Simultaneous analysis of methionine- and leucine-enkephalin from rat brain: quantification by liquid chromatography-electrochemistry

This study focuses on the application of liquid chromatography with electrochemical detection (LC-ED) for the analysis of methionine-enkephalin (ME) and leucine-enkephalin (LE) extracted from rat brain regions. The high applied potentials necessary for enkephalin detection required the development of an efficient sample processing protocol. Brain extracts were processed using chromatographic mode sequencing (CMS). The decrease in electroactive interfering substances by CMS improved the chromatographic resolution of ME and LE and the electrode performance. Other qualitative and analytical methods were used to evaluate the enkephalin data obtained by LC-ED for rat brain regions. This study demonstrates that LC-ED provides both the sensitivity and specificity necessary for the analysis of enkephalins from rat brain regions.     

An amperometric detector for use with small-bore liquid chromatographic columns

A miniaturized electrochemical flow cell for amperometric detection in liquid chromatography has been designed to meet the requirement of low volumetric dispersion when using small-bore columns. The volume of the thin-layer cell is in the range 10-50 nl, depending on the spacer dimensions, with the electrode area being about 1 mm2. The cell is constructed from glass and designed in such a way that the working electrode can be mounted without alterations to its surface state. The flow-rate dependence, contribution to peak dispersion and linear dynamic range were investigated. After separation on a 250 X 1 mm I.D. column, a detection limit for dopamine of about 0.15 pg was achieved with a linear dynamic range of 10(6). The variance of the extra-column dispersion was determined to be less than 0.3 microliter2, including injector and connecting capillaries. The applicability of the detector is illustrated with the detection of dopamine and its major metabolites, dihydroxyphenylacetic acid and homovanillic acid, and of Met- and Leu-enkephalin in samples of biological origin.     

Determination of drug-protein interactions by microdialysis coupled with liquid chromatography and electrochemical detection based on a nickel hexacyanoferrate modified electrode

Microdialysis sampling coupled with liquid chromatography-electrochemical detection (LC-ED) was developed and applied to determine the interaction of thiopurine (TP) with bovine serum albumin (BSA). A nickel hexacyanoferrate (NiHCNFe) modified electrode was fabricated and used as the working electrode in LC-ED for the determination of TP. Cyclic voltammetric experiments demonstrate that this chemically modified electrode (CME) can effectively catalyze the electrooxidation of TP. The mechanism of the catalytic oxidation of TP at the CME involves an 'EC' process. In the LC-ED, the CME also shows good stability and reproducibility for the determination of TP. The limit of detection is 5.0 x 10(-7) mol l(-1) for 6-TP with a signal-to-noise ratio of 3. The utility of microdialysis as a quantitative sampling technique for in vitro studies of drug-protein interactions was studied. The microdialysis experiments were performed in phosphate buffer solution (pH 7.4) containing different molar ratios of the drug and protein at 37 degrees C. The collected microdialysis sample with unbound TP was analyzed at the NiHCNFe CME in the LC-ED. The relative recovery of TP determined in vitro is 18.3% at a perfusion rate of 1.0 microl min(-1) and the RSD is about 2.1%. The association constant and the number of binding sites on a BSA molecule calculated with the Scatchard equation are 3.72 x 10(-3) (1 mol(-1)) and 1.51, respectively. This method provides a fast, sensitive and simple technique for the study of drug-protein interactions.     

Positron-emitting N-[18F]fluoroalkyl and [18F]fluoropyrrolidinyl analogues of eticlopride as potential in vivo radioligands for dopamine D2 receptors.

N-Fluoroalkyl and 4-fluoropyrrolidinyl eticlopride analogues with high affinity toward central nervous system dopamine D2 receptors in vitro were labelled with positron emitting fluorine-18 (t1/2 = 110 min), and their in vivo biodistribution was investigated in rats. N-[18F]Fluoro-ethyl and -propyl eticlopride derivatives showed poor in vivo selectivity in the rat brain. On the other hand, 4-[18F]fluoropyrrolidinyl eticlopride exhibited almost constant and relatively high striatal concentration. The striatal/cerebellar radioactivity ratio, which corresponds to the ratio of a brain D2 receptor-rich to poor region, gradually increased to 5.2-6.4, 90 min after the injection. The striatal accumulation was selectively inhibited by pre-injection of haloperidol, a dopamine D2 antagonist, without affecting accumulation in other tissues. Thus, the selective striatal accumulation of 4-[18F]fluoropyrrolidinyl eticlopride in striatal tissue appears to be due to the specific binding to dopamine D2 receptors.     

