Direct injection high-performance liquid chromatographic method with electrochemical detection for the determination of ethanol and methanol in plasma using an alcohol oxidase reactor

A highly sensitive reversed-phase high-performance liquid chromatographic assay for ethanol and methanol in plasma, using a post-column enzymic reactor with electrochemical detection, has been developed. The alcohols, separated on the column, were converted by immobilized alcohol oxidase into their respective aldehydes with formation of stoichiometric amounts of hydrogen peroxide, detected via oxidation at a platinum electrode. As the chromatographic column, two glass cartridges (150 mm x 3 mm I.D.) in series, packed with 10 microns HEMA-S 1000 packing, were used. Alcohol oxidase from Candida boidinii was immobilized onto HEMA-BIO 1000 VS-L (10 microns), packed in a 30 mm x 3 mm I.D. glass cartridge. The reaction product, hydrogen peroxide, was detected with an amperometric detector with a platinum electrode, operated at +500 mV vs. an Ag/AgCl reference electrode. A 20-microliters volume of ten-fold diluted plasma was injected without any pre-treatment. Under the described conditions, methanol and ethanol were well resolved from each other and from the "front" of the chromatogram. The limit of detection was ca. 2.5 nmol for ethanol and 0.6 nmol for methanol in plasma, at a signal-to-noise ratio of 3. Excellent linearity was observed for ethanol, in the range 0.125-4 micrograms injected (r = 0.9999). In contrast, the response for methanol was markedly non-linear above 500 micrograms injected, presumably owing to progressive saturation of the reactor. The precision and accuracy of the assay were satisfactory, as was the reactor life (one month).     

Simple method for the simultaneous determination of acetylcholine, choline, noradrenaline, dopamine and serotonin in brain tissue by high-performance liquid chromatography with electrochemical detection

A simple method for the simultaneous determination of acetylcholine, choline, noradrenaline, dopamine and serotonin in brain tissue was developed by using high-performance liquid chromatography with electrochemical detection. These compounds are analysed in a single chromatographic run within 30 min with a simple sample clean-up procedure. The detection system consists of two electrochemical detector cells aligned in series: a glassy-carbon electrode for catecholamines and serotonin, and a platinum electrode for acetylcholine and choline. For the detection of the latter compounds, they were converted enzymatically into hydrogen peroxide through a column reactor with immobilized acetylcholinesterase and choline oxidase. A column of boronic acid gel was placed just ahead of the immobilized enzyme column to remove catecholamines, which caused interfering responses on the platinum electrode. Two equivalent analytical columns and a column switching were employed to speed up the serotonin assay. Simultaneous determination of these major neurotransmitters in rat brain regions was successfully carried out with the system described.     

Characterization of mediated and non-mediated oxidase enzyme based glassy carbon electrodes

The performance and analytical characteristics of a glassy carbon glutaraldehyde immobilized glucose oxidase electrode have been established with regard to the direct detection of hydrogen peroxide produced from the reaction of glucose with oxygen. Measurements were performed at + 1.1 V vs. SCE, and selectivity was obtained by casting the surface with a cellulose acetate membrane. Results compared favorably with the classical platinum-enzyme probe. The mechanism of ascorbic acid interference in hydrogen peroxide detection is reported. Mediated detection was also investigated for oxidase enzymes (glucose oxidase and xanthine oxidase) immobilized on the bare glassy carbon electrode. The probes were characterized using a specific enzyme mediator in solution (phenazine methosulfate or dichlorophenol-indophenol) plus hexacyanoferrate(III) as an electrochemical mediator. The electrode was poised at + 0.36 V vs. SCE for the detection of hexacyanoferrate(II). The advantages of this dual mediator configuration include high stability and sensitivity of the electrochemical signal and the ability to use less positive potentials for increased selectivity. Application to other enzymes, such as hydrogenases, using such a binary redox configuration is suggested.     

