Rapid estimation of catecholamines, octopamine and 5-hydroxytryptamine in biological tissues using high-performance liquid chromatography with coulometric detection

A rapid, convenient procedure is described for the simultaneous determination of catecholamines, monohydroxyphenolamines and 5-hydroxytryptamine in biological tissues. The procedure involves homogenization of tissue in perchloric acid, addition of heparin and centrifugation followed by direct injection of the supernatant onto a C18 reversed-phase high-performance liquid chromatographic column. The mobile phase employed sodium dodecyl sulfate as ion pair reagent with 20% acetonitrile and 10-12% methanol as organic modifier. Eluted fractions were detected electrochemically using dual coulometric electrodes operated in screen mode. The procedure has been applied to the analysis of norepinephrine, epinephrine, dopamine, octopamine, tyramine, 5-hydroxytryptamine and tryptophan in a variety of tissues including mammalian heart and brain and insect nerve cord.     

Fluorimetric detection of octopamine in high-performance liquid chromatography and its application to the assay of dopamine beta-monooxygenase in human serum.

A high-performance liquid chromatographic procedure is described for the determination of octopamine. The method, which is based on the separation on a microparticulate bonded strong cation-exchange resin and measurement of the native fluorescence, has been applied to give a sensitive assay of dopamine beta-monooxygenase (EC 1.14.17.1) activity in human serum with tyramine as the substrate. The procedure, which has been designed for use with an automatic sampler, has a detection limit of about 50 pmoles of octopamine, and the analysis time is approximately 10 min per sample.     

Analysis of biogenic amines in the brain of the American cockroach (Periplaneta americana) by gas chromatography-negative ion chemical ionisation mass spectrometry

Biogenic amines in the brain of the American cockroach have been identified and quantified by an extraction-derivatisation procedure involving their reaction with ditrifluoromethylbenzoyl chloride (DTFMB) in the aqueous phase followed by extraction into an organic solvent, hydrolysis of phenolic esters and conversion of free hydroxyl groups to trimethylsilyl (TMS) ethers and subsequent analysis by gas chromatography-negative ion chemical ionisation mass spectrometry. The molecular ion of these DTFMB-TMS derivatives carried most of the ion current which made the method highly specific and gave a potential limit of detection below the picogram level. This method establishes unequivocally that the principal amines in cockroach brain are tyramine, p-octopamine, dopamine, 5-hydroxytryptamine and noradrenaline. In contrast to mammalian nervous tissue, the other positional isomers of octopamine, together with the isomeric synephrines, are absent.     

Determination of gamma-glutamyl conjugates of monoamines by means of high-performance liquid chromatography with electrochemical detection and application to gastropod tissues.

Catabolism of aminergic neurotransmitters in gastropods appears to be primarily by means of gamma-glutamyl conjugation rather than by oxidative deamination as is typical of vertebrates. High-performance liquid chromatography with electrochemical detection was used to develop a method for the routine measurement of gamma-glutamyl conjugates of dopamine and 5-hydroxytryptamine in gastropod tissues.     

In vivo monitoring of the monoamine neurotransmitters in the rat brain by microdialysis coupled with the liquid chromatography dual electrochemical detector

The carbon film based ring-disk dual electrodes in the thin-layer radial flow cell are used as the dual electrochemical detector (DECD) for liquid chromatography (LC) to determine the monoamine neurotransmitters. Cyclic voltammetric experiments show there has great difference in the reversibility of the oxidative reactions of dopamine and ascorbate. Therefore the ring-disk dual electrode arrangement in the radial flow cell can effectively remove the interference of ascorbate and determine dopamine in the LC-DECD. In order to obtain the better collection efficiency (CE) and better peak current of analytes in the LC-DECD, several operational parameters have been investigated: flow rate, pH and the working potentials. Under the optimum conditions, the method shows a good stability and reproducibility to determine dopamine (DA), norepinephrine (NE), 5-hydroxytryptamine (5-HT), epinephrine (E) and 3,4-dihydroxyphenylacetic acid (DOPAC). The limits of detection are 0.1 pmol for DA, 0.1 pmol for NE, 0.1 pmol for 5-HT, 1.0 pmol for E and 0.1 pmol for DOPAC. The application of this method, coupled with microdialysis sampling, for the in vivo determination of the monoamine neurotransmitters in the striatum of the rat brain is satisfactory.     

