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.     

Selective solid-phase extraction of catecholamines by the chemically modified polymeric adsorbents with crown ether

A simple and selective one-step solid-phase extraction procedure using chemically modified polymer resin (Amberlite XAD-4) with crown ether was investigated for the measurement of urinary catecholamines. After loading the urine samples (adjusted to pH 4) on the synthesized adsorbent cartridge, the column was washed with methanol followed by water and then the adsorbed catecholamines were eluted by 1.0 mL of 6.0 M acetic acid. The effectiveness of sample clean-up method was demonstrated by reversed-phase ion-pair high-performance liquid chromatography with electrochemical detection. Under optimal condition, the recoveries of epinephrine, norepinephrine, and dopamine from spiked urine sample were >86% for all catecholamines. The detection limits (n=5) for epinephrine, norepinephrine, and dopamine were 37, 52, and 46 nmol/L, respectively.     

A Simplified HPLC-ECD Technique for Measurement of Urinary Free Catecholamines

Abstract

A simplified HPLC assay is described for quantification of free urinary catecholamines. The procedure involves exraction of catecholamines, (norepinephrine, epinephrine and dopamine) from urine, using columns filled with Biorex-70. The catecholamines from the extract were separated on a high performance liquid chromatographic system using reverse phase C18, 5 u column and determined by electrochemical detection. Integration and calculations are achieved by a data module using area ratio method with dihydroxybenzylamine as internal standard. Recovery of more than 90% was achieved for each catecholamine. A linear relationship between a wide range of concentrations and ratio of the area of amines to that of internal standard was observed. The method is simple and rapid and therefore can be used to analyze a large number of samples in one day and should prove useful in studies involving the role of catecholamines in different psychiatric disorders.     

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.     

1,2-Diarylethylenediamines as pre-column fluorescence derivatization reagents in high-performance liquid chromatographic determination of catecholamines in urine and plasma

Meso- and dl-1,2-diarylethylenediamines (14 species) were evaluated for pre-column fluorescence derivatization reagents in the high-performance liquid chromatographic determination of catecholamines (norepinephrine, epinephrine and dopamine) in human urine and plasma. Of the compounds, meso-1,2-bis(4-methoxyphenyl)ethylenediamine was most preferable for all the catecholamines in terms of sensitivity and selectivity. The detection limit for each catecholamine is approximately 0.5 fmol in a 50-microliters injection volume.     

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.     

Microanalysis of catecholamines in human plasma by high-performance liquid chromatography with amperometric detection as compared with a radioenzymatic method

A high-performance liquid chromatographic procedure is described for the quantitative determination of epinephrine, norepinephrine, and dopamine in human plasma. The method, which is based on adsorption of the catecholamines to alumina and, after liberation, separation on a microparticulate bonded strong cation-exchange resin and amperometric detection, has been optimized to give complete baseline separation of the substances of interest. Dihydroxybenzylamine, a nonendogenous catecholamine, is used as the internal standard. The detection limit is about 0.1 pmol for dopamine. Analysis of data obtained for norepinephrine and epinephrine from a total of 59 plasma samples showed a good correlation to the corresponding values obtained with a radioenzymatic method. Some results from normal and pathological conditions are compared.     

An improved approach for the determination of plasma-free catecholamines by HPLC-ED: Application to normal and hypertensive patients

Summary A dual-step procedure for the rapid, quantitative isolation of free catecholamines (norepinephrine, epinephrine and dopamine) from plasma, using a little column of CM-Sephadex and alumina adsorption, is described. Sensitive high performance liquid chromatography is also discussed, employing an amperometric detector for the quantitative determination. The recovery of the three catecholamines, and of N-methyldopamine used as the internal standard, was about 70–80%; the detection limits were 2pg for norepinephrine, 3pg for epinephrine and 3pg for dopamine. The combination of the rather specific and easy to handle two-step sample clean-up procedure, the high resolving power of the chromatography and the high sensitivity of electrochemical detection provided a simple method for the determination of free catecholamines in plasma samples of normal and essential hypertensive subjects under different conditions (supine position for 45 min, standing for 5 and 10 min). It was found that a significant increase in epinephrine levels (P<0.01) occurred in hypertensive patients under the three conditions studied.     

