Catecholamines In Human Plasma Research Articles

Fast determination of catecholamines in human plasma using carboxyl-functionalized magnetic-carbon nanotube molecularly imprinted polymer followed by liquid chromatography-tandem quadrupole mass spectrometry

A novel, simple and sensitive method based on the use of dispersive micro-solid-phase extraction (d-μ-SPE) procedure combined with ultra-fast liquid chromatography–tandem quadrupole mass spectrometry (UFLC–MS/MS) for the determination of catecholamines, i.e., dopamine (DA), norepinephrine(NE) and epinephrine (E), was developed and validated. The novel catecholamines molecularly imprinted polymer (MIP) on the surface of carboxyl-functionalized magnetic-carbon nanotube (CF@m-CNTs-MIP) was synthesized and characterized by vibrating sample magnetometer (VSM), scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). The CF@m-CNTs-MIP was used as the d-μ-SPE sorbent to extract catecholamines from human plasma samples. The obtained results demonstrated the higher extraction capacity of CF@m-CNTs-MIP with recoveries between 87.5–110%. The limits of quantification (LOQs) for NE, E and DA were 76ng/L, 18ng/L and 10ng/L, respectively. Validation results on linearity, specificity, accuracy, precision and stability, as well as on application to the analysis of catecholamines in 120 healthy volunteers demonstrated the applicability to clinical studies.

Separation of free amino acids and catecholamines in human plasma and rabbit vitreous samples using a new fluorogenic reagent 3‐(4‐bromobenzoyl)‐2‐quinolinecarboxaldehyde with CE‐LIF detection

A sensitive and efficient analysis of amino acids and catecholamines is currently presented with 3-(4-bromobenzoyl)-2-quinolinecarboxaldehyde as a fluorogenic derivatization reagent using CE separation with LIF detection. For good derivatization conditions, the reagent concentrations, pH value, temperature, and reaction time were explored, which were followed by the derivatization reaction in stable yield. The optimal running buffer was composed of mixtures involving 120 mM, pH 9.1 boric acid, 38.5 mM SDS, and 19% ACN v/v. The LOD (S/N=3) was found as low as 0.65 nM. The proposed method was validated by the two-order-magnitude linearity and correlation coefficient ranging from 0.9957 to 0.9998. The accuracy and specificity of this assay were also assured from the spiking of real samples with a standard known concentration. In order to demonstrate its wide range of applications, the method has been applied to the analysis of both human plasma and rabbit vitreous samples.

Simultaneous analysis of human plasma catecholamines by high-performance liquid chromatography with a reversed-phase triacontylsilyl silica column

The clinical importance of simultaneous analysis of 3,4-dihydroxyphenylglycol with other human plasma catecholamines has been investigated to better understand the sympathetic nervous system. However, previous reports have had analytical difficulties with both resolution and extraction. The current study uses a reversed-phase triacontylsilyl silica (C30) column under the mobile phase condition without ion-pair reagents to separate catecholamines and their metabolites, with above 91% recoveries for intra-assay, above 85% for inter-assay, and less than 10% (n=5) coefficient of variation. Lower detection limits (S/N=4) and quantification limits (S/N=6) were 40 and 100 pg/mL for norepinephrine, 3,4-dihydroxyphenylglycol, and 3,4-dihydroxyphenylalanine, 10 and 20 pg/mL for epinephrine, 10 and 40 pg/mL for dopamine. Linear ranges were from 40 to 5000 pg/mL for norepinephrine and 3,4-dihydroxyphenylalanine, from 100 to 5000 pg/mL for 3,4-dihydroxyphenylglycol, and from 10 to 2000 pg/mL for epinephrine and dopamine. The C30 column may prove clinically useful, as it provides a convenient and simultaneous method of evaluation of human plasma catecholamines.

Determination of catecholamines in human plasma by high-performance liquid chromatography with electrochemical detection

In the present study, assays were improved for the determination of catecholamines in human plasma. High-performance liquid chromatography with electrochemical detection was employed for quantitative analysis. The influence of various parameters on chromatographic performance, such as the composition and the pH of the mobile phase, and the detection potential, was investigated. An accurate solid-phase extraction procedure, after catecholamine complexation with diphenylborate, was developed. The efficiency yield for all catecholamines was in the range 92–98%. Relative standard deviation values for repeatability and for intermediate precision were less than 2% and 3%, respectively, for all three analytes.

