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.     

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.     

Ion pairing high-performance liquid chromatography of catecholamines

Catecholamine standards have been separated by ion pairing reversed-phase high-performance liquid chromatography (HPLC). A trace substance was separable from four standards when low concentrations of methanol were used in the elution buffer. This method has allowed separation of an unknown uterine catecholamine, partially purified on a boronate affinity gel column, from the standards: norepinephrine, epinephrine, normetanephrine and metanephrine.     

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.     

Oxidative stress increases 6-nitronorepinephrine and 6-nitroepinephrine concentrations in rat brain

6-Nitronorepinephrine (nitroNE) and 6-nitroepinephrine (nitroE) are reaction products of nitric oxide and norepinephrine and epinephrine, respectively. The authors have previously reported a method for determination of nitroNE and nitroE in rat brain using high-performance liquid chromatography-peroxyoxalate chemiluminescence reaction detection. In this study, the effect of oxidative stress on nitroNE and nitroE concentrations in rat brain was examined using this method. After kainic acid administration in rats for 2 days, the concentrations of both nitroNE and nitroE in rat brains were found to have increased by 400-600%, which was partly suppressed by the co-administration of a superoxide dismutase mimetic. This indicates that oxidative stress might increase nitroNE and nitroE concentrations in rat brains.     

The automated analysis of catecholamines: An improved procedure for the simultaneous differential analysis of epinephrine and norepinephrine in tissues, blood and gland perfusates

The automated method of Andersson et al. (2) for the determination of epinephrine and norepinephrine in urine has been modified to allow the simultaneous quantitation of the two amines in a sample during a single run through the AutoAnalyzer. The differentiation of epinephrine and norepinephrine was based on the use of two stabilizing agents and oxidation of the catecholamines at two quite different pHs. As a result epinephrine could be measured directly from the recorded fluorescence peak. Norepinephrine fluorescence was accompanied by a minimal contamination by epinephrine fluorescence, amounting to less than 4% of the total fluorescence.The method has been applied to the determination of the amines in tissues, adrenal vein blood, and perfused adrenal effluents.     

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.     

Optical fiber biosensor coupled to chromatographic separation for screening of dopamine, norepinephrine and epinephrine in human urine and plasma

An optical fiber biosensor has been developed for the determination of catecholamines (dopamine, norepinephrine and epinephrine) based on the recognition capacity of the enzyme laccase. In this study, a glass tube constituted by a fused silica fiber coated with a film of polystyrene/divinylbenzene resin (PS/DVB) was used for catecholamines separation. Firstly, the analyzer was tested for calibration and its analytical performance for catecholamines detection was compared with a classical analytical method, namely high performance liquid chromatography-electrochemical detector (HPLC-ED). The developed analytical device shows a high potential for catecholamines quantification with a detection limit of 2.1, 2.6 and 3.4 pg mL⁻¹ for dopamine, norepinephrine and epinephrine, respectively. The analytical sensitivity, inferred from the slope of the calibration curves established for a range of concentrations between 5 and 125 pg mL⁻¹, was found to be 0.344, 0.252 and 0.140 dB/pg mL⁻¹ for dopamine, norepinephrine and epinephrine, respectively. Furthermore, catecholamines speciation with the PS/DVB fiber was completely achieved in 3 min. The analytical performance of the reported sensor was also evaluated and found adequate for catecholamines determination in human urine and plasma samples.     

Study of the acute cardiovascular effects of several antihypertensive agents with the measurement of plasma catecholamines in mice

A highly sensitive determination method was established for catecholamines (norepinephrine (NE), epinephrine, and dopamine) with high-performance liquid chromatography-peroxyoxalate chemiluminescence reaction detection. In this study, the method was applied to mouse plasma, and it was determined that only 10 μl of mouse plasma was necessary for the selective and reproducible determination of catecholamines. Studies were then conducted in acute cardiovascular effects of sodium nitroprusside, nicardipine, captopril (angiotensin-converting enzyme (ACE) inhibitor), candesartan, and olmesartan (type 1 angiotensin receptor antagonists (AT₁ antagonists)) by this method. Sodium nitroprusside and nicardipine elevated plasma NE concentrations significantly, whereas the ACE inhibitor and the AT₁ antagonists did not change plasma NE concentrations in anesthetized mice. These results suggested that angiotensin II-induced augmentation may be mainly carried through the central baroreflex pathway.     

Facile synthesis of Fe3O4@polyethyleneimine modified with 4‐formylphenylboronic acid for the highly selective extraction of major catecholamines from human urine

The levels of catecholamines, especially dopamine, epinephrine and norepinephrine in urine and plasma have been used to assist the diagnosis and treatment of psychosis. Due to their low endogenous concentrations, the determination of the three major catecholamines is very difficult. Boronate adsorbents are often employed to extract these cis‐diol compounds from complex matrices. In this work, a novel type of magnetic nanoparticles modified with 4‐formylphenylboronic named Fe₃O₄@PEI‐FPBA was synthesized by a facile two‐step approach. The abundant amino groups of polyethyleneimine provided the rich binding sites for boronate ligands. Herein, the adsorption capacity of Fe₃O₄@PEI‐FPBA is greatly improved with a value of 3.45 mg/g towards epinephrine, which is much larger than that of analogous material without polyethyleneimine. The magnetic nanoparticles also exhibited high magnetization (72.25 emu/g) and specific selectivity towards the catecholamines. Finally, a liquid chromatography tandem mass spectrometry method based on Fe₃O₄@PEI‐FPBA nanoparticles was successfully used to determine the three catecholamines from human urine samples. The linearity, limit of quantitation, recovery and precision of the method were satisfactory. Based on the method, it is found that the levels of dopamine, epinephrine and norepinephrine in depressive patients are higher than those in healthy controls.     