分子印迹纳米管膜的制备及对人体尿液中儿茶酚胺类药物的检测

以多巴胺(DA)为模板, 多孔阳极氧化铝膜(AAO)为反应载体, 合成了多巴胺分子印迹聚合物纳米管膜(AAO@MIP). 利用扫描电子显微镜对分子印迹纳米管膜的形貌进行了表征, 并用高效液相色谱(HPLC)研究了其对儿茶酚胺类(CLs)药物的吸附性能. 实验结果表明, 在最优萃取条件下, AAO@MIP 纳米管膜对多巴胺、 肾上腺素和去甲肾上腺素具有较高的选择性, 3种儿茶酚胺类药物在0.50~300 μmol/L浓度范围内呈良好的线性关系(r²>0.9970); 检出限(S/N=3)分别为15.5, 12.6和22.5 ng/L. AAO@MIP纳米管膜对多巴胺的最大吸附容量可达82.1 μmol/g; 6次吸附-解吸附重复利用后, 吸附容量仅降低3.3%. 将AAO@MIP 纳米管膜应用于萃取人体尿液中3种儿茶酚胺, 样品加标回收率为74.0%~100.4%, 相对标准偏差(RSD)为3.6%~6.8%. 该方法简便、 快速、 选择性高, 适用于检测人体尿液中的儿茶酚胺类药物的含量.     

Determination of catecholamines in urine using aminophenylboronic acid functionalized magnetic nanoparticles extraction followed by high‐performance liquid chromatography and electrochemical detection

A new method was developed for the simultaneous determination of three catecholamines in urine using aminophenylboronic acid functionalized magnetic nanoparticles extraction followed by high‐performance liquid chromatography with electrochemical detection. Novel aminophenylboronic acid functionalized magnetic nanoparticles were prepared by multi‐step covalent modification, and characterized by transmission electron microscopy, Fourier‐transformed infrared spectroscopy, X‐ray diffraction, and vibrating sample magnetometry. With the help of the high affinity between the boronate and cis‐diol group, the particles were used for the highly selective separation and enrichment of three major catecholamines, norepinephrine, epinephrine, and dopamine. Effects of the pH of the feed solution, the extraction time, the composition of the buffer solution, the amount of the magnetic particles, the elution conditions, and the recycling of aminophenylboronic acid functionalized magnetic nanoparticles were explored. Under the optimized conditions, 13–17‐fold enrichment factors were obtained. The linear ranges were 0.01–2.0 μg/mL for the studied analytes. The limits of detection and quantification were in the range of 2.0–7.9 and 6.7–26.3 ng/mL, respectively. The relative recoveries were in the range of 92–108%, with intraday and interday relative standard deviations lower than 6.8%. This method was successfully applied to analysis of catecholamines in real urine.     

Simultaneous determination of catecholamines and their metabolites related to Alzheimer's disease in human urine

A simple and specific high-performance liquid chromatography method coupled with fluorescence detection (HPLC-FL) has been developed for the simultaneous determination of L-3,4-dihydroxyphenylalanine, norepinephrine, dopamine, epinephrine and 3,4-dihydroxyphenylacetic acid in human urine. The samples were derivatized by 1,2-diphenylethylenediamine with isoprenaline as internal standard. The factors affecting the fluorescence yield were investigated, including the reaction and separation conditions. The catecholamine derivatives were separated on a Kromasil C(18) column with methanol and sodium acetate buffer as mobile phase. The limits of detection for all catecholamines ranged from 0.2 to 1.1 ng/mL. The linear ranges were from 2.5 to 200 ng/mL except 3,4-dihydroxyphenylacetic acid from 5 to 200 ng/mL. The intra- and interday RSDs for all catecholamines were 1.0-8.0 and 2.1-14%, respectively. The method was successfully applied to determine the catecholamines in human urine from 14 Alzheimer's disease patients and 14 healthy volunteers. It was concluded that the mean levels of catecholamines in urine of Alzheimer's disease patients were all lower than those in healthy volunteers. The cluster analysis and independent samples T-test were used to distinguish the Alzheimer's disease patients and healthy volunteers.     

