Determination of Plasma Norepinephrine and Epinephrine by High Performance Liquid Chromatography Using a Two-Column System and an Electrochemical Detector

Abstract

A method for the simultaneous plasma norepinephrine (NE) and epinephrine (E) determination by reversed-phase ion-pair liquid chromatography with electrochemical detection has been developed. Catecholamines were extracted from a 4 ml plasma sample using an alumina adsorption procedure. A two-pump, two-injection valve, two-column system allowed both to detect plasma NE and E with a good sensitivity due to large injected volumes of extract without any electrochemical detector disturbance and to eliminate uric acid and dopa the low k' of which would prevent the NE detection. Using this method, NE and E would be detected in respective injected amounts down to 30 and 50 picograms. Plasma NE and E determinations were found to be linear in the range of 288 to 788 pg/ml and 24 to 274 pg/ml respectively. The reproducibility, expressed as the coefficients of variation, varied from 2.1% for NE to 10.8% for E.     

Simultaneous determination of catecholamines by ion chromatography with direct conductivity detection

A simple method for simultaneous determination of three catecholamines using ion chromatography (IC) with direct conductivity detection (CD) based on the ionization of catecholamines in acidic medium without chemical suppression is developed in the present paper. The method could be used for the determination of these catecholamines in pharmaceutical preparations for the purpose of drug quality control. The recovery of catecholamines was more than 97% (n=3) and the relative standard deviation (R.S.D.) (n=11) was less than 2.1%. In a single chromatographic run, norepinephrine (NE), epinephrine (E) and dopamine (DA) can be determined in less than 10 min. The detection limits were found to be 0.001 μg/ml for NE, 0.01 μg/ml for E and DA respectively. Linear ranges were 0.01–50 μg/ml for NE (r²=0.9998), 0.1–50 μg/ml for E (r²=0.9995) and DA (r²=0.9999), respectively.     

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。     

Selective extraction of catecholamines by packed fiber solid‐phase using composite nanofibers composing of polymeric crown ether with polystyrene

For the first time, electrospun composite nanofibers comprising polymeric crown ether with polystyrene (PCE‐PS) have been used for the selective extraction of catecholamines – dopamine (DA), norepinephrine (NE) and epinephrine (E) – prior to their analysis by high‐performance liquid chromatography–electrochemical detection. Using a minicartridge packed with PCE‐PS composite nanofibers, the target compounds were extracted effectively from urine samples to which diphenylborinic acid 2‐aminoethyl ester was added as a complexing reagent. The extracted catecholamines could be liberated from the fiber by the addition of acetic acid. A good linearity was observed for catecholamines in the range of 2.0–200 ng mL^?1 (NE, E and DA). The detection limits of catecholamines (signal‐to‐noise ratio = 3) were 0.5 ng mL^?1 (NE), 0.2 ng mL^?1 (E) and 0.2 ng mL^?1 (DA), respectively. Under the optimized conditions, the absolute recoveries of the above three catecholamines were 90.6% (NE), 88.5% (E) and 94.5% (DA). The repeatability of extraction performance was from 5.4 to 9.2% (expressed as relative standard deviation). Our results indicate that the proposed method could be used for the determination of NE, E and DA in urine. Copyright © 2014 John Wiley & Sons, Ltd.     

Determination of Catecholamine Concentration by Reverse-Phase Liquid Chromatography with Elecrtrochemical Detector in Plasma

The plasma catecholamine include mainly norepinephrine(NE),epinephrine(E) and dopamine (DA). The catecholamines(CA) are nervous mediator which are synthesized and release from sympathetic system. It is significant for diagnosing disease and researching surgical stress in clinic that determine the concentration of catecholamine in plasma.The analytic method had traditionally radioactive enzyme, fluorescent spectrometry and high performance liquid chromatography(HPLC). This paper describe an analytical technique that determine the concentration of NE, E and DA in plasma using HPLC-ED. The method is sensitive, fast and accuracy,and can be applied for the clinical diagnosis and laboratory research.     

Determination of femtomole concentrations of catecholamines by high-performance liquid chromatography with peroxyoxalate chemiluminescence detection.