Flexible dopamine-sensing fiber based on potentiometric method for long-term detection in vivo

Achieving real-time, continuous and long-term monitoring of dopamine(DA) in vivo is essential for revealing brain functions and preventing and treating neurogenic diseases. However, it remains challenging to achieve a low limit of detection(LOD) and high neuron-compatibility at the same time for the current microsensors, resulting in the failure of long-term and accurate detection of DA in vivo. A DA-sensing fiber was achieved by the potentiometric method to possess a low LOD of 5 nM, 1-3 orders of magnitude lower than amperometry and differential-pulse voltammetry. The sensing fiber showed a wide linear range from 5 to 185 nM that well matched the DA concentration(26-40 nM) in vivo. After implantation, the sensing fiber showed no influence on the firing rates of neurons with the potentiometric test, indicating high neuron-compatibility. It was then integrated with electrophysiology to simultaneously monitor DA variation and electrical signal in the brain, with stable monitoring of DA change in vivo for 8 weeks. The sensing fiber was flexible and stably worked after hundreds of bending, and it showed high sensitivity even after protein adsorption, thus offering a reliable tool for neuroscience.     

Determination of cis-diamminedichloroplatinum(II) in human plasma using ion-pair chromatography with electrochemical detection

A thin-layer gold/mercury electrode and a hanging mercury drop electrode are compared as part of an evaluation of liquid chromatography (LC) with electrochemical detection (ED) for the determination of platinum species in human plasma ultrafiltrate. The platinum species, derived from an aged aqueous solution of the antineoplastic agent cis-diamminedichloroplatinum(II) (CDDP), can be separated by ion-pair chromatography. Variation of a number of parameters is described along with the limitations and advantages of each kind of electrode system. We have used our LC-ED technique to separate CDDP from its hydrolysis products and other non-platinum-containing species in human plasma ultrafiltrate with a detection limit of 62 ng/ml (ppb).     

In vivo electrochemistry with carbon fibre electrodes: Principles and application to neuropharmacology

In vivo voltammetry involves the stereotaxic implantation into the brain of a miniaturised version of the sensitive electrochemical detection system normally used with high-performance liquid chromatography. This article describes the use of working electrodes made from 8 or 30 μm carbon fibres. Their small size makes these electrodes ideally suited for use in small brain nuclei. Chemical and/or electrical pretreatment of these electrodes allows them to be used to monitor the extracellular levels of either neurotransmitter amines such as serotonin and dopamine or their metabolites 5-hydroxyindoleacetic acid and dihydroxyphenylacetic acid.     

快捷两步法制备金纳米电极用于活体多巴胺检测

用微电极进行活体检测神经化学物质属于侵入式分析,会对脑组织产生不可避免的损伤,进而在生理上产生一些信号干扰检测过程. 减小电极的尺寸对于减小对脑组织的损伤非常重要. 该研究报道了一种新型制备金纳米电极的方法并将其用于活体鼠脑内多巴胺分析研究. 这种金纳米电极的制备过程包含两步：1）通过离子溅射在毛细管的尖端覆盖一层金种子;2）把覆盖有金种子的毛细管浸入氯金酸和盐酸羟胺混合溶液中湿法沉积生成连续导电金膜. 制备好的纳米电极尖端约300 ~ 400 nm. 该金纳米电极可以应用于多巴胺的检测,并且在多巴胺浓度1.0 ~ 56.0 μmol·L^-1范围内有很好的线性响应,最低检测限低至0.14 μmol·L^-1（信噪比=3）. 该金纳米电极具有优异的电化学性能,可以成功的应用于检测鼠脑纹状体儿茶酚胺的释放.     

