Development of post-column enzymic reactors with immobilized alcohol oxidase for use in the high-performance liquid chromatographic assay of alcohols with electrochemical detection

The development of a very sensitive, direct injection high-performance liquid chromatographic method, using a post-column reactor with immobilized alcohol oxidase, was undertaken with the aim of determining methanol and ethanol levels in microlitre volumes of biological samples. After reversed-phase chromatography to separate methanol and ethanol, the analytes were enzymically converted into the respective aldehydes with formation of stoichiometric amounts of hydrogen peroxide, which could be measured via electrochemical oxidation at a platinum electrode. Some problems were encountered in the development of solid-phase enzymic reactors, using a delicate enzyme, that is prone to lose activity, such as alcohol oxidase. Owing to the slightly alkaline pH required for the optimum activity of alcohol oxidase, polymeric columns seemed to be preferable for the chromatography. HEMA copolymer was chosen as the stationary phase, but the methanol and ethanol peaks eluted close together and posed severe problems of limiting post-column band spreading. Reactors based on coarse supports for enzyme immobilization gave unacceptable band spreading, causing the methanol and ethanol peaks to overlap. On the other hand high-performance liquid chromatographic packings maintained the efficiency of the chromatographic separation, quite independently of the reactor volume. Polymeric supports proved superior to silicas in maintaining the enzyme activity. However, relevant changes in the enzyme substrate specificity were observed after immobilization.     

Nonionic micellar liquid chromatography coupled to immobilized enzyme reactors

Immobilized enzyme reactors are used as post-column reactors to modify the detectability of analytes. An immobilized amino acid oxidase reactor was prepared and coupled to an immobilized peroxidase reactor to detect low level of amino acids by fluorescence of the homovanilic dimer produced. A cholesterol oxidase reactor was prepared to detect cholesterol and metabolites by 241 nm UV absorbance of the enone produced. The preparation of the porous glass beads with the immobilized enzymes is described. Micellar liquid chromatography is used with non-ionic micellar phases to separate the amino acids or cholesterol derivatives. It is demonstrated that the non ionic Brij 35 micellar phases are very gentle for the enzyme activity allowing the reactor activity to remain at a higher level and for a much longer time than with hydro-organic classical chromatographic mobile phases or aqueous buffers. The coupling of nonionic micellar phases with enzymatic detection gave limits of detection of 32 pmol (4.8 ng injected) of methionine and 50 pmol (19 ng injected) of 20alpha-hydroxy cholesterol. The immobilized enzyme reactors could be used continuously for a week without losing their activity. It is shown that the low efficiency obtained with micellar liquid chromatography is compensated by the possibility offered by the technique to easily adjust selectivity.     

Determination of purine nucleosides and their bases by high-performance liquid chromatography using co-immobilized enzyme reactors

A selective and sensitive assay of inosine, guanosine, hypoxanthine, guanine and xanthine by high-performance liquid chromatography with immobilized enzyme reactors was developed. The separation was achieved on a Capcell Pak C18 column (15 cm x 0.46 cm I.D.) with a mobile phase of 0.1 M phosphate buffer (pH 8.0) containing 7 mM sodium 1-hexanesulphonate and 0.1 mM p-hydroxyphenylacetic acid. The fluorimetric detection of hydrogen peroxide using immobilized peroxidase and p-hydroxyphenylacetic acid was applied to the assay of these compounds, which were oxidized to yield hydrogen peroxide in the presence of immobilized enzyme (purine nucleoside phosphorylase, guanase and xanthine oxidase). Enzyme reactions occurred sufficiently without post-column addition of reagents. Enzymes that catalysed the conversion of purine compounds were co-immobilized on aminopropyl controlled-pore glass packed in stainless-steel tubing. The detection limits were 30-200 pg per injection.     