Determination of Catecholamines in Human Plasma by HPLC with Electrochemical Detection

An assay procedure is described to quantitate low picogram concentrations of catecholamines in human plasma using reversed phase ion-pair HPLC with electrochemical detection. Optimization of chromatographic conditions allows use of samples as small as 0.25 mL. The overall recovery of 78% offers high precision and accuracy at low concentrations. The lower limit of detection is 0.87 pg/mL for epinephrine, 3.5 pg/mL for norepinephrine and 8.3 pg/mL for dopamine.

Plasma free dopamine: physiological variability and pathophysiological significance.

Dopamine (DA) is the most abundant catecholamines in human plasma and exists mostly in the sulfo-conjugated form (DA sulfate), a biologically inactive metabolite. The paucity of unconjugated DA (PDA) in plasma throws doubt on its physiological significance. However, PDA, when measured with a highly sensitive radioenzymatic method, showed quite different features from norepinephrine and epinephrine in some types of clinical hypertension, lower in essential hypertension and higher in primary aldosteronism and pheochromocytoma. There was a weak but significant correlation between the values of PDA and DA sulfate measured in the same specimens, but DA sulfate was more susceptible to impaired renal function. Upright posture, high salt diets and an intravenous injection of metoclopramide (MCP, 10 mg), a DA receptor antagonist, induced a slight but significant increase in PDA in normal and hypertensive subjects. An intravenous dexamethasone (2 mg) caused a gradual increase in PDA over 150 min after medication, which was completely blocked by concomitant administration of alpha-methyl-p-tyrosine, a tyrosine hydroxylase inhibitor. The responses of PDA to both high salt diets and MCP were blunted in salt-sensitive patients with uncomplicated essential hypertension. The results suggest that DA is not only a precursor of norepinephrine biosynthesis but also plays an inherent role as an active neurotransmitter in the peripheral sympathoadrenal system, and that PDA is a sensitive marker of peripheral dopaminergic activity, which may operate to modulate the cardiovascular and endocrine functions and participate in the pathogenesis of some types of hypertension.

Measurement of Conjugated Catecholamines in Human Plasma. A Low Cost HPLC-ECD Method

Abstract We present an HPLC assay with electrochemical detection of conjugated catecholamine levels in human plasma. This method allows the direct assessment of the conjugated catecholamine values without the subtraction of the free catecholamine amounts: the assay is performed in the residual supernatant after the extraction (and the assay) of free catecholamines. The results are comparable with the classic enzymatic and acid-neat hydrolysis methods (which yield the sum of free and conjugated catecholamine concentrations). This technique shows good reproducibility (within-run and between-run CVs < 8% in the physiological range), and sensitivity (< 20 pg per each deconjugated catecholamine per extract). This method allows about 30 low cost determinations of plasma free and conjugated catecholamines to be done in a working day.

HPLC with Electrochemical Detection of Catecholamines in Human Plasma. A Mini-Review

Abstract Since 1970s HPLC received growing attention among catecholamine (CA) assay methods. Earlier methods such as colorimetric (1) or fluorimetric (2) nowadays seem obsolete. Radiotracer methods are usually effective for the determination of norepi-nephrine (NE) and epinephrine (Epi) in human plasma, although the high cost per sample, the poor sensitivity regarding plasma dopamine (DA), the involvement of radiolabelled compounds and the te-

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-diphenylethylenediamine prior to chromatography. Optimal conditions for extraction, derivatization and chromatography were investigated. With α-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.

Sample clean-up method for determination of catecholamines in plasma and heart by HPLC.

A modified sample clean-up method for the HPLC of catecholamines (CA) in biological samples was proposed. At the adsorption procedure the loss of alumina was neglected by placing a disk filter on the bottom disk in the Mini column, and also a top filter on the surface of the sample solution. Plasma and heart extract were adsorbed onto alumina in Sepacol Mini tube. In order to obtain a constant and efficient adsorption of CA, the authors introduced the improved rotary mixer for the rotation of alumina column. By mixing the CA sample solution and alumina with rotary mixer, the recovery and reproducibility of CA were considerably improved comparing with the conventional method. HPLC with the clean-up procedure was applied to the determination of CA in human plasma and rat heart. The utility of the proposed clean-up method of CA (norepinephrine, epinephrine and dopamine) was sufficiently recognized.