Real‐Time Monitoring of Mass‐Transport‐Related Enzymatic Reaction Kinetics in a Nanochannel‐Array Reactor

To understand the fundamentals of enzymatic reactions confined in micro‐/nanosystems, the construction of a small enzyme reactor coupled with an integrated real‐time detection system for monitoring the kinetic information is a significant challenge. Nano‐enzyme array reactors were fabricated by covalently linking enzymes to the inner channels of a porous anodic alumina (PAA) membrane. The mechanical stability of this nanodevice enables us to integrate an electrochemical detector for the real‐time monitoring of the formation of the enzyme reaction product by sputtering a thin Pt film on one side of the PAA membrane. Because the enzymatic reaction is confined in a limited nanospace, the mass transport of the substrate would influence the reaction kinetics considerably. Therefore, the oxidation of glucose by dissolved oxygen catalyzed by immobilized glucose oxidase was used as a model to investigate the mass‐transport‐related enzymatic reaction kinetics in confined nanospaces. The activity and stability of the enzyme immobilized in the nanochannels was enhanced. In this nano‐enzyme reactor, the enzymatic reaction was controlled by mass transport if the flux was low. With an increase in the flux (e.g., >50 μL min^?1), the enzymatic reaction kinetics became the rate‐determining step. This change resulted in the decrease in the conversion efficiency of the nano‐enzyme reactor and the apparent Michaelis–Menten constant with an increase in substrate flux. This nanodevice integrated with an electrochemical detector could help to understand the fundamentals of enzymatic reactions confined in nanospaces and provide a platform for the design of highly efficient enzyme reactors. In addition, we believe that such nanodevices will find widespread applications in biosensing, drug screening, and biochemical synthesis.     

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.     

微柱液相色谱和电化学检测法测定鼠脑微透析液中神经传递物质

报道了使用微柱色谱和电化学检测器测定鼠脑微透析液中神经传递物质的方法。儿茶酚胺，５－羟色胺和代谢物的检测限为 ０． １～０． ５ ｐｇ。含有乙酰胆碱酯酶和胆碱氧化酶的柱后反应器使分离后的乙酰胆碱和胆碱转化成过氧化氢．再用铂电极或过氧化物酶修饰过的玻碳电极检测过氧化氢。乙酰胆碱的检测限当使用怕电极时是５０× １０－１５ｍｏｌ，当使用酶修饰电极时是１０×１０－１５ｍｏｌ。     

High performance liquid chromatography of biological polyamines using immobilized enzyme as post-column reactor followed by electrochemical detection

A novel analytical method for biological polyamines (putrescine, spermidine and spermine) was developed. Polyamines were separated by ion-pair reversed phase chromatography using a polymer-based octadecyl bonded column. A polyamine oxidase immobilized column worked effectively as a post-column reactor to convert polyamines to hydrogen peroxide which was eventually detected by electrochemical oxidation on platinum electrode. This method required neither tedious derivatization nor gradient elution, permitting us to perform simple and rapid analysis of polyamines. The detection limits were 0.3, 0.6, and 4 pmol injected for putrescine, spermidine, and spermine, respectively with a linear range of two to three orders of magnitude. Chromatograms obtained with samples from human urine and rat brain homogenates demonstrated the high sensitivity and selectivity of the method.     

A flow system with a conventional spectrophotometer for the chemiluminescent determination of lactic acid in yoghurt