A highly sensitive method for determination of the plasma catecholamines, norepinephrine (NE), epinephrine (E) and dopamine (DA) is described. The method consists of the extraction of the catecholamines, using 3,4-dihydroxybenzylamine as internal standard, from plasma with alumina (5 mg), followed by a reversed-phase column separation, on-column fluorogenic derivatization with ethylenediamine (ED) and post-column peroxyoxalate chemiluminescent reaction detection utilizing bis[4-nitro-2-(3,6,9-trioxadecyl-oxycarbonyl)phenyl] oxalate (TDPO) and hydrogen peroxide. In order to optimize the reaction conditions for high-performance liquid chromatography to obtain highly sensitive detection, the effects of changing reagent compositions on the chemiluminescence yield were investigated. The following are the optimized conditions. Eluent, a mixture of 50 mmol l-1 potassium acetate (pH 3.20)-50 mmol l-1 potassium phosphate (pH 3.20)-acetonitrile (90.15 + 4.85 + 3 v/v/v) containing 1 mmol l-1 sodium hexanesulfonate (40 degrees C) and flow rate, 0.5 ml min-1. Fluorogenic reagent solution, 105 mmol l-1 ED and 175 mmol l-1 imidazole in acetonitrile-ethanol (90 + 10 v/v) and flow rate, 0.25 ml min-1. Reaction coil (15 m x 0.5 mm i.d.) heated at 80 degrees C. Chemiluminogenic reagent solution, 0.25 mmol l-1 TDPO, 150 mmol l-1 hydrogen peroxide and 110 mmol l-1 trifluoroacetic acid in dioxane-ethyl acetate (50:50 v/v) and flow rate, 1.4 ml min-1. The detection limits for all the catecholamines were 1 fmol (signal-to-noise ratio at 2). The standard deviations of the method for the determination of NE, E and DA added to rat plasma (2.5 nM) were 3, 3 and 4%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of l-dopa on dopamine and norepinephrine concentrations in rat brain assessed by gas chromatography

A highly specific and sensitive gas chromatographic method has been developed which is capable of determining picogram amounts of dopamine (DA) and norepinephrine (NE) simultaneously. The catecholamines are converted to the N-2,6-dinitro-4-trifluoromethylphenyl, O-trimethylsilyl derivatives, which are analyzed by gas chromatography with electron-capture detection. The method has been applied to the assay of catecholamines in rat brain extracts. One hour after an acute dose (150 mg/kg i.p.) of l-3,4-dihydroxyphenylalanine, the rat brain concentration of DA increased by 130% while the concentration of NE was unchanged.     

Fully automated high-performance liquid chromatographic assay for the analysis of free catecholamines in urine

A totally automated and reliable high-performance liquid chromatographic method is described for the routine determination of free catecholamines (norepinephrine, epinephrine and dopamine) in urine. The catecholamines were isolated from urine samples using small alumina columns. A standard automated method for pH adjustment of urine before the extraction step has been developed. The extraction was performed on an ASPEC (Automatic Sample Preparation with Extraction Columns, Gilson). The eluate was collected in a separate tube and then automatically injected into the chromatographic column. The catecholamines were separated by reversed-phase ion-pair liquid chromatography and quantified by fluorescence detection. No manual intervention was required during the extraction and separation procedure. One sample may be run every 15 min, ca. 96 samples in 24 h. Analytical recoveries for all three catecholamines are 63-87%, and the detection limits are 0.01, 0.01, and 0.03 microM for norepinephrine, epinephrine and dopamine, respectively, which is highly satisfactory for urine. Day-to-day coefficients of variation were less than 10%.     

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。     

A simple and sensitive CE method for the simultaneous determination of catecholamines in urine with in-column optical fiber light-emitting diode-induced fluorescence detection