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.

Liquid-chromatographic determination of norepinephrine and epinephrine in human plasma.

We describe a "high-performance" liquid-chromatographic method for measuring picogram amounts of norepinephrine and epinephrine simultaneously in human plasma. Alumina-treated samples are injected onto a strong cation-exchange column (Zipax SCX) for the separation of catecholamines, with a sodium phosphate buffer/acetonitrile mixture as mobile phase. The separated catecholamines then enter a continuous-flow system and the reagents for the trihydroxyindole reaction are added sequentially; the fluorescent products are then measured with a spectrofluorophotometer. Analytical recoveries from plasma averaged 66% for norepinephrine, 68% for epinephrine. Amount and response (peak height) were linearly related up to 1000 pg. For catecholamines in human plasma, within-run and day-to-day CV's were 0.2% and 1.0%, respectively, for norepinephrine, and 0.3% and 0.9%, respectively, for epinephrine. Average values for norepinephrine and epinephrine in apparently normal human plasma were 185 +/- 29 ng/L and 32 +/- 8 ng/L, respectively (mean +/- SEM, n = 10). This relatively rapid and highly sensitive system is suitable for routine use.

Liquid-chromatographic determination of norepinephrine and epinephrine in human plasma.

Abstract We describe a “high-performance” liquid-chromatographic method for measuring picogram amounts of norepinephrine and epinephrine simultaneously in human plasma. Alumina-treated samples are injected onto a strong cation-exchange column (Zipax SCX) for the separation of catecholamines, with a sodium phosphate buffer/acetonitrile mixture as mobile phase. The separated catecholamines then enter a continuous-flow system and the reagents for the trihydroxyindole reaction are added sequentially; the fluorescent products are then measured with a spectrofluorophotometer. Analytical recoveries from plasma averaged 66% for norepinephrine, 68% for epinephrine. Amount and response (peak height) were linearly related up to 1000 pg. For catecholamines in human plasma, within-run and day-to-day CV's were 0.2% and 1.0%, respectively, for norepinephrine, and 0.3% and 0.9%, respectively, for epinephrine. Average values for norepinephrine and epinephrine in apparently normal human plasma were 185 +/- 29 ng/L and 32 +/- 8 ng/L, respectively (mean +/- SEM, n = 10). This relatively rapid and highly sensitive system is suitable for routine use.

Single-isotope enzymatic derivative method for measuring catecholamines in human plasma.

The radioenzymatic determination of plasma catecholamines with a modification of the method of da Prada & Zürcher ((1976), Life Sci 19, 1161-1174) is described. The several reaction steps were optimized with respect to the quantities of substrate and enzyme, and reaction time. There were particular methodological difficulties concerning the blanks, which were determined by using sodium metaperiodate-oxidized plasma. The reliability criteria of the method were determined. Coefficients of variation between 3.4 and 8.6% were found for the intra-assay variability of 10 pg of norepinephrine and 3 pg of epinephrine or dopamine, resp. The recoveries o the three catecholamines ranged from 88-93%. The detection limits were calculated from the standard deviation of the blanks and amounted to 12 ng/l (norepinephrine), 6 ng/l (dopamine) and 3 ng/l (epinephrine). The method was used for the analysis of plasma samples from patients. In a further investigation we examined the stability of plasma catecholamines stored at different temperatures. It was found that samples can be stored for 1-2 hours at room temperature and for several weeks at -27 degrees C without losses in catecholamine content.

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.

Comparison of double- and single-isotope enzymatic derivative methods for measuring catecholamines in human plasma.

