The Determination of Catecholamines,Indoleamines, Metabolites,and Related Enzymatic Activities Using Three Micron Liquid Chromatography Columns

Abstract

Liquid chromatography with electrochemical detection has become an established technique for the determination of catechol-amines, indoleamines, precursors, metabolites, and related enzymatic activities in tissues and fluids. Previously available instrumentation, however, has limited the number of individual species readily and simultaneously accessible with reasonable throughput to only a few. Determinations of other species required either extended amounts of time per individual chromatogram or the use of an entirely separate chromatographic setup employing different columns and eluting solvents. Using reversed-phase columns packed with 3 micron particles, we have been able to produce the separation of 16 different catecholamine and in-doleamine related species and two different internal standard compounds in 5 or 7 minutes. Samples may be analyzed directly after only homogenization, centrifugation, and clarification by filtration. No further purification steps are required. The enzymatic activities of 6 separate enzymes may be determined using the same chromatographic apparatus and simply monitoring selected metabolites following appropriate incubation of pretreatment. The metabolites and transmitters currently accessible with this apparatus include norepinephrine, dopamine, epinephrine, serotonin, 3,4-dihydroxyphenylacetic acid, 3,4-dihydroxyphenylalanine, normetanephrine, metanephrine, 3-methoxytyramine, 3,4-dihydroxy-phenylethyleneglycol, vanillylmandelic acid, homovanillic acid, 5-hydroxytryptophan, 5-hydroxyindoleacetic acid, 5-hydroxytryptophol, and N-acetyl-5-hydroxytryptamine. The enzymatic activities include tyrosine hydroxylase, tryptophan hydroxylase, dopa decarboxylase, 5-hydroxytryptophan decarboxylase, monoamine oxidase, and catechol-O-methyltransferase.     

A Fast HPLC Analysis Of Catecholamines and Indoleamines in Avian Brain Tissue

Abstract

A fast HPLC method for the determination of monoamine levels in avian brain tissue, using a short 3 micron column and electrochemical detection, is presented. This system simultaneously analyzes 18 catecholamines, indoleamines, metabolites, and internal standards, without further sample preparation, in 20 minutes. Contrary to reports involving the mammalian species, incubation of the tissue homogenate with ascorbate oxidase and/or urate oxidase does not significantly alter the analysis under the reported conditions. The assay was used to evaluate differences in the neurochemical content of brain tissue from chickens divergently selected for 42 day exponential growth rate.     

高效液相色谱法测定尿液中儿茶酚胺类物质

本文报道用高效液相色谱法(HPLC)分离,荧光检测器检测儿茶酚胺含量的新方法。该法可同时分离测定去甲肾上腺素(NE)、肾上腺素(E)和多巴胺(DA)三种组分,三种组分的浓度与其峰面积均呈良好的线性关系。方法灵敏度高,选择性好,测定结果令人满意,可在临床推广应用。     

生物样品中神经递质含量的测定

用反相高效液相色谱 -电化学检测法 ,同时测定了小鼠脑组织以及人血清中的 6种神经递质的含量。以柠檬酸钠缓冲溶液和甲醇作流动相 ,优化后得到最佳色谱条件。在 1～ 2 0 0× 1 0 - 3mg/ L范围内 ,浓度与响应的线性关系良好 ,各待测物的检出限可达 0 .4～ 5.0× 1 0 - 3mg/ L。     

Simultaneous HPLC Analysis of Catecholamines and Indoleamines in Mouse Brain Tissue Following Acetate Extraction and Treatment with Ascorbate Oxidase

Abstract

A refined HPLC Method for the determination of monoamine levels in six brain regions is presented. Analyses are made for the olfactory tubercles, prefrontal cortex, septum, striatum, amygdala and hypothalamus of adult male ICR mice. This system permits the simultaneous analysis of norepinephrine, dopamine, serotonin and their major metabolites during a single run of approximately twenty-five minutes without prior clean-up of samples.     

