts is described. In the present method, enzyme and substrate were individually introduced into the capillary as distinct plugs, and then mixed for a short time. Afterwards the voltage was reapplied to separate the product from the unreacted substrate and the natural extract. The measured peak area of the product at 214 nm represents the enzyme activity. The electrophoretic conditions for the enzyme reaction and separation of substrate and product were optimized in this study. Under the optimal conditions, the Michaelis–Menten constant and the inhibitive mechanism of zanamivir were studied, which agreed with the literature data. Furthermore, the inhibitory ratios of enzymatic activity of depolymerized glycosaminoglycan and traditional Chinese drugs were determined. The EMMA method has superiority over traditional assay methods, in not only minimizing the false-positive results but also in simplifying the experimental procedure. Therefore, it could be employed to screen inhibitors from natural sources.     

Field-amplified sample injection and in-capillary derivatization for sensitivity improvement of the electrophoretic determination of histamine

The feasibility of the combination of field-amplified sample injection (FASI) and in-capillary derivatization was explored for improving sensitivity of histamine in capillary electrophoresis (CE). Naphthalene-2,3-dicarboxaldehyde (NDA) was used as derivatization reagent. The reagent and sample was introduced by tandem mode. The derivatization was accomplished by at-inlet mode with standing time of 1.5 min. The combination of FASI and in-capillary derivatization was successfully achieved with about 400-fold concentration sensitivity enhancement compared to pre-capillary derivatization at the same set-up. The detection limit of concentration for histamine reached 1.25 x 10(-11) M by CE and fluorescence detection with S/N = 3. Parameters affecting FASI and in-capillary derivatization process including sample matrix, buffer concentration and reagent injection amount, were investigated.