Post-column derivatization in liquid chromatography using immobilized enzyme reactors and amperometric detection

The advantages to be gained by conducting enzyme assays of carbohydrates in liquid chromatography are discussed. The enzymes are contained in immobilized enzyme reactors and used in the post-column mode. The product formed in the reactor is selectively detected amperometrically at a chemically modified electrode mounted in a flow-through detector. Selected examples are given to illustrate the advantages obtained.     

Enzyme amplification as detection tool in continuous-flow systems. II. On-line coupling of liquid chromatography to enzyme-amplified biochemical detection after pre-column derivatization with biotin.

Enzyme-amplified biochemical detection (EA-BCD) was used as a post-column detection technique, coupled on-line with high-performance liquid chromatography (HPLC). The enzyme detection system was developed to detect biotin or biotin containing compounds. Biotinylation is widely used to label analytes of interest ranging from small molecules to proteins and DNA. Naphthalene aldehyde and anthracene aldehyde were used as model compounds. Both compounds were biotinylated off-line with biotin aminocaproic hydrazide (BACH). On-column biotinylation was performed by preconcentration of anthracene aldehyde on copper phthalocyanine. After biotinylation, samples were introduced to the HPLC system. Enzyme-labeled streptavidin, which possesses high affinity to biotin, was added post-column to the HPLC effluent. Excess of enzyme-labeled affinity protein was removed by means of an immobilized biotin column. After separation of free and bound fraction, substrate was added, which was converted to a fluorescent product by the enzyme label. Using alkaline phosphatase as an enzyme label, a mass detection limit after on-column preconcentration and biotinylation of 250 fmol was achieved.     

Determination of aldehydes in food by high-performance liquid chromatography with biosensor coupling and micromembrane suppressors

A high-performance liquid chromatography system for the determination of aldehydes in food was developed incorporating an Cation MicroMembrane Suppressor (CMMS) and enzyme reactors packed with VA-Epoxy on which aldehyde dehydrogenase from bakers yeast and NADH oxidase from Bacillus licheniformis were immobilized. The method was based on the principle that the separation efficiency of HPLC is combined with the sensitivity of electrochemical detection and the specificity of enzymes. Main attention was directed to the determination of 5-hydroxymethyl-2-furaldehyde and 2-furaldehyde, the occurence of which is an indication of quality deterioration in several food products. The efficiency of the method has been shown by the analysis of honey, coffee and related beverages, refreshments, sherry, port, dry fruits and breakfast cereals.     

Simultaneous assay of hypoxanthine, xanthine and allopurinol by high-performance liquid chromatography and activation of immobilized xanthine oxidase as an enzyme reactor

A selective and sensitive assay of substrates (hypoxanthine, xanthine and allopurinol) of xanthine oxidase by reversed-phase liquid chromatography coupled with the use of immobilized enzyme reactors is described. These compounds were oxidized by immobilized xanthine oxidase and produced hydrogen peroxide, which was determined fluorometrically using immobilized peroxidase and p-hydroxyphenylacetic acid. The detection limits of hypoxanthine, xanthine and allopurinol were approximately 50, 120 and 130 pg per injection, respectively. Immobilized xanthine oxidase inhibited by oxipurinol during the assay was reactivated by 2,6-dichlorophenolindophenol and could be used for a long period without a significant activity loss. These methods were applied to plasma and urine samples.     

Specific detection of acetyl-coenzyme A by reversed-phase ion-pair high-performance liquid chromatography with an immobilized enzyme reactor

A selective chromatographic detection system for the determination of acetyl-coenzyme A (CoA) is reported. The short-chain acyl-CoA thioesters were separated by reversed-phase ion-pair high-performance liquid chromatography (HPLC), and then acetyl-CoA was selectively detected on-line with an immobilized enzyme reactor (IMER) as a post-column reactor. Thio-CoA liberated enzymatically from acetyl-CoA was determined spectrophotometrically after reaction with Ellman's reagent in the reagent stream. The IMER with phosphotransacetylase had a substrate specificity sufficient to determine acetyl-CoA and was active and stable in the mobile phase containing methanol and the ion-pair reagent. The calibration graph was linear between 0.2 and 10 nmol, with a detection limit of 0.05 nmol. This HPLC system with detection by IMER allows the selective identification and determination of acetyl-CoA in a mixture of acetoacetyl-CoA and 3-hydroxy-3-methylglutaryl-CoA, which are difficult to separate with ion-pair HPLC.     