Simultaneous HPLC Determination of Oxcarbazepine,Carbamazepine and Their Metabolites in Serum

Abstract

We propose a simple procedure for the simultaneous determination of the anticonvulsants oxcarbazepine, carbamazepine and three of their metabolites (10-hydroxy-10, 11-dihydro-carbamazepine, trans-10, 11-dihydroxy-10, 11-dihydro-carbamazepine and 10, 11-epoxy-carbamazepine) in serum or plasma. The alkalinized sample is extracted with ethyl acetate. The extract is evaporated to dryness and taken up with the mobile phase. An aliquot is injected into the liquid chromatograph and eluted with water/methanol/acetonitrile (55/40/5, by vol.) on a 5-μm C-18 reversed-phase column. Eluent is monitored at 254 nm. No interference by other anticonvulsants or by endogenous constituents from the sample is observed. Owing to its good precision, specificity, sensitivity, and selectivity, this method is well adapted to the therapeutic monitoring of oxcarbazepine or carbamazepine treated patients, as well as for pharmacokinetic studies.     

Simultaneous Determination of Catecholamines and Related Metabolites by Capillary Electrophoresis with Amperometric Detection

An electrophoretic method was developed for the determination of several important catecholamine markers, namely norepinephrine, epinephrine, dopamine, metanephrine, vanillylmandelic acid and homovanillic acid in urine samples. Under the optimum conditions, the six marker compounds could be well separated with the major coexisting interference compound uric acid within 24 min at a separation voltage of 16 kV in a borate running buffer (80 mmol/L, pH=9.48). Highly linear response can be obtained over three orders of magnitude for the above markers with the low limits of detection ranging from 1.0 ng/mL to 5.0 ng/mL(S/N=3). The proposed capillary electrophoresis with amperometric detection(CE-AD) method has been used to simultaneously determine the six catecholamine markers in urine samples of healthy volunteers and patients suffering from different diseases avoiding redundant measurements and high assay cost; and the electrochemical profiles can suggest more diagnostic information of multiple diseases, which provides a promising and convenient entry into primary diagnoses of several clinical diseases.     

Electrochemical Oxidations of Central Nervous System Indoleamines,Catecholamines and Alkaloids: Potential Significance into Neurodegenerative Diseases

The serotonergic neurotransmitter 5-hydroxytryptamine (5-HT), the catecholaminergic neurotransmittcr dopaminc (DA) and various tetrahydroisoquinoline (TIQ) and tetrahydro-β-carboline (THβC) alkaloids are all easily oxidized compounds. Aberrant oxidative transformations of 5-HT and other central indoles might be involved in neurodegenerative Alzheimer's Disease (AD). Changes in the oxidation chemistry of DA appear to be fundamental in substantia nigra neurons in Parkinson's Disease (PD). Various TIQ and THβC alkaloids are elevated in the brain as a result of ethanol drinking. Recent studies into the electrochemical oxidation chemistry of 5-HT, DA and various TIQ and THβC alkaloids have been reviewed. The potential roles of the oxidation chemistry of these compounds in neurodegenerative AD, PD and alcoholism are discussed.     

高效液相色谱法测定聚丙烯腈基碳纤维原丝中的二甲基亚砜残留

建立了聚丙烯腈（PAN）基碳纤维原丝中二甲基亚砜（DMSO）残留量的高效液相色谱（HPLC）检测方法。采用的色谱柱为Phenomenex C18柱（150 mm×4.6 mm,5 μm）,流动相为甲醇-三氟乙酸-水（体积比为29∶1∶70）混合溶液,流速为1.0 mL/min,进样量为10 μL,检测波长为205 nm,柱温为常温。研究结果表明,在DMSO的质量浓度为1.00~100.00 mg/L范围内,峰面积与质量浓度的线性关系良好,相关系数r=0.9989,最低检出限为0.3 mg/L,平均回收率为98.4%,相对标准偏差（RSD）为2.6%。该方法简便、快速、准确。另外,利用建立的HPLC方法,对原丝中残留的DMSO在水中的溶出规律也进行了研究。     