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.     

Monitoring of dopamine and its metabolites in brain microdialysates: method combining freeze-drying with liquid chromatography-tandem mass spectrometry

A sensitive assay method was developed for a parallel, rapid and precise determination of dopamine and its metabolites, homovanillic acid, 3-methoxytyramine and 3,4-dihydroxyphenylacetic acid, from brain microdialysates. The method consisted of a pre-treatment step, freeze-drying (lyophilization), to concentrate dopamine and its metabolites from the microdialysates, and a detection step using liquid chromatography combined with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). In particular, the reaction monitoring mode was selected for its extremely high degree of selectivity and the stable-isotope-dilution assay for its high precision of quantification. The developed method was characterized by the following parameters: the precision of the developed method was determined as ≥88.6% for dopamine, ≥89.9% for homovanillic acid, ≥86.1% for 3-methoxytyramine and ≥88.1% for 3,4-dihydroxyphenylacetic acid; the mean accuracy was determined as ≥88.2% for dopamine, ≥88.3% for homovanillic acid, ≥85.9% for 3-methoxytyramine and ≥88.6% for 3,4-dihydroxyphenylacetic acid. The developed method was compared to (1) other combinations of pre-treatment methods (solid phase extraction and nitrogen stripping) with LC-MS and (2) another detection method, liquid chromatography, with electrochemical detection. The novel developed method using combination of lyophilization with LC-ESI-MS/MS was tested on real samples obtained from the nucleus accumbens of rat pups after an acute methamphetamine administration. It was proven that the developed assay could be applied to both a simultaneous analysis of all four substrates (dopamine, homovanillic acid, 3-methoxytyramine and 3,4-dihydroxyphenylacetic acid) in microdialysis samples acquired from the rat brain and the monitoring of their slight concentration changes on a picogram level over time following methamphetamine stimulus.     

Determination of phenols in water by on-line solid-phase disk extraction and liquid chromatography with electrochemical detection

Poly(styrene-divinylbenzene) (PS-DVB) membrane extraction disks were used as sorbents for the on-line solid phase extraction of 13 phenols (nitro and chlorophenols) in river and tap waters. Determination was performed by liquid chromatography with electrochemical detection (LC-ED). An acetate buffer-acetonitrile-methanol mixture as mobile phase and amperometric detection at +1100 mV were used. High water volumes, up to 250 ml, can be preconcentrated without loss of phenols (recoveries between 80% and 100%) except for the more polar ones. Moreover, detection limits between 0.01 and 0.1 μg l⁻¹ in tap water and between 0.1 and 1.0 μg⁻¹ in river water were obtained. The method has been applied to the analysis of two river water samples.     

Microelectrodes with gold nanoparticles and self-assembled monolayers for in vivo recording of striatal dopamine

Electrochemical determination of in vivo dopamine (DA) using implantable microelectrodes is essential for monitoring the DA depletion of an animal model of Parkinson's disease (PD), but faces substantial interference from ascorbic acid (AA) in the brain area due to similar electroactive characteristics. This study utilizes gold nanoparticles (Au-NPs) and self-assembled monolayers (SAMs) to modify platinum microelectrodes for improving sensitivity and specificity to DA and alleviating AA interference. With appropriate choice of ω-mercaptoalkane carboxylic acid chain length, our results show that a platinum microelectrode coated with Au-NPs and 3-mercaptopropionic acid (MPA) has approximately an 881-fold specificity to AA. During amperometric measurements, Au-NP/MPA reveals that the responsive current is linearly dependent on DA over the range of 0.01-5 μM with a correlation coefficient of 0.99 and the sensitivity is 2.7-fold that of a conventional Nafion-coated electrode. Other important features observed include fast response time (below 2 s), resistance to albumin adhesion and low detection limit (7 nM) at a signal to noise ratio of 3. Feasibility of in vivo DA recording with the modified microelectrodes is verified by real-time monitoring of electrically stimulated DA release in the striatum of anesthetized rats with various stimulation parameters and administration of a DA uptake inhibitor. The developed microelectrodes present an attractive alternative to the traditional options for continuous electrochemical in vivo DA monitoring.     