A flow-based analytical procedure for lactate determination in yoghurt by chemiluminescence using a conventional UV-Vis spectrophotometer as detector is described. The radiation source was switched off. The flow cell was machined in acrylic with a 1 mm path length and a 80 mm(2) surface and was positioned 2 mm distal of the photodetector (100 mm(2) sensible area) in order to improve detection. The flow network computer-controlled comprised a set of three-way solenoid valves assembled to implement the multicommutation approach. The chemiluminescence was obtained by using the reaction of luminol with hydrogen peroxide, catalyzed by hexacyanoferrate (III) after enzymatic reaction with lactate. The lactate oxidase enzyme was immobilized on porous silica beads (glass aminopropyl, SIGMA). The signal generated by the spectrophotometer reaction was read by the microcomputer and stored as a function of time for further treatment. Immobilization condition, enzyme concentration, temperature, pH, stability of the enzymatic reactor, and flow rates were investigated. The feasibility of the system was ascertained by analyzing a set of yoghurt samples. Results were in agreement with those obtained by a conventional method (Boehringer UV-Kit), and no significant difference at 95% confidence level was observed. A linear response within 10-125 mg l(-1)l-lactate, a 1.9% standard deviation (n=10), and an analytical throughput of 55 determinations per hour were achieved.     

The stable o‐phthalaldehyde‐ferrocene/6‐ferrocenyl‐1‐hexanethiol pre‐column derivatization high‐performance liquid chromatography of dimethylarginine by novel dual fluorescence and electrochemical detector

Monomethylarginine, asymmetric dimethylarginine and symmetric dimethylarginine were separated on a Wakopak Combi ODS with an acetonitrile–100 mm potassium phosphate buffer (pH 7.0; 1:1, v/v). Dimethylarginines were derived from o‐phthalaldehyde for the fluorescence detector and from 6‐ferrocenyl‐1‐hexanethiol for the electrochemical detector. The detection limits of the dimethylarginines in spiked plasma were 0.3–0.5 pmol by electrochemical detection and 1–2 pmol by fluorescence detection. The detection limits were improved over 30 times by electrochemical detection and 10 times by fluorescence detection compared with previous reports. In previous derivatization liquid chromatography, the reaction solutions, o‐phthalaldehyde, 2‐mercaptethanol and dimethylarginines were unstable and required quick derivatization at 4°C. By our proposed pre‐column methods, the dimethylarginines were derivatized at room temperature and the fluorescent products were stable for 6 h. The manipulation performance was greatly advanced compared with previous LC reports. This is the first report on stable and sensitive dimethylarginines by dual detection. The selectivity was also improved by dual detection. The proposed method was applied to preliminary monitoring of dimethylargines in plasma and urine. Copyright © 2012 John Wiley & Sons, Ltd.     

Amperometric Determination of Galactose,Lactose and Dihydroxyacetone Using Galactose Oxidase in a Flow Injection System with Immobilized Enzyme Reactors and On-Line Dialysis

Abstract

A flow injection manifold containing a dialyzer and reactors with immobilized galactose oxidase and peroxidase was used for the determination of galactose in urine, lactose in milk and dihydroxyacetone in a biotechnological reaction medium. The hydrogen peroxide which is formed by the galactose oxidase reaction was detected by amperometric reduction of a mediator. The latter had been produced from hydrogen peroxide in a peroxidase catalyzed reaction. The hydrogen peroxide detection step was studied with several mediators and hexacyanoferrate (II) was selected. An ion exchange HPLC procedure was used to purify the galactose oxidase, in particular from catalase, and the kinetics and the selectivity of a reactor containing the immobilized enzyme was investigated. Columns for removal of certain interferents such as ascorbic acid were used in the determination of galactose in urine. The response to galactose standards was linear from the detection limit of 2 μM to 60 mM. The throughput was 45 samples per hour and the relative standard deviation 0.4%.     

High performance liquid chromatography of glucose using a post-column reactor of immobilized enzyme followed by electrochemical detection (glucose-LCEC)

A sensitive and selective, reasonably fast method for the determination of glucose content has been developed. A glucose oxidase immobilized column was coupled to a small-size anion exchange column/borate buffer chromatograph. The hydrogen peroxide produced in the enzyme reaction was detected directly by an amperometric detector using a platinum working electrode. The detection limit was 0.03 ppm (1.5 x 10(-7) M, 3 pmol/injection). The linear dynamic range was three orders of magnitude at least. The system was stable and reproducible both in short- and long-term operation. The proposed method is suitable for analysis of complicated matrices of biological samples because of its good selectivity and sensitivity.     