A strategy for screening trypsin inhibitors from traditional Chinese medicine based on a monolithic capillary immobilized enzyme reactor coupled with offline liquid chromatography and mass spectrometry

A novel strategy was successfully developed for screening trypsin inhibitors in traditional Chinese medicines based on monolithic capillary immobilized enzyme reactors combined with liquid chromatography‐tandem mass spectrometry. Organic polymer based monolithic enzyme reactors were firstly prepared by covalently bonding trypsin to a poly(glycidyl methacrylate‐co‐poly (ethylene glycol) diacrylate) monolith by the ring‐opening reaction of epoxy groups. The activity and kinetic parameters of the obtained monolithic trypsin reactors were systematically evaluated using micro‐liquid chromatography. Fourier transform infrared spectroscopy and scanning electron microscopy were also used to characterize the monolithic trypsin reactors. The resulting functional and denatured monolithic trypsin reactors were applied as affinity solid‐phase extraction columns, and offline coupled with a liquid chromatography‐tandem mass spectrometry system to construct a binding affinity screening platform. Subsequently, the proposed platform was applied for screening trypsin binders in a Scutellaria baicalensis Georgi extract. Three compounds, namely scutellarin, baicalin, and wogonoside were identified, and their inhibitory activities were further confirmed via an in vitro enzymatic inhibition assay. Additionally, molecular docking was also performed to study the interactions between trypsin and these three compounds.     

Electrochemical detection of cyanogenic glycosides after enzymatic post-column cleavage

Crude and partly purified extracts from Helix pomatia and linamarase from cassava were immobilized on columns packed with porous glass or silica and used as post-column reactors in the high-performance liquid chromatography of cyanogenic glycosides. Sodium hydroxide (2 M) was added to the flowstream after the enzyme-reactor resulting in the formation of cyanide, which was then detected at a silver electrode by an amperometric measurement at 0 V with reference to a silver-silver chloride electrode. The selective detection of cyanide allows measurements in a complex matrix. The response is linear and the detection limit is in the low picomole range.     

Evaluation and application of liquid chromatographic columns coated with "intelligent" ligands. (III). Immobilized phospholipid column.

Immobilized enzyme columns have been developed for use as high-performance liquid chromatographic enzyme reactors. Enzyme reactors were prepared by immobilizing trypsin or cytochrome-c on phospholipid columns. Dynamic coating was employed to prepare the reactors by recycling a buffer solution containing trypsin or cytochrome-c through a phospholipid-coated column, on which the enzymes were immobilized by hydrophobic binding. The immobilized trypsin column displayed hydrolytic activity which catalyzed the hydrolysis of L-amino acid esters to amino acid. The immobilized cytochrome-c column exhibited oxidation activity which catalyzed N-demethylation of N,N-dimethylaniline, codeine, and dihydrocodeine in the presence of hydrogen peroxide as an oxygenating agent. The enzyme reaction proceeded rapidly in the column; both product and substrate could be separated and detected simultaneously. The immobilized enzyme columns could be readily regenerated using the original phospholipid column by repeating the dynamic coating. These immobilized enzyme columns could be utilized as enzyme reactors in the high-performance liquid chromatographic mode. Complete hydrolysis of amino acid ester was observed with the trypsin column. Demethylation of codeine and of dihydrocodeine were observed with the cytochrome-c column.     