A simple and sensitive method has been developed for simultaneous analysis of three catecholamines: dopamine (DA), epinephrine (EP) and norepinephrine (NE) in urine by capillary electrophoresis (CE) coupled with in-column fiber-optic light-emitting diode-induced fluorescence detection (ICFO-LED-IFD). Fluorescein isothiocyanate was used as the fluorescence tagged reagent for derivatization of DA, EP and NE. The CE conditions for separation of these catecholamines were systematically investigated. It was found that catecholamines could be more effectively separated by adding β-cyclodextin (β-CD) and acetonitrile (ACN) to a background electrolyte (BGE) of sodium borate. The migration times are 10.61, 10.83 and 11.14 min for DA, EP and NE, respectively and the catecholamines are completely separated within 11.5 min under the optimal condition of a BGE containing 10% v/v ACN, 20 mM β-CD and 20 mM sodium borate (pH 9.5), and an applied voltage of 13 kV. The relative standard deviations of migration time and peak area for these catecholamines are less than 0.16 and 2.0%, respectively. The limit of quantifications (LOQs) for DA, EP and NE are 3.5, 1.0 and 3.1 nM whereas the limit of detections (LODs) for DA, EP and NE are 1.0, 0.3 and 0.9 nM, respectively. Our proposed CE method provides low LOQ and LOD values. This CE-ICFO-LED-IFD methodology has been successfully applied to analyze catecholamines in human urine samples with good accuracy and satisfactory recovery.     

High performance liquid chromatography coupled to an optical fiber detector coated with laccase for screening catecholamines in plasma and urine

An analytical method based on separation by high performance liquid chromatography (HPLC) and detection by optical fiber (OF) coated with an enzyme (laccase), has been developed for separation and quantification of catecholamines, namely epinephrine, dopamine and norepinephrine. The application of OF as a detector in this analytical system relies on the variation of the reflected optical power detected when the catecholamines eluted from the HPLC column act as the substrate of the laccase immobilized on a tip of a single-mode OF. The developed method shows a high linearity in a range between 5 and 125 pg/mL and detection limits of 3.5, 2.9 and 3.3 pg/mL for epinephrine, dopamine and norepinephrine, respectively. The analytical performance of the proposed method was compared with a classical analytical method, namely high performance liquid chromatography-electrochemical detector (HPLC-ED) regarding catecholamines detection, showing great analytical advantages such as low cost of equipment. Additionally, the proposed method was applied to catecholamines determination in actual samples of plasma and human urine.     

Determination of heat-exposure effects on the concentration of catecholamines in bovine plasma and milk

A reversed-phase high-performance liquid chromatographic method with electrochemical detection has been adapted for the determination of picogram concentrations of norepinephrine and epinephrine in bovine plasma and milk. This method has been used to monitor the levels of these catecholamines when lactating cows are exposed to heat stress under controlled conditions. In response to heat stress, epinephrine concentrations in milk and plasma were similar. However, norepinephrine concentrations in milk were one tenth of that in plasma.     

无泵负压进样-芯片电泳分离-激光诱导荧光光谱法测定儿茶酚胺类物质

提出了用芯片电泳分离-激光诱导荧光光谱法测定儿茶酚胺类物质的方法。采用自制的无泵负压进样系统,避免了进样歧视效应。在优化的条件下,去甲肾上腺素(NE)、多巴胺(DA)和肾上腺素(E)可在1 min内完全分离。3种儿茶酚胺的平均迁移时间依次为30.59,37.23,46.43 s,其相对标准偏差(n=7)依次为1.10%,1.28%,0.45%。3种物质的线性范围为0.3～5.0 mg.L-1(NE及DA)和0.05～4.0 mg.L-1(E),检出限(3S/N)依次为30,30,10μg.L-1。     

Rapid determination of catecholamines in urine samples by nonaqueous microchip electrophoresis with LIF detection

A method was developed for the rapid separation of catecholamines by nonaqueous microchip electrophoresis (NAMCE) with LIF detection, A homemade pump‐free negative pressure sampling device was used for rapid bias‐free sampling in NAMCE, the injection time was 0.5 s and the electrophoresis separation conditions were optimized. Under the optimized conditions, the samples were separated completely in <1 min. The average migration times of the epinephrine (E), dopamine (DA), and norepinephrine (NE) were 34.26, 43.81, and 50.07 s, with an RSD of 1.05, 1.26, and 0.89% (n = 7), respectively. The linearity of the method ranged from 0.0125 to 2.0 mg/L for E and 0.025～4.0 mg/L for DA and NE, with correlation coefficients ranging between 0.9978 and 0.9986. The detection limits of E, DA, and NE were 2.5, 5.0, and 5.0 μg/L, respectively. The recoveries of E, DA, and NE in spiked urine samples were between 86 and 103%, with RSDs of 4.5～6.8% (n = 5). The proposed NAMCE with LIF detection combined with a pump‐free negative pressure sampling device is a simple, inexpensive, energy efficient, miniaturized system that can be successfully applied for the determination of catecholamines in urine samples.     