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.     

别嘌呤醇对黄嘌呤氧化酶的抑制作用研究

用聚苯胺黄嘌呤氧化酶电极（生物传感器）研究了别嘌呤醇对黄嘌呤对氧化酶的抑制作用，实验结果表明：别嘌呤醇对黄嘌呤氧化酶有明显的抑制作用，但这种抑制作用是可逆的，抑制剂在存在使固定酶的表观米氏常数增大但并没有影响催化反应的最大速率，所以别嘌呤是黄嘌呤氧化酶的一种可逆竞争抑制剂，抑制剂对固定酶的最适ｐＨ值没有影响，而对反应活化能略能影响。     

Diamine oxidase and putrescine oxidase immobilized reactors in flow injection analysis: a comparison in substrate specificity

Enzyme reactors for the determination of biogenic amines have been developed using diamine oxidase (DAO) from porcine kidney and from lentil and putrescine oxidase (PUO) from microorganism (Micrococcus roseus). Determination is based on the electrochemical oxidation of enzymatically produced H(2)O(2) at platinum electrode poised at 600 mV versus Ag/AgCl. The enzymes are immobilized on controlled pore glass beads activated by glutaraldehyde in a small reactor (diameter 5 mm, length 50 mm) and included in a flow injection analysis assembly. The reactor using DAO from porcine kidney as the biochemical component responds mainly to histamine (with a detection limit of 0.5 muM), and it can be used for the evaluation of fish spoilage. The PUO reactor shows a significant response only to putrescine. It is linear in the range 0.07-500 muM. The reactor using DAO from lentil is sensitive to several amines and it could be useful to evaluate a total value. The buffer used for both types of oxidase based sensors is phosphate 0.10 M pH 7.0 containing 0.10 M NaCl.     

Determination of fructosyl amino acids and fructosyl peptides in protease-digested blood sample by a flow-injection system with an enzyme reactor.

A flow-injection system with an enzyme reactor was proposed for the measurement of fructosyl amino acids and fructosyl peptides in protease-digested blood samples. A fructosyl-amino acid oxidase (FAOX-TE) and two fructosyl-peptide oxidases (FPOX-CE and FPOX-CET) were covalently immobilized onto an inert support. They were used as the enzyme reactor in a FIA system with a hydrogen peroxide electrode. In particular, the FPOX-CET reactor possessed high selectivity for the detection of fructosyl valine (FV) and fructosyl valyl histidine (FVH) and an excellent operational stability. The proposed FIA system with the FPOX-CET reactor responded linearly to the concentration of FV over the dynamic range of 7.8 x 10(-6) to 5.8 x 10(-4) M. The present method could be successfully applied to the assay of FV and FVH in the protease-digested blood samples.     

Amperometric Biosensor Based on Enzymatic Reactor for Choline Determination in Flow Systems

A simple, selective and stable biosensor with the enzymatic reactor based on choline oxidase (ChOx) was developed and applied for the determination of choline (Ch) in flow injection analysis with amperometric detection. The enzyme ChOx was covalently immobilized with glutaraldehyde to mesoporous silica powder (SBA‐15) previously covered by NH₂‐groups. This powder was found as an optimal filling of the reactor. The detection of Ch is based on amperometric monitoring of consumed oxygen during the enzymatic reaction, which is directly proportional to Ch concentration. Two arrangements of an electrolytic cell in FIA, namely wall‐jet cell with working silver solid amalgam electrode covered by mercury film and flow‐through cell with tubular detector of polished silver solid amalgam were compared. The experimental parameters affecting the sensitivity and stability of the biosensor (i. e. pH of the carrier solution, volume of reactor, amount of the immobilized enzyme, the detection potential, flow rate, etc.) were optimized. Under the optimized conditions, the limit of detection was found to be 9.0×10^?6 mol L^?1. The Michaelis‐Menten constant for covalently immobilized ChOx on SBA‐15 was calculated. The proposed amperometric biosensor with the developed ChOx‐based reactor exhibits good repeatability, reproducibility, long‐term stability, and reusability. Its efficiency has been confirmed by the successful application for the determination of Ch in two commercial pharmaceuticals.     