A Hafnium Oxide Based Voltammetric Platform for the Sensitive Determination of Octopamine

An electrochemical sensor was constructed by modification of a glassy carbon electrode (GCE) with nanoparticles of hafnium oxide (HfO₂) and multi-walled carbon nanotubes (MWCNTs) for the sensitive determination of octopamine. The platform (HfO₂NPs/MWCNTs/GCE) presented an improved anodic peak for octopamine at 0.65 V. The combination of HfO₂ and MWCNTs resulted in outstanding catalytic activity and enhanced the magnitude of the peak response. Results suggest that a three-electron oxidation occurs for the process of octopamine. Voltammetry of octopamine exhibited a dynamic linear response in the concentration range of 1.6×10⁻⁶∼4.8×10⁻⁵ M with a detection limit of 5.4×10⁻⁷ M for octopamine.     

Simultaneous measurement of tyrosine, tryptophan and related monoamines for determination of neurotransmitter turnover in discrete rat brain regions by liquid chromatography with electrochemical detection

Concomitant measurement of monoamine neurotransmitter turnover in discrete rat brain areas with the use of radiolabeled amino acid precursors permits simultaneous evaluation of interacting transmitter systems. [3H]Tyrosine and [3H]tryptophan were administered via indwelling catheters to unrestrained rats. Content and specific activity of norepinephrine, dopamine, 5-hydroxytryptamine, and the metabolites dihydroxyphenylacetic acid, homovanillic acid, and 5-hydroxyindoleacetic acid in addition to tyrosine and tryptophan were quantified by liquid chromatography with electrochemical detection and liquid scintillation counting. The method employs a simple extraction procedure without prior cleanup for chromatography. Neurotransmitter turnover rates that incorporated tyrosine- or tryptophan-specific activities were found to be two to four times greater than those that did not include them.     

Simultaneous determination of biogenic amines and morphine in discrete rat brain regions by high-performance liquid chromatography with electrochemical detection

A simple and sensitive method has been developed for the simultaneous determination of norepinephrine, epinephrine, dopamine, 5-hydroxytryptamine, 5-hydroxyindoleacetic acid, and morphine in discrete rat brain regions by reversed-phase high-performance liquid chromatography with electrochemical detection. Perchloric acid extracts of the tissue were directly injected into the chromatographic system. Each of these compounds gave a linear response over the range of 20-160 ng/ml cerebellar homogenate (0.4-3.2 ng on column). Recoveries of these compounds, added to the homogenates, were complete when compared with standards dissolved in perchloric acid. The average between-run coefficients of variation for all these compounds were lower than 7.4% over the range of 20-160 ng/ml, and the within-run coefficients of variation at 20 ng/ml were lower than 8.7%. The present method has been applied to a study of the effects of intraperitoneal administration of morphine on biogenic amines in several discrete rat brain regions.     

High-performance liquid chromatography of monoamines on phenyl-bound sorbents using organic free mobile phases

Tryptophan and its metabolites, 5-hydroxytryptophan, 5-hydroxytryptamine, 5-hydroxyindolacetic acid, as well as dopamine, homovanilic acid and 2,3-dihydroxyphenylacetic acid, were separated on phenyl bound silica gel using isocratic elution with phosphate buffer. The method was successfully transferred to several other phenyl HPLC columns from different manufacturers simply by adjusting the pH of the buffer. The method has been validated by the determination of the level of monoamines in rat hypothalamus.     

