A rapid method for simultaneous estimation of 5-hydroxy-3-indole acetic acid (5HIAA) and 5-hydroxytryptamine (5HT) in rat brain

The authors describe a method to separate 5-hydroxy-3-indole acetic acid (5HIAA) from 5-hydroxyindole derivatives by means of a batch procedure using Dowex 50 WX 12 resins. These compounds were assayed by the Maickel and Miller procedure, and 5-hydroxytryptamine (5HT) was then calculated as the difference between total 5-hydroxyindoles and 5HIAA. This procedure allows a rapid assay of 5HT and 5HIAA in about 60 mg of rat cerebral tissue, but it may not be used when large amounts of 5-hydroxytryptophan are present.     

Microassay for N-acetyltransferase activity using high-performance liquid chromatography with electrochemical detection

A rapid, sensitive procedure has been developed for determination of N-acetyltransferase activity against octopamine, dopamine and 5-hydroxytryptamine. The assay, which is performed in a volume of 10 microliters, is based upon the separation and detection of monoamine substrates and their N-acetylated derivatives using high-performance liquid chromatography with electrochemical detection. The method has been used to measure N-acetyltransferase activity against octopamine, dopamine and 5-hydroxytryptamine in the cerebral ganglion of the American cockroach, Periplaneta americana and to study bi-substrate kinetics of the enzyme.     

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.     

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。     

Rapid determination of 5-hydroxytryptamine in whole blood by liquid chromatography with fluorimetric detection.

A rapid and simple reversed-phase (using muBondapak C18 as the stationary phase) liquid chromatographic method with fluorimetric detection is described for the quantitation of 5-hydroxytryptamine in whole blood. The rapidity and simplicity of the method are explained by the absence of a pretreatment. 5-Fluoro-dl-tryptophan was used as internal standard. The mobile phase was 0.01 M phosphate buffer (pH 4.5) with 0.0025 M 1-heptanesulfonic acid and 20% methanol. The detection wavelength were 302 nm for excitation and 340 nm for emission. Analysis time was 10 min with retention times for 5-hydroxytryptamine of 9 min and for 5-fluoro-dl-tryptophan of 7 min. This method is proposed for biological exploration of psychiatric disorders involving 5-hydroxytryptamine and would be useful for tryptophan.     

Determination of the biogenic amines and their major metabolites in single human brain tissue samples using a combined extraction procedure and high-performance liquid chromatography with electrochemical detection

A combined extraction system for the selective and quantitative isolation of the monoamines norepinephrine, epinephrine, dopamine, serotonin (5-hydroxytryptamine) and their metabolites 3-methoxy-4-hydroxyphenylethylene glycol, 3,4-dihydroxyphenylacetic acid, 5-hydroxyindoleacetic acid, homovanillic acid and 3-methoxytyramine from one single brain tissue sample is described. The extraction system is a combination of an ethyl acetate extraction for 3-methoxy-4-hydroxyphenylethylene glycol, 3,4-dihydroxyphenylacetic acid, 5-hydroxyindoleacetic acid and homovanillic acid, and two successive ion-pair extractions. In a first step, the catecholamines are quantitatively isolated by extracting with heptane--octanol (99:1) containing 0.25% tetraoctylammonium bromide as an ion-pairing agent in the presence of 0.2% diphenylborate. In a second step, 3-methoxytyramine and 5-hydroxytryptamine are isolated from the aqueous phase with di(2-ethylhexyl)phosphoric acid as counter-ion in chloroform. Dihydroxybenzylamine, isohomovanillic acid and 5-hydroxy-N-methyltryptamine are used as the internal standards.     

In vivo microdialysis determination of collagen-induced serotonin release in rat blood

We developed a sensitive microbore HPLC method coupled with an on-line microdialysis system to simultaneously measure endogenous 5-hydroxytryptamine (serotonin; 5-HT) and its major metabolite 5-hydroxyindoleacetic acid (5-HIAA) in the rat blood in vivo. A dialysis tube was placed in the right jugular vein. The validity of the procedure is demonstrated because analysis of the aggregating agents, collagen (I mg/kg) plus epinephrine (0.3 mg/kg) after intravenous injection, showed that they induced an increase in 5-HT and 5-HIAA levels in the jugular vein of the rat.     