Effect of antigen size on optimal ligand density of immobilized antibodies for a high-performance liquid chromatographic support

The antigen binding capacities for purified polyclonal antibodies immobilized onto a silica-based high-performance liquid chromatographic (HPLC) affinity support are described for three serum proteins over a range of antibody ligand densities. The rate of decline in the specific activity of the immobilized antibodies with respect to increasing ligand density was found to increase with the molecular weights of the antigens. The antibodies used were purified from whole antiserum using high-performance affinity chromatography and were examined using HPLC on an SCX stationary phase. Conditions are also described for efficient coupling of the ligand to the support.     

A Selective Determination of Hypoxanthine,Xanthine and Inosine by Reversed-Phase Liquid Chromatography Coupled with Enzyme Reactors

Abstract

A Selective and sensitive assay of hypoxyanthine, xanthine and inosine by reversed-phase liquid chromatography coupled with immobilized enzyme reactors is described. The flourometric detection of hydrogen peroxide using immobilized peroxidase and p-hydroxyphenylacetic acid was applied to the assay of hypoxanthine, xanthine and inosine, which were oxidized to hydrogen peroxide in the presence of the immobilized enzymes (purine nucleoside phosphorylase and/or xanthine oxidase. The enzymes were immobilized the the intermolecular cross-linking method on controlled pore-glass. The method established was applied to serum and urine samples. The detection limits of hypoxanthine, xanthine and inosine were approximately 130, 300 and 650 pg per injection, respectively.     

Porous graphitic carbon chromatography/tandem mass spectrometric determination of cytarabine in mouse plasma

A high-performance liquid chromatography (HPLC) system using a porous graphitic carbon (PGC) stationary phase interfaced with an electrospray ionization (ESI) source and a tandem mass spectrometer (MS/MS) for the analysis of cytarabine (ara-C) in mouse plasma samples has been developed in support of a pharmacodynamic study. The graphitized carbon column was adopted for the separation of ara-C and endogenous peaks from mouse plasma samples under the reversed-phase phase mode in liquid chromatography. The retention characteristics of the PGC column and the ionization efficiencies of all analytes based on the experimental factors such as the composition of mobile phases were investigated. The potential of ionization suppression resulting from the endogenous biological matrices on the PGC column during HPLC/ESI-MS/MS was investigated using post-column infusion. The concentrations of ara-C in mouse plasma obtained by using PGC-HPLC/MS/MS and ion-pairing HPLC/MS/MS were found to be in good agreement in terms of analytical accuracy.     

Selective detection in flow analysis based on the combination of immobilized enzymes and chemically modified electrodes

The combination of immobilized enzymes and amperometry to build selective detection devices in flow-injection analysis and liquid chromatography is described. The pros and cons of enzyme electrodes and of immobilized enzyme reactors are discussed. The paper concentrates on the use of immobilized dehydrogenases, oxidases, peroxidases, and on electrodes on which these enzyme reactions can be selectively followed. The work in the field by the authors is reviewed.     

On-Line HPLC with Biochemical Detection for Screening Bioactive Compounds in Complex Matrixes

On-line high-performance liquid chromatography (HPLC) coupled with biochemical detection (BCD) has been developed to screen compounds showing antioxidant action, enzyme inhibition and receptor affinity in complex matrixes. This review summarizes HPLC methods combining different post-column detection methods, such as diode-array detection (DAD), mass spectrometry (MS), chemiluminescence (CL) and nuclear magnetic resonance, for antioxidant screening. The methods based on a single relatively stable reagent such as DPPH^• and ABTS^•+ were the most popular. Oxygen free radical scavengers mainly depended on post-column CL detection. The on-line hyphenated HPLC–BCD systems based on post-column UV/DAD fluorescence and MS detection were also widely applied to screen enzyme- and receptor-active compounds. These strategies provide a convenient tool for quick identification and quantification of active compounds in complex matrixes.