We directly compared the reliability of a single-isotope enzymatic derivative technique for measurement of plasma catecholamines with that of the well-established double-isotope method. A significant (p less than 0.001) correlation was observed between measurements (n = 52) in the two assays, both for norepinephrine (r = 0.97) and epinephrine (r = 0.80). Means and coefficients of variation for the two analytes in a pooled specimen of plasma, measured repeatedly during six months, were virtually identical by each assay method. Basal plasma catecholamine concentrations in two different groups of apparently healthy subjects were also similar by each method. Dopamine concentrations in plasma were consistently below the limits measurable by either technique. The single-isotope assay requires half the assay time and 1/200th the sample as the double-isotope method. We conclude that this assay is just as reliable as the double-isotope technique and gives virtually identical values for norepinephrine and epinephrine concentrations in the physiological range.

Gas-liquid chromatographic and mass fragmentographic determination of catecholamines in human plasma

Quantitative analytical methods for plasma catecholamines and their conjugates by the use of gas-liquid chromatography have been developed. Epinephrine and dopamine have also been determined by mass fragmentography. The contents of catecholamines in the plasma of normal adults and patients with hypertension, neuroblastoma and pheochromocytoma have been demonstrated.

Gas-liquid chromatographic and mass fragmentographic determination of 3- O-methylated catecholamines in human plasma

Plasma 3- O-methylated catecholamines, i.e. 3-methoxytyramine, normetanephrine and metanephrine, were separated from catecholamines by passing through alumina and further purified by adsorbing on weakly acidic resin and Amberlite XAD-4. The amines were trifluoroacetylated and determined by gas chromatography or mass fragmentography. Tracer quantities of tritiated 3-MT, NMN or MN were used as internal standards for total recovery estimations. The contents of 3- O-methylated catecholamines in the plasma of normal persons and patients with hyperthyroidism, hypertension, neuroblastoma and pheochromocytoma were measured.

Changes in human plasma catecholamines and dopamine-beta-hydroxylase produced by prostaglandin F 2α

The intravenous infusion of prostaglandin F 2α to pregnant humans produces an increase in circulating norepinephrine, without significantly altering plasma epinephrine or dopamine-β-hydroxylase. Plasma dopamine-β-hydroxylase activity at 10–16 weeks gestation is not significantly greater than that in non-pregnant controls of a similar age range, and central venous enzyme activity is not significantly different from that in arterial plasma.

Rapid and sensitive method for determination of catecholamines in human plasma

Various methods1-4 using trihydroxyindol reaction for the measurement of catecholamines in human plasma have been designed by workers hitherto. However, in these methods1, 3, 4, isolation of the catecholamines with an adsorbent presents considerable difficulties. Thus, in spite of the availability of these methods1, 3, 4 reproduction by other laboratories are difficult. The main objective of the present work was to improve the method for simultaneous determination of epinephrine (Ep.) and norepinephrine (NE.) in human plasma. A preliminary experiment was set up in order to run a comparison against these methods with respect to the recovery, accuracy and rapidity. Of these methods PRICE's method3 showed satisfactory recovery and rapidity. Thus several improvements of PRICE's methods3 were made: 1) Purification of acid-washed aluminum oxide (Merck acid washed #71695) was made. 2) A large amount of EDTA disodium was used in several steps. 3) The timing procedure becomes essential due to the fact that fluorophor of both Ep. and to a smaller degree NE. decays at a considerable rate. Thus, a means with sufficiently high accuracy and rapidity was obtained. Material and Method A preparation of acid-washed aluminum oxide (for chromatographic use). a) 300-500 g of alumina (Merck-71695 acid washed) was boiled using a reflux condenser with frequent shaking in 1 L 2N HCl for 1 hour. The slurry was allowed to cool to room temperature. This was transferred to a 1 L chromato-graphic tube with a fritter disc (pyrex #38450). and was cycled with deionized water until the pH of the supernatant rose to 5.0 after which a stirring of the column was followed by cleaning of the supernatant for 2 minutes. This rapid cleaning indicates that most of the fine grain alumina is removed. Next collect the alumina in a suction funnel with filter paper, allow to dry overnight on large sheet of filter paper (Whatman No.3) at room temperature. Then heat in an oven at 200°C for two hours, place in desiccator. b) All solutions of mineral acids, bases, and buffers were prepared from reagent grade chemicals with water previously distilled and passed through a commercial deionizer. Water purified in this manner is used throughout the procedure.