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。     

Determination of Acetylcholine and Choline in Rat Brain Tissue by Liquid Chromatography/Electrochemistry Using an Immobilized Enzyme Post Column Reactor

Abstract

Following separation by conventional LC, acetylcholine and choline are converted to hydrogen peroxide in a packed bed reactor consisting of covalently bound acetylcholinesterase and choline oxidase. Hydrogen peroxide, the ultimate enzymatic product, is detected amperometrically at a platinum electrode thin-layer cell. This method is simple and highly sensitive to the detection of acetylcholine and choline, with detection limits of about 100 femtomoles. The immobilized enzyme columns were stable for at least 60 days and conserved precious and expensive supplies of enzyme relative to continuous addition schemes. The apparatus is generally applicable to other enzymatic reactions which yield electroactive products.     

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.     

Highly sensitive assay for choline acetyltransferase activity by high-performance liquid chromatography with electrochemical detection

A highly sensitive assay for choline acetyltransferase activity by high-performance liquid chromatography with electrochemical detection was devised. This assay method is based on the separation of acetylcholine and choline on a Develosil Ph-5 reversed-phase column (a phenyl column), followed by their enzymatic conversion to hydrogen peroxide through post-column reaction with acetylcholinesterase and choline oxidase. The sensitivity of the system is excellent and 5 pmol of acetylcholine enzymatically formed could be detected. The linearity between the peak height and the amount of acetylcholine was observed over the range of 5 pmol to 5 nmol. Some enzymatic properties were investigated by using a soluble fraction of bovine caudate nucleus as enzyme. The Michaelis constants of the enzyme for choline and acetyl coenzyme A were 0.3 mM and 0.03 mM, respectively. The enzyme exhibited the maximum activity over the pH range 7.4-9.5. The regional distribution of choline acetyltransferase activity in rat brain was examined. The order of the activity from the highest to the lowest agreed with the reported brain distribution of the enzyme: striatum, pons plus medulla oblongata, cerebral cortex, thalamus plus hypothalamus, olfactory bulb and cerebellum.     

Flow-injection determination of glutathione with amperometric monitoring of the enzymatic reaction

Glutathione sulfhydryl oxidase is immobilized on oxirane-acrylic beads (Eupegit-C) and packed in a small column. The simple system for glutathione comprises the immobilized enzyme column and a flow-through membrane-covered platinum/silver/silver chloride electrode pair for detection of hydrogen peroxide. The calibration graph for glutathione was linear from 0.05 to 1.0 mM for 200-μl samples. The assay took 3 min. The relative standard deviation for 0.5 mM glutathione was 2% (n=10).     

Amperometric determination of cholinesterase with use of an immobilized enzyme electrode

A choline-sensitive electrode consisting of an immobilized choline oxidase layer and an oxygen electrode is described. Cholinesterase (0.5–60 I.U. l^-1) is measured by addition of acetylcholine, and detection of the choline produced. The precision is 3%, and the electrode is stable for more than 2 weeks (140 assays).     

Analysis of acetylcholine and choline in microdialysis samples by liquid chromatography/tandem mass spectrometry

A sensitive liquid chromatography/electrospray ionisation tandem mass spectrometric (LC/ESI-MS/MS) method was developed for the analysis of acetylcholine and choline in microdialysis samples. A Ringer's solution that contains high (150 mM) concentrations of inorganic salts was used to extract acetylcholine and choline from a rat or mouse brain. The separation of acetylcholine, choline, an internal standard acetyl-beta-methylcholine, endogenous compounds and inorganic cations was achieved with hydrophilic interaction chromatography using a diol column. The eluent consisted of 20 mM ammonium formate (pH 3.3) and acetonitrile (20:80) which is favourable for the ESI process. Limits of detection (signal-to-noise (S/N) ratio = 3) of 0.02 nM (0.2 fmol) for acetylcholine and 1 nM (10 fmol) for choline were observed using standards diluted in Ringer's solution. A good linearity was obtained from the limit of quantitation: 0.1 nM (S/N ratio = 10) to 50 nM (r = 0.999) for acetylcholine and within the concentration range of 100-3500 nM (r = 0.998) for choline. The between-day repeatability of the method was good; RSD was 3.1% at 1 nM level of acetylcholine and 3.5% at 1000 nM level of choline. The recoveries for addition of 1 or 2.5 nM acetylcholine and 0.2 or 1 microM choline in microdialysis balancing samples were between 93 and 101% indicating that no suppressing endogenous compounds were co-eluting with acetylcholine or choline. The developed method was applied to the analysis of microdialysis balancing samples collected from rat and mouse brains.     