Simultaneous Determination of Fourteen Catecholamines and Their Metabolites by High Performance Liquid Chromatography with Electrochemical Detection

Abstract

A simple method has been developed for simultaneous determination of 14 catecholamines and their metabolites in cerebrospinal fluid and brain tissue by reversed-phase, ion-pair high-performance liquid chromatography with electrochemical detection. The time required for complete separation and analysis of all compounds was less than 35 min. Quantitation was based on the use of an internal standard isoproterenol. The mobile phase consisted of a 91:9 (v/v) mixture of 0.1 M formic acid and acetonitrile containing sodium-1-octane sulfonic acid. Using this method, analysis of neurotransmitters in brain tissue can be accomplished without a clean-up procedure.     

HPLC—ECD法检测小鼠体内多巴胺及其代谢产物

建立了HPLC-电化学检测法同时测定小鼠血浆及脑组织中多巴胺及其代谢产物的方法。采用安捷伦水相柱,流动相为V（甲醇）＋V（水）=10＋90,其中水相中含NaH2PO4·H2O,KCl,EDTA,辛烷磺酸钠,流量0．25mL·min^-1。,检测电压0．52V。结果表明,线性范围：多巴胺（DA）25～750ng,多巴烯（DOPAC）25～750ng,高香草酸（HVA）50～1000ng,线性关系与精密度良好;该法操作简单、快速、准确,可用于检测小鼠血浆及脑组织中多巴胺及其代谢产物的含量。     

Routine Analysis of Catecholamines and Metabolites in Urine by a Liquid Chromatographic Column Switching System

Abstract

A reversed-phase liquid chromatographic technique including a column switching system has been adapted for the routine measurement of catecholamines and their metabolites (14 compounds) in urine. From 1 ml of urine all the compounds and the internal standards were obtained according to combined extraction procedures involving organic solvent, anionic and weakly cationic resins. Finally four extracts (catecholamines, methoxamines, acidic and neutral derivatives) had to be chromatographed throughout a wholly automated apparatus. For each run, the column switching system determined the analytical columns to be used to obtain the separation of the compounds from interferences due to other co-extracted endogenous substances, while the analysis times remained between 20 and 40 min. Such a system allowed the rapid clean-up of columns (in direct- and back-flush mode) carried out between two consecutive injections. By coupling on-line fluorimetric and electro-chemical detections the specificity of the technique could be checked, since the ratio of the responses of both detectors was an index of the purity of the peaks. Finally the advanced automation of the equipment allowed weekly the evaluation of catecholamines and the whole range of their known metabolites in 36 urine samples.     

高效液相色谱法分离和鉴别棉花中13种糖

应用一根Ｓｈｉｍ－ｐａｃｋＣＬＣ－ＮＨ２柱（６０ｍｍ×１５０ｍｍ）和一根Ｌｉｃｈｒｏｓｏｒｂ－ＮＨ２柱（５ｍｍ×２５０ｍｍ）串联组合，用ＲＩＤ－６Ａ折光检测器检测，以ＣＨ３ＣＮ＋Ｈ２Ｏ（７５＋２５）作流动相，成功地分离了鼠李糖、木糖、树胶醛糖、果糖、葡萄糖、半乳糖、蔗糖、纤维二糖、海藻糖、乳糖、蜜二糖、松三糖、棉籽糖，建立一种快速简便的测糖方法。     

Fluorometric Determination of Tryptophan and Its Brain Indoleamine Metabolites by Ion-Pair HPLC

Abstract

The reverse phase HPLC separation of tryptophan, serotonin, 5-hydroxyindoleacetic acid, tryptamine and indoleacetic acid using two solvent systems (one of them containing an ion-pairing reagent) is reported. The two low concentration eluents employed are either formic acid/water or 0.005 mol/L solution of 1-pentane sulfonn ic acid. In both cases chromatographic separation was achieved through a linear gradient elution using methanol/water (7/3) as the high concentration eluent. We also describe the variation of retention volumes of these compounds as a function of the pH of the mobile phase. pH values ranged from 1.5 to 4.0 and were obtained by adding either formic acid or NaOH respectively to the low concentration eluent. Tryptophan and its metabolites were detected fluorometrically. All compounds show a linear response in the pg to μg range. The chromatographic separation achieved allows a concurrent measurement of tryptophan and its main indoleamine metabolites which coupled to the high sensitivity of fluorometric detection permits a direct estimation of these metabolic pathways in brain tissue.     