High-performance liquid chromatographic assay of free norepinephrine, epinephrine, dopamine, vanillylmandelic acid and homovanillic acid

A procedure is described for the concurrent assay of free norepinephrine, epinephrine, dopamine, vanillylmandelic acid and homovanillic acid in physiological fluids using high-performance liquid chromatography with electrochemical detection. The column packing is an octadecyl-bonded silica. A single mobile phase containing 1-octanesulphonate is used for the assay of catecholamines and for the assay of the acidic metabolites. An efficient sample preparation scheme is presented for the isolation of the catecholamines and their acidic metabolites from the same sample aliquot. Catecholamines are extracted by ion exchange on small columns and adsorption on alumina, using dihydroxybenzylamine as an internal standard. Vanillylmandelic acid and homovanillic acid are recovered from the combined loading and washing effluents of the ion-exchange column by a solvent extraction procedure. Recovery of catecholamines averages 67%. The limit of detection for individual catecholamines is ca. 30 pg. Recoveries of vanillylmandelic acid and homovanillic acid average 77% and 87%, respectively. The use of the same mobile phase for the concurrent assay of catecholamines and their acidic metabolites considerably increases the throughput of samples in the chromatographic system by eliminating the time-consuming column-equilibration periods.     

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.     

Quantitative determination of epinephrine and norepinephrine in the picogram range by flame ionization gas-liquid chromatography.

A gas-liquid chromatographic method has been developed using the hydrogen flame detector to determine epinephrine (E) and norepinephrine (NE) in blood plasma, red blood cells, serum, and urine. The chromatographic method presents several advantages over other existing techniques. The derivatives enable separation of E and NE and are stable at room temperature with no signs of decomposition. The detection limit for the catecholamines with the hydrogen detector was approximately 0.1 pg. The catecholamines can be determined simultaneously from the same gas-liquid chromatogram. Purification of the catecholamines using the conventional procedure of chromatographing on alumina has been eliminated. With this gas chromatographic method, no by-products are formed that interfere with E and NE determinations. Dopamine, which constitutes the major source of interference in the commonly used fluorometric methods, does not interfere with the E and NE determinations. Norepinephrine and epinephrine values for several physiological fluids are given with the analysis expanded to include red blood cells, the contents of which have not been previously reported.     

Plasma Sample Cleanup for Catecholamines Analysis Using Weak Cation Exchange Solid Phase Extraction

Abstract

Norepinephrine and epinephrine were isolated from human plasma at picogram levels by solid phase extraction (SPE), on a proprietary weak cation exchange silica based packing. Plasma samples were spiked with catecholamines at two concentrations to simulate body response to varying levels of stress. Recoveries of norepinephrine and epinephrine at both concentrations exceeded 95%.

An analysis of catecholamines by ion pair high-performance liquid chromatography with amperometric detection is described.     

Selective liquid-chromatographic determination of native fluorescent biogenic amines in human urine based on fluorous derivatization

A liquid chromatographic (LC) derivatization method for simple and selective determination of catecholamines and indoleamines in human urine has been developed. This method uses "fluorous interaction" in which perfluoroalkyl compounds show affinity with each other. The amino groups of native fluorescent analytes are precolumn derivatized with a non-fluorescent fluorous isocyanate, 2-(perfluorooctyl)ethyl isocyanate, and the fluorous-labeled analytes are retained in the fluorous LC column, whereas underivatized substances are not. Only the retained fluorous-fluorescent analytes are detected fluorometrically at appropriate retention times, and retained amines without fluorophores are not detected. In this study, 3,4-dihydroxyphenylalanine, dopamine, norepinephrine, epinephrine, and metanephrine were used as the representative of catecholamines. Tryptophan, 5-hydroxytryptophan, and 5-hydroxytryptamine were used as the representative indoleamines. This method was applied to determine eight biogenic amines in urine from healthy humans. The fluorous-labeled amines could be separated by fluorous LC column under conditions of isocratic elution within 35 min and simultaneously determined without interference from contaminants in biological samples. The detection limits for eight biogenic amines were 31-640 fmol on column. Calibration curves of them were linear over the range of at least 10-100 nmol/mL urine (r2 > 0.9989) with good repeatability.     