Selective detection of extracellular glutamate in brain tissue using microelectrode arrays coated with over-oxidized polypyrrole

We describe the selective measurement of extracellular glutamate concentration in rodent brain using ceramic-based platinum microelectrode arrays (MEAs) coated with electropolymerized, over-oxidized polypyrrole (OPP) as a permselective barrier to interference from dopamine and ascorbate. OPP-coated MEAs displayed similar sensitivity (97 +/- 9 nA microM(-1) cm(-2)) and response time (ca. 1 s) to glutamate as previously described Nafion-coated MEAs but, unlike Nafion-coated MEAs, were unresponsive to 5 microM dopamine. Such OPP-coated MEAs afforded selective detection of evoked glutamate release in the dopamine-rich striatum of the mouse brain.     

Characterization of amperometry for in vivo measurement of dopamine dynamics in the rat brain

Constant potential amperometry with Nafion-coated carbon-fiber electrodes has been evaluated as a technique for in vivo detection of the neurotransmitter dopamine. The results of this technique have been compared to results obtained with fast-scan cyclic voltammetry at the same electrode during release of dopamine into the extracellular space of the brain during electrical stimulation of neurons. The data indicate that constant potential amperometry is a viable technique for detecting low concentrations of dopamine. Dopamine permeates the film more quickly with constant-potential amperometry than with repeated fast-scan cyclic voltammetry as predicted by diffusion equations. For the case of cyclic voltammetry, it is demonstrated that the temporal delay caused by diffusion through Nafion film can be removed by deconvolution procedures. Despite the suitability of constant potential amperometry as an in vivo monitoring technique, it does have several disadvantages when compared to fast-scan cyclic voltammetry. The diffusion layer extends outside of the Nafion film making determination of concentration based on in vitro calibrations more difficult to interpret. The reported concentrations are larger than obtained by cyclic voltammetry, a technique with the diffusion layer restricted to the Nafion film, and this result is likely an underestimation of the effect of the catalytic reaction between the o-quinone of dopamine and ascorbate. Amperometry was found to provide only slightly improved signal-to-noise ratios than cyclic voltammetry despite the use of greater filtering. This was because the advantage of dopamine accumulation in the film was lost. In addition, the small magnitude of the amperometric signal makes it more susceptible to electrical interference.     

Capillary electrophoresis‐laser‐induced fluorescence detection of rat brain catecholamines with microwave‐assisted derivatization

In this study, a rapid and sensitive method is described for the catecholamines detection in rat brain. CE with LIF detection for the determination of FITC derivatized catecholamines (dopamine, epinephrine, and norepinephrine) was demonstrated. Conventional water bath and microwave‐assisted derivatization methods were employed and a significant reduction in the derivatization time from 2 h for the conventional water bath at room temperature (ca. 25°C) to 2 min for the microwave‐assisted derivatization was achieved. Online sample concentration of field‐amplified sample stacking (FASS) method was employed to achieve higher sensitivities (the detection limits obtained in the normal injection mode ranged from 2.6 to 4.5 ng L^?1 and in the FASS mode ranged from 22 to 34 pg L^?1). Furthermore, this microwave‐assisted derivatization CE–LIF method successfully determined catecholamines in rat brain with as low as 100 ng L^?1 (FASS mode) to 10 μg L^?1 (normal injection mode). This CE–LIF method provided better detection ability when compared to the best reports on catecholamines analyses.     

Ceramic-based multisite microelectrode array for rapid choline measures in brain tissue

A ceramic-based multisite microelectrode array to measure choline in vivo in brain tissues is described. The microelectrodes were linear to 200 μM choline (R²=0.999±0.001) with a detection limit of approximately 0.4 μM (S/N of 3) in both single microelectrode and self-referencing amperometric recording modes. The 90% rise time of the sensor was 1.4 s, allowing for rapid measures of choline. Good selectivity (>300:1) was observed over interferents such as ascorbic acid, uric acid, and DOPAC in the single microelectrode mode. However, a self-referencing recording mode was needed to remove potassium-evoked dopamine signals in rat striatum. In vivo measurements of choline in the rat brain are presented.     