Superior Performance of a MoS2‐RGO Composite and a Borocarbonitride in the Electrochemical Detection of Dopamine,Uric Acid and Adenine

A MoS₂‐RGO composite and borocarbonitride (BC₅N) have been used as electrodes to selectively detect dopamine and uric acid in the presence of ascorbic acid. Both the electrodes show excellent eletrocatalytic activity towards the detection of dopamine, the detection limits being 0.55 μM and 2.1 μM in the case of MoS₂‐RGO and BCN respectively. MoS₂‐RGO shows a linear range of current over the 1–110 μM concentrations of dopamine, while BCN shows over the 2.3–20 μM range. BCN also exhibits satisfactory performance in the oxidation of uric acid with a detection limit of 3.8 μM and the linear range from 4 to 40 μM. The MoS₂‐RGO has also been used to detect adenine as well.     

Simultaneous determination of morphine and monoamine transmitters in a single mouse brain

A simple procedure for the simultaneous determination of morphine and monoamine transmitters was developed. The procedure consisted of (1) n-butanol extraction and (2) separation and quantitative determination by means of high-performance liquid chromatography combined with electrochemical detection. The maximum intracerebral concentration (210 +/- 35 ng/g wet tissue) of morphine was detected 30 min after intramuscular injection (10 mg/kg), which agreed with previous research. Noradrenaline was significantly decreased by morphine injection, while dopamine and 5-hydroxytryptamine were unchanged. However, 3-methoxytyramine, a metabolite of dopamine, was increased, suggesting that the drug increased the turnover rate of dopamine. The procedure used revealed a direct correlation between pharmacokinetics (e.g., distribution of morphine) and pharmacodynamics (e.g. changes of monoamine concentrations) of the drug in vivo.     

Simultaneous determination of monoamine transmitters, precursors and metabolites in a single mouse brain

A simple and sensitive procedure for simultaneous determination of monoamine transmitters and related substances including precursors and metabolites has been developed for a single mouse brain. The proposed procedure involves (1) primary butanol extraction, (2) separation of the substances into either acid or alkaline aqueous layers according to their physicochemical properties, and (3) determination by means of high-performance liquid chromatography with electrochemical detection. Three transmitters (noradrenaline, dopamine and 5-hydroxytryptamine) and their precursors (tyrosine, 3,4-dihydroxyphenylalanine and tryptophan) and major metabolites (normethanephrine, 3-methoxy-4-hydroxyphenylethylene glycol, 3-methoxytyramine, 3,4-dihydroxyphenylacetic acid, 3-methoxy-4-hydroxyphenylacetic acid and 5-hydroxyindoleacetic acid) were selectively separated and sensitively detected in mouse whole brain sample. Although 3-methoxy-4-hydroxymandelic acid was also separated from other substances by authentic chromatography, the substance was not detected in mouse brain. Changes in levels of these substances were examined for drugs whose effects had been previously confirmed. These changes reflected putative effects of the drugs and confirmed that the procedure is effective for neurochemical research into the transmitter system.     

高效液相色谱/电化学法测定大鼠血液和脑组织中单胺类物质的含量

建立了高效液相色谱／电化学检测法测定大鼠脑组织和血液中单胺递质及其代谢产物的方法。能同时测定去甲肾上腺素（NE）、肾上腺素（E）、3,4-二羟基苯乙酸（DOPAC）、多巴胺（DA）、高香草酸（HAV）、5-羟色胺（5-HT）,并能和内标3,4-二羟卞胺（DHBA）分离良好。本方法快速、简便,回收率为92％-105％,线性范围2．8-680ng／mL,检出限为0．05ng（S／N=3）。本方法已应用在服用中药的大鼠下丘脑组织及血液的测定中,数据显示,本法能满足测定要求。     

Electrocatalytic response of the modified ZnO-G electrodes towards the oxidation of serotonin with multi metallic corrosion protection