Electrochemical behavior of tryptophan and its derivatives at a glassy carbon electrode modified with hemin

The electrochemical behavior of tryptophan (Trp) and its derivatives, such as indole-3-acetic acid (IAA), 5-hydroxytryptamine (5-HT), 5-hydroxy-indole-3-acetic acid (5-HIAA) and glycyl-tryptophan (Gly-Trp) peptide at a glassy carbon electrode modified with hemin (hemin/GC electrode) by electropolymerization have been investigated in detail. The results showed that the hemin/GC electrode would catalyze the electrochemical oxidation of Trp and its derivatives, based on which a differential pulse voltammetric procedure has been proposed for determination of Trp and its derivatives. Meanwhile, the electrochemical reaction mechanism for these compounds at hemin/GC electrode has been also investigated, and the results indicated that a two electron and two proton transfer was involved in the electrode reaction process.     

Characterisation and determination of indole alkaloids in frog-skin secretions by electrospray ionisation ion trap mass spectrometry

The characterisation of selected indole alkaloids in a quadrupole ion trap mass spectrometer is presented. Fragmentation profiles for tryptamine, 5-hydroxytryptamine (5-HT), N'-methyl 5-hydroxytryptamine (N'-methyl 5-HT), N',N'-dimethyl 5-hydroxytryptamine (bufotenine), N',N',N'-trimethyl 5-hydroxytryptamine (5-HTQ), and N',N'-dimethyl 5-methoxytryptamine (5-MeODMT) are presented with proposed structures given for each product ion observed. Such MS(n) experiments can be used to differentiate the isobaric molecular ions of the compounds 5-HTQ (M(+)) and 5-MeODMT (MH(+)). The quantitative determination of certain indole alkaloids in the skin secretions of the Australian Golden Bell frog, Litoria aurea, by LC/ESI-ion trap MS is also presented. The concentrations of 5-HT, N'-methyl 5-HT and 5-HTQ were found to be 2.68, 0.26 and 0.54 microg per mg of skin secretion, respectively.     

Revised method for the quantitative determination of 5-hydroxytryptamine in human plasma by gas chromatography--mass spectrometry--selected ion monitoring

To estimate the 5-hydroxytryptamine level in human plasma by gas chromatography--mass spectrometry--selected ion monitoring (GC--MS--SIM), a recovery method from plasma and derivatization conditions with pentafluoropropionic anhydride were investigated. By heating 5-hydroxytryptamine at 140 degrees C for 4 h in the presence of pentafluoropropionic anhydride the peak intensity of derivatized 5-hydroxytryptamine increased more than three times in comparison with the reported method. There was a marked difference in the plasma levels of 5-hydroxytryptamine obtained using the GC--MS--SIM method compared to those obtained using fluorometric assay.     

High-Performance Liquid Chromato- Graphic Assay for Kinetic and Inhibition Properites of Monoamine Oxidase form A

Abstract

A rapid and convinient method to study the kinetic and inhibition properties of rat brain monoamine oxidase form A by high-performance liquid chromatography is described. The procedure consists of homogenization of the brain tissue, enzymatic reaction with or without inhibitors using 5-hydroxytryptamine as the substrate, termination of the reaction with perchloric acid, removal of protein by centrifugation and detection of reaction products at 280 nm after HPLC separation. The method was applied to determine K_m and V_max values of 5-HT for MAO-A and to study the inhibition properties of MAO-A using clorgyline and 1-deprenyl as inhibitors.     

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 measurement of serotonin and 5-hydroxyindoleacetic acid in rat brain using a liquid chromatographic method with electrochemical detection.