Sample Preparation for the Analysis of Catecholamines and their Metabolites in Human Urine

Abstract

A specific, sensitive, qualitative and quantitative extraction procedure followed by an high pressure liquid chromatography equipped with electrochemical detector assay of catecholamines (CATs) and their metabolites from human urine has been developed. Using an unique multiple interaction bonded silica gel disposable solid phase extraction (SPE) column, various analytes were selectively isolated from the urine components. After following three different extraction procedures, the presence of free CATs (epinephrine, norepinephrine and dopamine) and their basic (normetanephrine, metanephrine, and 3-methoxydopamine) and acidic (vanillylmandelic acid, homovanillic acid and 5-hydroxyindoleacetic acid) metabolites was confirmed and quantitated by electrochemical detector.     

毛细管电泳安培测定鼠单个交感神经细胞中儿茶酚胺类神经递质

用自组装的毛细管电泳微电极安培检测系统，分析了鼠的单个交感神经细胞。大白鼠经麻醉、微解剖及酶解分离出新鲜的单个交感神经细胞。采用全细胞进样，用异丙肾上腺素 作内标。测定了儿茶酚胺类神经递质去甲肾上腺素和肾上腺素。检测限分别达 ５７ ａｍｏｌ和 ５９ ａｍｏｌ。八个活细胞分析结果，每个细胞平均含去甲肾上腺素 ４５５ ａｍｏｌ及肾上腺素 ６５ ａｍｏｌ。     

An HPLC Method for the Determination of Theophylline and Its Metabolites in Serum and Urine

Abstract

A urine and a serum assay have been developed to quantitate theophylline and its major metabolites:1,3-dimethyluric acid, 3-methylxanthine and 1-methyluric acid. Reverse phase chromatography follows a serum acetone extraction procedure and a urine anion exchange clean-up procedure. Lower limits of sensitivity are 0.04 μg/ml for serum metabolites and 1 μg/ml for urine metabolites. Both assays are free of interference from endogenous substances. These assays have been tested successfully in pharmacokinetic and metabolic studies of theophylline.     

Determination of Triazine Herbicides and Metabolites by Solid Phase Extraction with HPLC Analysis

An efficient solid phase extractive preconcentration/separation method was developed for the trace determination of herbicides in aqueous samples using Amberlite XAD-4 resin as the adsorbent. The retained herbicides were eluted with methanol at a flow rate of 1.0 mL min^?1 and determined by HPLC-DAD (wavelength of 220 nm) using water (pH:4.7, phosphoric acid) and methanol (ratio 35:65) as the mobile phase with a flow rate of 1.0 mL min^?1. Quantitative recoveries of simazine, atrazine and its metabolities were achieved at optimized analysis conditions that included 0.75 g of resin; a pH of 3.0; an eluent volume of 3.0 mL; an eluent flow rate of 1.0 mL min^?1; and a sample flow rate of 4.0 mL min^?1. The limits of detection, preconcentration factor, and linear ranges for the herbicides were 0.084–0.121 µgL^?1, 1000, and 0.5–20 mg L^?1, respectively. The performance of the method was evaluated by analysis of spiked water samples. The recoveries of simazine, atrazine and their metabolities were found to be quantitative (99.6–104.8%) with RSDs of 2.2–4.8% and 2.8–4.7% for intra-day and inter-day precision, respectively. The proposed method was successfully applied for trace determination of studied analytes in waste water, apple juice, and red wine samples.