Serotonin receptors, also known as 5-hydroxytryptamine (5-HT), play a significant part in neurotransmission and regulate various neurological activities. Researchers are working on finding the appropriate material for sensing 5-HT. Herein, we developed a dual role ZnO-G nanocomposites with electrocatalytic sensing and anticorrosive behavior against Mg, Al, and Cu alloy. ZnO has an oxidative response of 4.55 μA at 0.378 V, and So-Zn-G electrode has reduced overpotential and enhanced current response. The electroactive surface area was found to be ～596 cm²/g. The calculated limit of detection (LOD) was 6.48 μM and linear range of 20–100 μM. Positive corrosion protection activity of ZnO-G was noted for all three alloys, opening opportunities for its integrated effects (anticorrosive and serotonin sensing) in electronic devices.     

Improved method for the determination of 5,6-dihydroxytryptamine and 5,7-dihydroxytryptamine in tissue using high-performance liquid chromatography with electrochemical detection.

A liquid chromatographic method with electrochemical detection is described for the determination of the 5-hydroxytryptamine (5-HT) neurotoxins 5,6-dihydroxytryptamine (5,6-DHT) and 5,7-dihydroxytryptamine (5,7-DHT) in rat brain tissue. This method has also been used for the determination of 5-hydroxyindoleacetic acid, homovanillic acid and 5-HT in other tissue samples. The method is based on extraction of the indoles from brain samples with perchloric acid followed by reversed-phase liquid chromatography with electrochemical detection. The detection limit is 1 ng per 100 mg of tissue. This paper describes a quick and reliable method of assaying the 5-HT neurotoxins 5,6-DHT and 5,7-DHT in brain tissue, which is improved compared to currently available assays.     

Identification and quantitation of N-acetyl metabolites of biogenic amines in the thoracic nervous system of the locust, Schistocerca gregaria, by gas chromatography-negative-ion chemical ionisation mass spectrometry

The N-acetylated metabolites of p-tyramine, p-octopamine and dopamine were identified unambiguously and quantitatively determined in a single ventral thoracic nerve cord of the locust, Schistocerca gregaria, by gas chromatography-negative-ion chemical ionisation mass spectrometry (GC-NICIMS). Deuterium-labelled analogues of each compound were added to a single ventral thoracic nerve cord in acetonitrile: the tissue was homogenised and the suspension centrifuged. The solvent was removed from the supernatant and the resultant residue was derivatised with trifluoroacetic anhydride. Under negative-ion chemical ionisation conditions, the trifluoroacetyl derivatives produced ions which were sufficiently abundant to be suitable for selected-ion monitoring. This method is highly specific and gave a limit of detection below the picogram levels. N-Acetyl-5-hydroxytryptamine was determined using a previously published GC-NICIMS technique [S.P. Markey, R.W. Colburn and J.N. Johannessen, Biomed. Mass Spectrom., 7 (1981) 301]. The concentrations of N-acetyltyramine, N-acetyloctopamine, N-acetyldopamine and N-acetyl-5-hydroxytryptamine in locust thoracic nerve cords were, respectively, 1.86 +/- 0.71, 1.13 +/- 0.34, 6.77 +/- 8.48 and 0.07 +/- 0.02 ng per tissue.     

Fast-scan cyclic voltammetry for the detection of tyramine and octopamine

Tyramine and octopamine are biogenic amine neurotransmitters in invertebrates that have functions analogous to those of the adrenergic system in vertebrates. Trace amounts of these neurotransmitters have also been identified in mammals. The purpose of this study was to develop an electrochemical method using fast-scan cyclic voltammetry at carbon-fiber microelectrodes to detect fast changes in tyramine and octopamine. Because tyramine is known to polymerize and passivate electrode surfaces, waveform parameters were optimized to prevent passivation. No fouling was observed for octopamine when the electrode was scanned from 0.1 to 1.3 V and back at 600 V/s, while a small decrease of less than 10% of the signal was seen for 15 repeated exposures to tyramine. The technique has limits of detection of 18 nM for tyramine and 30 nM for octopamine, much lower than expected levels in insects and lower than basal levels in some brain regions of mammals. Current was linear with concentration up to 5 μM. This voltammetry technique should be useful for measuring tyramine and octopamine changes in insects, such as the fruit fly, Drosophila melanogaster.