Compact polytetrafluoroethylene assembly-type capillary electrophoresis with chemiluminescence detection

We have developed a compact polytetrafluoroethylene (PTFE) assembly-type capillary electrophoresis with chemiluminescence (CL) detection system. Luminol-microperoxidase-hydrogen peroxide chemiluminescence reaction was adopted. The device is rectangular in shape (60 mm x 40 mm x 30 mm) and includes three reservoirs (sample, migration buffer, and detection reservoirs) with electrodes. The detection reservoir includes an optical fiber to transport light at the capillary tip to a photomultiplier tube. Isoluminol isothiocyanate (ILITC) was analyzed as a model using this device with fused-silica or polytetrafluoroethylene capillary tubes 10 cm in length. We also used the sample reservoir as a reactor for an immune reaction between anti-human serum albumin immobilized on glass beads and isoluminol isothiocyanate-labeled human serum albumin. The present polytetrafluoroethylene assembly with the capillary tube was useful as a palm-sized analysis device for separation and detection, as well as a reactor.     

Nanoconfinement Effects: Glucose Oxidase Reaction Kinetics in Nanofluidics

Size‐tunable nanofluidic devices coupled to an electrochemical detector have been designed and then used to study glucose oxidase (GOx) reaction kinetics confined in nanospaces. The devices are fabricated via a photochemical decomposition reaction, which forms nanochannels covered with carboxyl groups. The generated carboxyl groups enable us to chemically pattern biological molecules on the polymer surfaces via covalent bonding. With this approach, the activity of the immobilized biological molecules confined in nanospaces with different sizes has been investigated. GOx species are chemically immobilized on the surface of the nanochannels, catalyzing the oxidation of substrate glucose as it flows through the channels. The enzyme reaction product, hydrogen peroxide, passing through the nanochannels, reaches an electrochemical detector, giving rise to an increase in anodic current. This steady‐state electrochemical current, which responds to various glucose concentrations, can be used to evaluate the GOx activity under confinement conditions. The results show significant nanoconfinement effects that are dependent on the channel size where the reaction occurs, demonstrating the importance of spatial confinement on the GOx reaction kinetics. The present approach provides an effective method for the study of enzyme activity and other bioassay systems, such as cell assays, drug discovery, and clinical diagnosis.     

Detection of Oxidase Generated Hydrogenperoxide by a Solid State Peroxyoxalate Chemiluminescence Detector

Abstract

The compatability of a solid state peroxyoxalate chemiluminescence detector for hydrogen peroxide with an immobilized oxidase reactor is investigated. As a model system glucose oxidase immobilized by electrostatic forces on an ion-exchanger or chemically bonded to glass beads were chosen. The former support is less suitable for immobilization of oxydases due to strong retention of hydrogenperoxide on the ion exchanger.

The relatively little flow dependence of these systems renders them suitable for low-cost manual sample injection monitors as well as in a flow injection analyses (FIA) mode with low-cost pumping systems. The system was operated with 80% acetonitrile water solutions. A detection limit of 8 × 10^?7M of glucose was achieved in directly injected samples.

Enzymes more sensitive to organic solvents can be operated with pure water and adjustment for optimal chemiluminescence condition is achieved with a make-up flow prior to detection. A detection limit of 5 × 10^?8M glucose is achieved under these conditions. The feasability of this approach to other oxidase based monitors and to detection in liquid chromatography is discussed.