Immobilized enzymes in clinical and biochemical analysis : Applications to the Simultaneous Determination of Acetylcholine and Choline and to Determination of Lipids

Uses of immobilized enzyme mini-columns in flow-injection systems are described Simultaneous determination of ? × 10^?5 M choline and acetylcholine is achieved by using acetylcholinesterase and choline oxidase columns. A home-made amperometric detector is used to detec the hydrogen peroxide produced enzymatically. An ion-exchange column is used on-line to remove interferences at the amperometric detector during analysis of blood and brain samples. Immobilization of the lipid enzymes phospholipase-C and -D is described. These enzymes are used for the determination of phospholipids. Total phospholipids (1– mM) are determined with a combination of phospholipase-D, lipase and glycerol-3-phosphate oxidase. All the methods described are simple and reproducible and the immobilized enzymes show good stability.     

On-column polymer-imbedded graphite inlet electrode for capillary electrophoresis coupled on-line with flow injection analysis in a poly(dimethylsiloxane) interface

A method for coupling an electrophoretic driven separation to a liquid flow, using conventional fused-silica capillaries and a soft polymeric interface is presented. A novel design of the electrode providing high voltage to the electrophoretic separation was also developed. The electrode consisted of a conductive polyimide/graphite imbedded coating immobilized onto the capillary electrophoresis (CE) column inlet. This integrated electrode gave the same separation performance as a commonly used platinum electrode. The on-column electrode also showed good electrochemical stability in chronoamperometric experiments. In addition, with this electrode design, the electrode position relative to the inlet end of the CE column will always be constant and well defined. The on-line flow injection analysis (FIA)-CE system was used with electrospray ionization (ESI)-time of flight (TOF)-mass spectrometry detection. The preparation of the PDMS (poly(dimethylsiloxane)) interface for FIA-CE is described in detail and used for initial tests of the on-column polymer-imbedded graphite inlet electrode. In this interface, a pressure-driven liquid flow, a make up CE electrolyte and a CE column inlet meet in a two-level cross (95 microm ID) in the PDMS structure, enabling independent flow characterization.     

Evaluation of the inhibition of choline uptake in synaptosomes by capillary electrophoresis with electrochemical detection

A direct method for evaluating choline uptake by the high-affinity choline transport system in synaptosomes was developed using capillary electrophoresis (CE) with electrochemical (EC) detection. On-column EC detection of choline and the internal standard, butyrylcholine, was accomplished with a 25 microm platinum electrode modified with the enzymes, choline oxidase and acetylcholinesterase. Choline uptake was evaluated as a function of choline concentration and a KM value of 1.7 microM was determined. The method was also used to evaluate a new class of redox affinity inhibitors of choline transport. In particular, the effectiveness of 3-[(trimethylammonio)methyl]catechol (TMC) as an inhibitor of choline uptake was examined independently and relative to the inhibition of the well-known inhibitor of choline transport, hemicholinium-3. The IC50 and KI for TMC were determined to be 30 microM and 14 microM, respectively. The combination of the selectivity and sensitivity afforded by CEEC provides a relatively straightforward approach for monitoring choline transport in synaptosomes.     

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).     

Biosensors based on polymer networks formed by gamma irradiation crosslinking

Water-soluble polymers immobilized by gamma radiation have been investigated as a means of developing electrochemical sensors. Enzyme-based sensors for glucose and lactate have been made by immobilizing glucose oxidase and lactate oxidase, respectively, on platinized graphite electrodes. The enzyme is entrapped in a polymeric network of poly(vinyl alcohol) that is formed by gamma radiation crosslinking. Electrodes coated with poly (N-vinylpyrrolidone) and its corresponding monomer and then crosslinked with gamma radiation show an extraction of catecholamines into the polymer film that enhances the analytical signal for their detection by electrochemical oxidation. Poly(dimethyldiallylammonium chloride) spin-coated on a screen-printed electrochemical cell provides sufficient ionic conductivity for the cell to function as a gas sensor for oxygen, which is detected by reduction at a platinum working electrode.     

Swept-potential oxidative detection in flow streams

A swept-potential electrochemical detector, operated in the oxidative staircase voltammetric metric mode, is demonstrated for the high-performance liquid chromatography of a mixture of catecholamines. Voltammetric limits of detection are approximately 30 pg or 1 nM and chromatographic limits of detection are approximately 250 pg. The use of a platinum working electrode in a wall-jet cell configuration, with potential pulses for cleaning and activation between each sweep, results in a cell that has maintained a constant response for over a year without mechanical refinishing of the electrode surface.     