Analysis of octopamine in human doping control samples

The biogenic amine octopamine [4‐(2‐amino‐1‐hydroxyethyl)phenol] is prohibited in sports owing to its stimulating and performance‐enhancing properties. Adverse analytical findings in athletes' doping control samples commonly result from surreptitious applications; however, the occurrence of octopamine in nutritional supplements and in selected invertebrates as well as the assumption that its N‐methylated analog synephrine [4‐(1‐hydroxyethyl‐2‐methylamino)phenol, not banned by anti‐doping authorities but currently monitored in prevalence studies) might be converted in‐vivo into octopamine have necessitated a study to investigate the elimination of synephrine and octopamine present in over‐the‐counter products. Urine samples collected after administration of nutritional supplements containing octopamine and/or synephrine as well as urine samples collected after therapeutic application of octopamine‐ or synephrine‐containing drugs were analyzed using a validated solid‐phase extraction‐based procedure employing a weak cation exchange resin and liquid chromatographic/tandem mass spectrometric detection with electrospray ionization and multiple reaction monitoring. In the case of therapeutic octopamine application, the urinary concentration of the target compound increased from baseline levels below the lower limit of detection to 142 µg/mL, while urine samples collected after synephrine as well as dietary supplement administration did not yield any evidence for elevated renal excretion of octopamine. Copyright © 2011 John Wiley & Sons, Ltd.     

Hexahomotrioxacalix[3]arene derivatives as ionophores for molecular recognition of dopamine, serotonin and phenylethylamine

The lower rim functionalized hexahomotrioxacalix[3]arene derivatives cone-3 and cone-5 bearing three benzyl and three N,N-diethyl-2-aminoethoxy groups, respectively, were synthesized from triol 1. Their complexation with 2-(3,4-dihydroxyphenyl)ethylamine (dopamine), 5-hydroxytryptamine (serotonin), and 2-phenylethylamine (phenethylamine), which have biologically important activities, has been studied by (1)H-NMR spectroscopy. The chemical shifts of the aromatic protons of the host and guest molecules and the up-field shifts of the ethyl protons of the guest molecules strongly suggest the formation of inclusion complexes in solution. The formation of the host-guest complexes is assisted by a hydrogen bond and/or an electrostatic interaction between the host and ammonium ion (RNH(3)(+)) of the guest. The structures of receptors cone-3 and cone-5 have been determined by X-ray crystallography.     

Chemiluminescence determination of melatonin and some of its derivatives using potassium permanganate and formaldehyde system

It was found that melatonin and its derivatives, such as N-acetyl- 5-methoxytryptamine (MT), N-acetyl-5-hydroxytryptamine (NAS), 5-Methoxytrypt- amine (5-MT), 5-Methoxyindolyl acetic acid (5-MIAA) and N-acetyl-5-methoxy- 6-hydroxytryptamine (6-HMT) would give chemiluminescence in the acidic potassium permanganate solution, and formaldehyde would enhance this chemiluminescent reaction greatly. The optimum conditions for this chemiluminescent reaction were studied in detail by a flow injection system. A new simple rapid method has been developed under the optimum conditions for determination of melatonin. This method has the advantages of high sensitivity, wide range of linear response and low detection limit. On the basis of investigation of chemiluminescent, fluorescent and UV spectra of melatonin in acidic solution containing potassium permanganate and formaldehyde, a possible mechanism of this reaction was proposed.