A simple and sensitive method for the simultaneous measurement of 5-hydroxytryptamine and its main metabolite 5-hydroxyindoleacetic acid in rat brain is described. Brain tissue samples were only deproteinated and, without further extraction, were injected directly onto a high-performance liquid chromatography column and detected electrochemically. The detection limit for 5-hydroxytryptamine and 5-hydroxyindoleacetic acid was 16 and 8 g per injection, respectively. Within 8 min, the total separation of 5-hydroxytryptamine and 5-hydroxyindoleacetic acid is achieved. With this method over 100 analyses can be performed in a single working day. Brain samples from young and old, male and female Brown Norway rats were analysed for indoles by this method. The ratio 5-hydroxytryptamine 5-hydroxyindoleacetic acid, an index for serotonin turnover, showed significant differences between age groups and genders in the cortex, and a significant difference between genders in the hypothalamus.     

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.     

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.     

Rapid, simultaneous determination of isomers of retinal, retinal oxime and retinol by high-performance liquid chromatography

Thirteen geometric isomers of retinol, retinal and retinal oxime were resolved in a single chromatographic run on two 250 X 4 mm 5-micron LiChrosorb Si 60 columns in series, using 11.2% ethyl acetate, 1.4% 1-octanol and 2% dioxane in hexane as the mobile phase. All the 11-cis and all-trans isomers of retinol and retinal oxime were completely resolved from each other and from the 9-cis and 13-cis isomers. This chromatographic procedure should be particularly useful for quantifying geometric isomers of retinoids in methylene chloride-hydroxylamine extracts of biological samples such as ocular tissues.     

Quantification of luminally released serotonin in rat proximal colon by capillary electrophoresis with laser-induced fluorescence detection

Serotonin (5-hydroxytryptamine, 5-HT) plays vital roles in regulating gastrointestinal functions. Thus, the detection of 5-HT in the gastrointestinal tract is of great importance for biomedical research, medical diagnosis, and pharmaceutical therapy. This paper presents a simple, sensitive, and fast method for the quantification of luminally released serotonin in the feces and tissues of the rat proximal colon by means of capillary electrophoresis with laser-induced fluorescence detection. 5-Carboxyfluorescein N-succinimidyl ester was used for precolumn derivatization of serotonin. The optimal separation and detection conditions were obtained with an electrophoretic buffer containing 60 mM borate (pH 8.90) and an air-cooled argon-ion laser (excitation at 488 nm, emission at 520 nm). The serotonin concentrations in the feces and tissues of proximal colons were analyzed with this method, and the average values of serotonin in the feces samples were 1.951 ± 0.446 ng/mg (male) and 2.095 ± 0.533 ng/mg (female) and 1.397 ± 0.267 ng/mg in rat proximal colon tissues. The results demonstrate that this method can accurately determine luminally released 5-HT in rats.     

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.     

反-4-羟甲基哌啶-3-醇的合成

以4-羟甲基吡啶为原料,经苄基保护、硼氢化钠的还原、硼氢化氧化和脱苄基保护等四步反应得到新型5-羟色胺受体激动剂的关键中间体反-4-羟甲基哌啶-3-醇。总收率达55. 7%。产物结构经IR、1H NMR和MS分析确证,并对合成工艺进行了优化。     

Simultaneous determination of 5-hydroxytryptamine, its amino acid precursors and acid metabolite in discrete brain regions by high-performance liquid chromatography with fluorescence detection

L-Tryptophan, 5-hydroxytryptophan (5-HTP), 5-hydroxytryptamine, and 5-hydroxyindoleacetic acid (5-HIAA) from discrete regions of brain can be resolved by isocratic elution on a C-18 reversed-phase column and quantitated by native fluorescence measurement. This method is rapid and simple and can reproduceably detect 5-hydroxytryptamine, tryptophan and 5-HIAA with picogram sensitivity. The detection of 5-HTP in brain extracts from untreated animals is difficult because of the extremely low endogenous levels of 5-HTP. Quantitation of 5-HTP presents no problem in animals injected with 5-HTP or with an inhibitor of L-aromatic amino acid decarboxylase. Brain tissue requires minimal treatment for assay and the hydroxyindoles are stable enough to allow automated processing.