Electrochemical studies on tetrathiafulvalene-tetracyanoquinodimethane modified acetylcholine/choline sensor

A sensor for acetylcholine/choline is described using a tetrathiafulvalene-tetracyanoquinodimethane (TTF-TCNQ) electrode modified with acetylcholine esterase (AChE) and choline oxidase (ChO) enzymes. DC cyclic voltammetry and impedance measurements of the enzyme-modified TTF-TCNQ electrode that indicate the regeneration of choline oxidase at the electrode surface are reported. Effective electrochemical rate constants for the present enzyme electrode are calculated using the expressions derived by Albery et al. (1), which show the enzyme kinetics as the rate-limiting step. The values of the effective electrochemical rate constants are close to those reported by Hale and Wightman (2). The application of the sensor is described for the determination of fluorode ion and nicotine based on the reversible inhibition of AChE activity. The range of detection of fluoride ion and nicotine is found to be 5×10^-6 to 5×10^-4M.     

Simultaneous high-performance liquid chromatographic determination of norepinephrine, serotonin, acetylcholine and their metabolites in the cerebrospinal fluid of anaesthetized normotensive rats.

A high-performance liquid chromatographic method with electrochemical detection was developed for the simultaneous determination of the levels of norepinephrine (NE), serotonin (5-HT), acetylcholine (ACh) and their metabolites in the cerebrospinal fluid (CSF) of anaesthetized rats. The response curve for each compound was linear for the concentration way of interest. The within- and between-day coefficients of variation (C.V.) for NE, 5-HT and their metabolites were less than 7.85% and 15.67%, respectively, and those for ACh and choline were less than 3.08% and 6.27%, respectively. This simultaneous determination should be useful for elucidating the noradrenergic, serotonergic and cholinergic nerve activity in the central nervous system.     

Electrodeposited Nickel/Platinum Alloy as a Biosensor for Acetyl Choline

Nickel and platinum find extensive use in preparation of biosensors. In the present work, Ni/Pt alloy was plated on graphite to make acetylcholine sensor. The microstructure, surface composition and electrochemical performance of the electrode was analyzed by different techniques. The sensing performance was evaluated by cyclic voltammetry. The prepared alloy plate exhibited very good linear relationship between acetyl choline concentration and response current. The sensitivity and reproducibility of the prepared electrode were found better than other nickel electrodes reported.     

An Enzyme Sensor For Determination of Acetylcholine and Choline In Rat Brain Extracts

Abstract

A choline enzyme sensor, recently developed by the authors, was used for choline and acetylcholine determination in rat brain extracts, using choline oxidase immobilized on cellulose triacetate membranes, and acetylcholinesterase in homogeneous solution. the method proved useful for assay of the acetylcholine content in a commercial pharmaceutical formulation used in ophthalmology.     

Liquid chromatography with electrochemical detection for the determination of choline and acetylcholine in plasma and red blood cells. Failure to detect acetylcholine in blood of humans and mice

An assay is described for the measurement of choline in plasma and red blood cells using liquid chromatography, an enzyme reactor and electrochemical detection after a simple sample pretreatment. The intra-assay coefficient of variation for choline was 6.2 and 3.8% in plasma and in red blood cells, respectively. Using this method we have re-investigated the presence of acetylcholine in blood constituents. We were not able to demonstrate acetylcholine with a limit of detection of 10 pmol per ml of plasma or per ml of red blood cells.     

Bioelectrochemical Response and Kinetics of Choline Oxidase Entrapped in Polyaniline—Polyacrylonitrile Composite Film

A novel choline oxidase electrode was constructed by entrapping choline oxidase into polyaniline-polyacrylonitrile composite film,The enzyme film was prepared by in situ electropolyme-ritztion of aniline into porous polyacrylonitrile-coated platinum electrode in the presence of choline oxidase ,the enzyme electrode exhibited sensitive and stable electrochemical response to choline ,The kinetics analysis,showed that the mass transport is partially rate-liniting.The influences of pH,applied potential and temperature on the response of the enzyme electrode were also desribed.     

Determination of choline and acetylcholine levels in rat brain regions by liquid chromatography with electrochemical detection

Regional choline (Ch) and acetylcholine (ACh) in rat brain were clearly determined by high-performance liquid chromatography with electrochemical detection. The method is based on that of Potter et al.: the hydrogen peroxide that is enzymatically produced from both compounds is measured and a successful improvement of the method, particularly for purification, is described. Recoveries were 96.1 +/- 1.4% for Ch and 95.6 +/- 2.2% for ACh and amounts as low as 10 pmol could be determined. Prior to measuring the compounds, a newly developed magnetic field microwave instrument (10 kW) was utilized for the rapid inactivation of brain enzymes. The levels of Ch and ACh in brain regions were compared with those reported elsewhere.