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.     

Gas chromatographic method for the microdetermination of barbiturates in blood using a nitrogen-selective flame ionization detector.

A rapid, quantitative gas-liquid chromatographic method for the simultaneous determination of as little as 10 ng of unmodified barbital, pentobarbital, secobarbital, and hexobarbital from whole blood is described. The method involves one extraction from whole blood into chloroform with subsequent injection into a gas chromatograph equipped with a nitrogen-sensitive flame ionization detector. This method has the advantages of small sample size high specificity, sensitivity, and rapidity.     

Routine determination of urinary free catecholamines by high-performance liquid chromatography with electrochemical detection

A simple and reliable high-performance liquid chromatographic method is described for the routine determination of the free catecholamines (norepinephrine, epinephrine and dopamine) in urine. The catecholamines are isolated from urine samples using small affinity chromatography columns prepacked with immobilised m-aminophenylboronic acid, separated by ion-pair reversed-phase liquid chromatography and quantified by electrochemical detection. Total analysis, including sample preparation time, is achieved in less than 30 min with analytical recoveries of 92-96% for all three catecholamines. Long-term stability and reproducibility of the liquid chromatographic system is attained by selection of optimised conditions for chromatographic separation with a formate mobile phase and produces detection limits of 1.4, 1.8 and 2.2 nmol/l for norepinephrine, epinephrine and dopamine, respectively, in urine samples and day-to-day coefficients of variation of less than 6%. Furthermore, the affinity isolation gels can be reused a minimum of ten times providing a rapid and cost-effective means of sample preparation.     

High-performance liquid chromatography/atmospheric pressure chemical ionization mass spectrometric method for the analysis of catecholamines and metanephrines in human urine

An assay of norepinephrine (NE), epinephrine (E), dopamine (DA), normetanephrine (NE) and metanephrine (MN) based on high-performance liquid chromatography (HPLC) in combination with atmospheric pressure chemical ionization mass spectrometry (APcI-MS) is described. The catecholamines and metanephrines were extracted from urine and aqueous samples using Bio-Rex 70 cation-exchange resin and subjected to analysis by HPLC/APcI-MS. The separation was performed on a C18 column in 50 mM ammonium formate buffer, pH 3.0, using a flow rate of 0.8 mL/min. Acetonitrile was added post-column at a flow rate of 0.2 mL/min via a post-column addition tee. The total analysis time was 6.5 min. The quantitative analysis was performed using 3,4-dihydroxybenzylamine (DHBA) as the internal standard (I.S.). Selected ion monitoring detection was applied: m/z 170 (for NE), 184 (for E and NM), 154 (for DA), 198 (for MN) and 140 (for DHBA, I.S.). The limits of quantitation were 5 ng/mL for NE, E and DA and 2.5 ng/mL for NM and MN. The recovery ranged from 75 to 83% for each analyte. The method was found to be simple and highly selective for the determination of catecholamines and metanephrines in the urine of patients suspected of pheochromocytoma.     

Simultaneous assay of 3,4-dihydroxyphenylalanine, catecholamines and O-methylated metabolites in human plasma using high-performance liquid chromatography

We devised a procedure for the simultaneous determination of 3,4-dihydroxyphenylalanine, catecholamines and O-methylated metabolites using a reversed-phase liquid chromatographic system. Detection is achieved by an electrochemical detector and a fluorescence detector connected in series. Sample preparation is kept to a minimum, and involves precipitation of proteins with trichloroacetic acid and perchloric acid, and subsequent neutralization, thus omitting the commonly adopted adsorption step. Chromatographic peaks were identified on the basis of retention behaviour and the ratio of responses at several oxidation potentials. The method was applied to the quantitative determination of 3,4-dihydroxyphenylalanine, catecholamines and O-methylated metabolites in human plasma.     

Quantitative assay of maprotiline in biological fluids by gas-liquid chromatography.

A specific and sensitive gas-liquid chromatographic method has been developed for the assay of maprotiline in biological fluids. Maprotiline is isolated from the biological sample by base-specific extraction followed by conversion into the heptafluorobutyramide. The derivative is determined quantitatively by gas-liquid chromatography with an electron capture detector, nortriptyline being used as the internal standard. Amounts below 10 ng per biological sample can be measured.