Determination of catecholamines in human plasma by high-performance liquid chromatography: comparison between a new method with fluorescence detection and an established method with electrochemical detection

We report a sensitive fluorimetric method, in which catecholamines are concentrated from plasma by liquid-liquid extraction and derivatized with the selective fluorescent agent 1,2-diphenylethyl-enediamine prior to chromatography. Optimal conditions for extraction, derivatization and chromatography were investigated. With alpha-methylnorepinephrine as internal standard, the chromatographic separations are complete within 6 min. Limits of detection are 0.3 pg for norepinephrine and epinephrine and 0.5 pg for dopamine. Coefficients of variation are low (3-7%). Comparison of plasma catecholamine values determined with this method and with an established method with electrochemical detection (n = 135) shows good correlation (r = 0.94-1.00), and regression lines are close to lines of identity.     

Determination of vanilmandelic acid in urine by coupled-column liquid chromatography combining affinity to boronate and separation by anion exchange

An automated liquid chromatographic method for assaying vanilmandelic acid in urine is described. Vanilmandelic acid and potential interfering substances, such as catechol compounds and their metabolites, have been tested for affinity to boronic acid-substituted silica at various pH values. Vanilmandelic acid and the internal standard, isovanilmandelic acid, were bound to the boronate matrix at an acidic pH, whereas for instance catecholamines were unretained and passed through the column. The alpha-hydroxycarboxylic acids were then desorbed by another mobile phase (pH 6.0) and transferred to an anion exchanger for chromatography and electrochemical detection. A relative standard deviation of 2.8% was obtained for the analysis of human urine samples containing 6.6 microM vanilmandelic acid.     

Estimation of catecholamines in human plasma by ion-exchange chromatography coupled with fluorimetry

Estimation of catecholamines in human plasma was made by ion-exchange chromatography coupled with fluorimetry. Catecholamines in deproteinized plasma were adsorbed onto Amberlite CG-50 (pH 6.5, buffered with 0.4 M phosphate buffer) and selectively eluted by 0.66 M boric acid. The catecholamine fraction was separated further on a column of Amberlite IRC-50 which was coupled with a device for the automated performance of the trihydroxyindole method (epinephrine and norepinephrine) or the 4-aminobenzoic acid-oxidation method (dopamine). One sample could be analysed within 25 min with either method. The lower detection limits were 0.02 ng for epinephrine and dopamine, and 0.04 ng for norepinephrine. Plasma catecholamine contents of healthy adults at rest were epinephrine 0.07 +/- 0.01 ng/ml (n = 19), norepinephrine 0.27 +/- 0.03 ng/ml (n = 19) and dopamine 0.22 +/- 0.03 ng/ml (n = 26). The procedure of adsorption and elution of the plasma catecholamines by ion-exchange resin was simple, the simplicity contributing to constant recovery. The catecholamine fraction could be analysed without evaporation of the eluate. The analytical column could be used for the analysis of more than 1000 samples before excessive back-pressure developed. Our method of continuous measurement of plasma catecholamine fulfils clinical requirements.     

Determination of free and total catecholamines in human urine by HPLC with fluorescence detection

A simple and highly sensitive method for the determination of free and total (free + conjugated) catecholamines (norepinephrine, epinephrine and dopamine) in human urine is described which employs HPLC with fluorescence detection. Conjugated catecholamines (sulfate form) are hydrolyzed by a sulfatase-mediated reaction to the corresponding free amines. After cation exchange chromatography on a Toyopak IC-SP S cartridge, catecholamines and isoproterenol (internal standard) in urine samples were converted into the corresponding fluorescent compounds by reaction with 1,2-diphenylethylenediamine. These compounds were separated within 8 min on a reversed phase column with isocratic elution using a mixture of water, methanol and acetonitrile containing a Tris-hydrochloric acid buffer (pH 7.0). The detection limit for each catecholamine is ca 2 fmol per 100 microL injection volume.