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.     

Disposable Carbon Electrodes for Liquid Chromatographic Detection of Catecholamines in Blood Plasma Samples

This paper describes a comparative evaluation of disposable carbon electrodes and conventional glassy carbon electrodes. The detection of catecholamines was demonstrated in processed blood plasma and in the presence of catecholamine metabolites. Calibration plots of norepinephrine, epinephrine, 3,4‐dihydroxybenzylamine and dopamine were linear over three to four orders of magnitude with detection limits of 0.50, 0.73, 1.06, and 1.13 pg, respectively. The relative standard deviation of peak areas was ±2.3% for norepinephrine and ±5.3% for epinephrine from 30 injections of a 10 ng/mL mixed standard. Spike recoveries for norepinephrine, epinephrine, and dopamine from human blood plasma were 86±6%, 81±5%, and 77±4%, respectively.     

Estimation of plasma catecholamines by high-performance liquid chromatography with electrochemical detection in patients with subarachnoid haemorrhage

Plasma catecholamine levels were determined by high-performance liquid chromatography with electrochemical detection following alumina extraction. Mean individual recoveries of 50.5, 49.9 and 48% were obtained for norepinephrine, epinephrine and dopamine, respectively, and the limits of detection for each catecholamine were 0.15, 0.34 and 0.6 pmol/ml. Total analysis time for each plasma sample was approximately 1 h. Catecholamine levels were measured in plasma from control subjects and the ranges obtained were: norepinephrine, 0.33-5.98 pmol/ml; epinephrine, 0-4.77 pmol/ml; dopamine, 0-0.8 pmol/ml. When patients with subarachnoid haemorrhage were investigated, the ranges were found to be: norepinephrine, 0.23-7.27 pmol/ml; epinephrine, 0-4.91 pmol/ml; dopamine, 0-0.23 pmol/ml.     

Is There a Novel Biosynthetic Pathway in Mice That Converts Alcohol to Dopamine,Norepinephrine and Epinephrine?

Previous studies in animals and humans have shown multiple types of interaction between alcohol (ethanol) intake and the catecholamine signaling molecules: dopamine, norepinephrine and epinephrine. This literature suggests that the administration of alcohol to rodents affects the central and peripheral (blood plasma) levels of these catecholamines. Two prior publications (Fitzgerald 2012, 2020) put forth the hypothesis that there may be a currently unidentified biosynthetic pathway, in a range of organisms, that actually converts alcohol to dopamine, norepinephrine and epinephrine. This publication describes the details for how to test this hypothesis in mice. Mice can be systemically injected with an intoxicating dose of commercially available stable isotope-labeled ethanol (ethanol-1-¹³C), and blood plasma samples and brains can be collected approximately two to 24 h post-injection. Liquid chromatography-mass spectrometry analysis can then be used to test whether some of the labeled ethanol molecules have been incorporated into new dopamine, norepinephrine, and epinephrine molecules, in plasma and brain samples. If confirmed, this hypothesis may have broadly reaching implications both for basic neuroscience and our understanding of alcohol abuse and alcoholism.     

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.     

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.     

In-line derivatization method for fluorometric determination of catecholamines by high performance liquid chromatography

Chromatographia - A fluorometric method has been developed for the determination of norepinephrine, epinephrine and dopamine by high-performance liquid chromatography. Catecholamines extracted from...     

Analysis of plasma catecholamines by high-performance liquid chromatography with fluorescence detection: simple sample preparation for pre-column fluorescence derivatization

Analysis of plasma catecholamines (norepinephrine, epinephrine and dopamine) by high-performance liquid chromatography using 1,2-diphenylethylenediamine as a fluorescent reagent is described. We have developed an automatic catecholamine analyser, based on pre-column fluorescence derivatization and column switching. The analysis time for one assay was 15 min. The correlation coefficients of the linear regression equations were greater than 0.9996 in the range 10-10,000 pg/ml. The detection limit, at a signal-to-noise ratio of 3, was 2 pg/ml for dopamine. A new method of sample preparation for the pre-column fluorescence derivatization of plasma catecholamines was used. In order to protect the catecholamines from decomposition, an ion-pair complex between boric acid and the diol group in the catecholamine was formed at a weakly alkaline pH. The stabilities of plasma catecholamines were evaluated at several temperatures. After complex formation, the catecholamines were very stable at 17 degrees C for 8 h, and the coefficients of variation for norepinephrine, epinephrine and dopamine were 1.2, 4.2 and 9.3%, respectively.     

Thin-layer chromatographic separation of serotonin from epinephrine and norepinephrine

A thin-layer chromatographic procedure is reported for the separation of serotonin from epinephrine and norepinephrine. The method involves the use of ethylenediaminetetraacetic acid-impregnated silica gel G plates and n-butanol-ethanol-acetic acid-water (8:2:1:3) as developer. Serotonin is well separated from both epinephrine and norepinephrine. Epinephrine and norepinephrine overlapped slightly but were both detectable. The procedure is thus applicable to the separation of all three biogenic amines in spite of the absence of an absolutely clear separation of epinephrine from norepinephrine.     

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.