     

Direct separation and detection of biogenic amines by ion-pair liquid chromatography with chemiluminescent nitrogen detector

Analysis of biogenic amines is critical to pharmaceutical and food industry due to their biological importance. For many years, the determination of biogenic amines has relied on high performance liquid chromatography (HPLC) coupling with pre-, on-, or post-column derivatization procedures to enable UV or fluorescent detections. In this study, 14 biogenic amines were separated on a Phenomenex Luna Phenyl-Hexyl column by an ion-pair liquid chromatography method using perfluorocarboxylic acids as ion-pair reagents and detected by a chemiluminescent nitrogen detector (CLND). This direct separation and detection HPLC method eliminated the time consuming and cumbersome derivatization procedures. Compared with HPLC-UV (post-column derivatization with ninhydrin) and HPLC-charged aerosol detector (CAD) methods, this HPLC-CLND technique provided narrower peaks, better baselines, and improved separations and detections. Excellent linearity was acquired by CLND for each of the 14 biogenic amines ranging from less than 1 ng to about 1000 ng (on-column weights). The relative response factors determined by this LC-CLND method were proportional to the numbers of nitrogen atoms in each compound, which has been the characteristic of the equimolar determinations by CLND. In addition, a number of samples including beer, dairy beverage, herb tea, and vinegar were analyzed by the LC-CLND method with satisfactory precision and accuracy.     

高效液相色谱中固定酶柱后反应器的应用

本文评述了固定酶柱后反应器在高效液相色谱中的应用。阐述了高效液相色谱与固定酶柱后反应器联用的原理,介绍了固定酶柱后反应器的主要类型。评述了固定酶柱后反应器在生化分析和临床分析中的应用。引文43篇。     

Development and characterization of an immobilized enzyme reactor (IMER) based on human glyceraldehyde-3-phosphate dehydrogenase for on-line enzymatic studies

Immobilized enzyme reactors (IMERs) for on-line enzymatic studies are useful tool to select specific inhibitors and may be used for direct determination of drug-receptor binding interactions and for the rapid on-line screening to identify specific inhibitors. This technique has been shown to increase the stability of enzymes. The enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays an important role in the life cycle of the Trypanosoma cruzi and it has become a key target in the drug discovery program for Chagas' disease. Crystallographic studies have indicated that there are significant inter-species differences in GAPDH activity and sensitivity. For example the active sites of GAPDH in T. cruzi and humans differ by a substitution of ASP(210) (T. cruzi) by Leu(194) in human. Based on this information we initiated the study to develop optimal conditions for the covalent immobilization of the human GAPDH enzyme on a modified capillary support (400 mm x 0.10 mm). The chromatographic separation of NAD from NADH was achieved using a RP-Spherex-diol-OH (10 cm x 0.46 cm, 10 microm, 100 A) column. By using multidimensional HPLC chromatography system it was possible to investigate the activity and kinetic parameters of the GAPDH-IMER. The values obtained for D-GA3P and NAD were K(m)=3.5+/-0.2 mM and 0.75+/-0.04 mM, respectively, and were compared with values obtained with the free enzyme. The activity of the immobilized GAPDH has been preserved for over 120 days.     

Chromatographic study of the adsorption kinetics of albumin on monoclonal and polyclonal immunoadsorbents

Summary High-performance liquid chromatography (HPLC) has been used to study the adsorption kinetics of proteins on immunoadsorbents. The adsorption rate constant of human serum albumin (HSA) on monoclonal and polyclonal anti-HSA antibodies immobilized on a silica HPLC support was determined by saturating the column with repeated pulse injections. Studies on polyclonal immunodsorbents of different capacities enable evaluation of the contribution of transport to the binding sites. The adsorption properties of two different monoclonal anti-HSA antibodies immobilized on a chromatographic support were characterized by different approaches. The location of the epitope on the HSA molecule was determined by enzyme-linked immunoassay (ELISA) with albumin fragments. The chromatographic method was used to determine the column capacity and the adsorption rate constant of HSA on the immunoadsorbent. To compare the affinity of the antibodies for the antigen, an indirect ELISA method was used to determine the equilibrium constant of antigen-antibody association in solution Presented at the 21^st ISC held in Stuttgart, Germany, 15^th–20^th september, 1996.