Biosensor Based on Electrocodeposition of Carbon Nanotubes/Polypyrrole/Laccase for Neurotransmitter Detection

A new biosensor has been developed by a simple electrocodeposition of multiwalled carbon nanotubes (MWCNT), polypyrrole (PPy) and laccase (Lac) on the platinum (Pt) electrode surface. The neurotransmitter biosensor was applied to the detection of dopamine in urine samples using differential pulse voltammetry (DPV). The Pt/MWCNT/PPy/Lac biosensor exhibited a detection limit of 0.14 µM, which is an adequate level for monitoring dopamine in urine. Reproducibility and repeatability values of 2.9 % and 1.7 %, respectively, were obtained compared to the conventional procedure. The proposed biosensor was successfully applied in the determination of dopamine in urine, and the obtained results were in full agreement with those from the HPLC procedure.     

Phase-selective alternating current polarographic assay of methotrexate in human serum

The a.c. polarographic behaviour of methotrexate (MTX) was studied and the nature of the process taking place at the dropping mercury electrode was elucidated. Both the molecule and its reduced product appeared to be absorbed at the surface of the electrode. The observed electrochemical behaviour was in close agreement with theoretical predictions for an absorbed species which is reversibly reduced. This permitted a very sensitive and selective determination of MTX in serum samples by phase-selective a.c. polarography. The method, which involves a very simple and rapid previous clean-up procedure, has a limit of detection of 3.0 × 10^?7 M in serum and the results compare well with those obtained by liquid chromatography with oxidative amperometric detection (LC-ED) at +1.1 V. Both methods form an interesting alternative to others previously reported for monitoring MTX in cancer patients under treatment with high doses of the drug.     

Highly Sensitive Dopamine Detection Using a Bespoke Functionalised Carbon Nanotube Microelectrode Array

Understanding how the brain works requires developing advanced tools that allow measurement of bioelectrical and biochemical signals, including how they propagate between neurons. The introduction of nanomaterials as electrode materials has improved the impedance and sensitivity of microelectrode arrays (MEAs), allowing high quality recordings of single cells in situ using electrode diameters of ≤20 μm. MEAs also have the potential to measure electroactive biological molecules in situ, such as dopamine, a neurotransmitter in the nervous system. Thus, this work focused on fabricating a functionalised carbon nanotube (CNT)‐based MEA to demonstrate its potential for future measurement of small signals generated from excitable cells. To this end, the functionalised CNT MEA has recorded one of the lowest electrochemical interfacial impedances available in the literature, 2.8±0.2 kΩ, for an electrode of its geometric surface area. Electrochemical detection of dopamine revealed again one of the best sensitivity values per area available in the literature, 9.48 μA μM⁻¹ mm⁻². Additionally, a limit of detection of 7 nM was recorded for dopamine using the functionalised CNT MEA, with selectivity against common electrochemical interferents such as ascorbic acid. These results indicate improvement beyond currently available MEAs, along with the feasibility of using these devices for multi‐site detection of physiologically relevant electroactive biomolecules.     

Capillary electrophoresis microchip coupled with on-line chemiluminescence detection

In the present work, chemiluminescence detection was integrated with capillary electrophoresis microchip. The microchip was designed on the principle of flow-injection chemiluminescence system and capillary electrophoresis. It has three main channels, five reservoirs and a detection cell. As model samples, dopamine and catechol were separated and detected using a permanganate chemiluminescent system on the prepared microchip. The samples were electrokinetically injected into the double-T cross section, separated in the separation channel, and then oxidized by chemiluminescent reagent delivered by a home-made micropump to produce light in the detection cell. The electroosmotic flow could be smoothly coupled with the micropump flow. The detection limits for dopamine and catechol were 20.0 and 10.0 μM, respectively. Successful separation and detection of dopamine and catechol demonstrated the distinct advantages of integration of chemiluminescent detection on a microchip for rapid and sensitive analysis.