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.     

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.     

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.     

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.     

Simultaneous determination of catecholamines and dobutamine in human plasma and urine by high-performance liquid chromatography with fluorimetric detection.

We report a reliable fluorimetric assay for the simultaneous determination of norepinephrine, epinephrine, dopamine and dobutamine in human plasma and urine, based on liquid-liquid extraction and derivatization with the fluorogenic agent 1,2-diphenylethylenediamine prior to chromatography. The method is sensitive (detection limit 0.3-0.8 pg injected) and reproducible (coefficients of variation 1-10%), and shows good accuracy (93-98%). The method should also be used when one only wants to measure the concentrations of the natural catecholamines, in order to avoid interference by metabolites of dobutamine and by the late-eluting dobutamine itself.     

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.     

Capillary electrophoresis‐laser‐induced fluorescence detection of rat brain catecholamines with microwave‐assisted derivatization

In this study, a rapid and sensitive method is described for the catecholamines detection in rat brain. CE with LIF detection for the determination of FITC derivatized catecholamines (dopamine, epinephrine, and norepinephrine) was demonstrated. Conventional water bath and microwave‐assisted derivatization methods were employed and a significant reduction in the derivatization time from 2 h for the conventional water bath at room temperature (ca. 25°C) to 2 min for the microwave‐assisted derivatization was achieved. Online sample concentration of field‐amplified sample stacking (FASS) method was employed to achieve higher sensitivities (the detection limits obtained in the normal injection mode ranged from 2.6 to 4.5 ng L^?1 and in the FASS mode ranged from 22 to 34 pg L^?1). Furthermore, this microwave‐assisted derivatization CE–LIF method successfully determined catecholamines in rat brain with as low as 100 ng L^?1 (FASS mode) to 10 μg L^?1 (normal injection mode). This CE–LIF method provided better detection ability when compared to the best reports on catecholamines analyses.     

Catecholamines derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole: characterization of chemical structure and fluorescence properties

4-Fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) was evaluated as a fluorogenic derivatization reagent for the analysis of the catecholamines, dopamine, epinephrine, norepinephrine, and their naturally occurring metabolites, metanephrine and normetanephrine, homovanillic acid, 3,4-dihydroxyphenyl acetic acid. These compounds reacted rapidly with NBD-F under mild conditions to form stable derivatives. The optimal reaction conditions were found to be 12.5 mM borate buffer pH 8.0 in water:acetonitrile (1:1) at 50 °C for 5 min. New NBD derivatives of all the catecholamines and metabolites were prepared and purified and were shown by electrospray mass spectrometry to be fully reacted at all available catechol and amine sites, resulting in di- or tri-substituted derivatives. Homovanillic acid and 3,4-dihydroxyphenyl acetic acid reacted with NBD-F but gave non-fluorescent derivatives. The fluorescence excitation wavelength maximum demonstrated a red shift for the derivatives with increasing polarity of the solvent and the fluorescence intensity increased linearly with increasing organic ratio in the solvent-aqueous buffer complex. The presence of electrolyte in the solvent and the electrolyte concentration in the solvent-electrolyte complex had little effect on the fluorescent intensity. The fluorescence quantum yields in acetonitrile were also obtained. The separation behavior of the NBD-catecholamines was determined by high-performance liquid chromatography (HPLC). The studies demonstrated good potential for the application of NBD-F derivatization to the quantitative analysis of catecholamines and related compounds in biological matrices.     

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.     

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.     

Simultaneous determination of polyamines and catecholamines in PC-12 tumor cell extracts by capillary electrophoresis with laser-induced fluorescence detection

A method to detect polyamines and catecholamines in PC-12 tumor cell extracts by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) is described for the first time. Both derivatization conditions and buffer concentrations and pH were optimized. Under optimized conditions the polyamines (putresine, spermine, spermidine) and catecholamines (dopamine, norepinephrine, epinephrine, serotonin) were derivatized with fluorescein isothiocyanate and separated at 25 kV in a fused-silica capillary (50 microm ID x 40 cm) with 0.1 M borate, pH 9.0, in less than 18 min. The influence of running buffer conditions, such as buffer pH and concentrations, were also investigated. Linearity of the analytes ranged from 0.05 to 1.0 micromol/L, and the detection limit (S/N = 3 ) ranged from 0.03 to 2.50 nmol/L. The concentrations of polyamines and catecholamines in PC-12 tumor cell extracts were determined with this method.     

Catecholamine analysis with strong cation exchange column liquid chromatography–peroxyoxalate chemiluminescence reaction detection

A liquid chromatography–chemiluminescence detection method was developed and validated for the determination of catecholamines (norepinephrine, epinephrine, and dopamine) in mouse brains. Chromatography was performed on a strong cation exchange column (150 × 2.0-mm id) using an isocratic mobile phase of 65 mM potassium acetate/75 mM potassium phosphate (95:5, pH 3.5) at a flow rate of 0.2 mL/min following post-column fluorescence derivatization of catecholamines with ethylenediamine and peroxyoxalate chemiluminescence reaction detection. The recovery of catecholamines added to mouse brain samples was more than 95.0%, while intra- and inter-day precision of the assay were <4.8%. The validated method was used to determine norepinephrine and dopamine concentrations in mouse brains without prior sample purification.     

Measurement of catecholamines, their precursor and metabolites in human urine and plasma by solid-phase extraction followed by high-performance liquid chromatography with fluorescence derivatization

A high-performance liquid chromatographic method is described for the determination in human urine and plasma of catecholamines, their precursor and metabolites [amino compounds (norepinephrine, epinephrine, dopamine, normetanephrine, metanephrine, 3-methoxytyramine and L-DOPA), acidic compounds (3,4-dihydroxymandelic acid, 3,4-dihydroxyphenylacetic acid, vanillylmandelic acid and homovanillic acid) and alcoholic compounds (3,4-dihydroxyphenylethyleneglycol and 4-hydroxy-3-methoxyphenylethyleneglycol)]. Urine (0.5 ml) containing 3,4-dihydroxybenzylamine and 4-hydroxy-3-methoxycinnamic acid (internal standards) is deproteinized with perchloric acid, and the resulting solution is fractionated by solid-phase extraction on a strong cation-exchange resin cartridge (Toyopak IC-SP S) into two fractions (amine fraction and acid-alcohol fraction), which include 3,4-dihydroxybenzylamine and 4-hydroxy-3-methoxycinnamic acid, respectively. Plasma (0.7 ml) is deproteinized in the presence of 3,4-dihydroxybenzylamine (internal standard) in the same manner, and the resulting solution is directly used as an acid-alcohol fraction, while an amine fraction is obtained as for urine. Each fraction is subjected to the previously established ion-pair reversed-phase chromatography with post-column derivatization involving coulometric oxidation followed by fluorescence reaction with 1,2-diphenylethylenediamine. The detection limits, at a signal-to-noise ratio of 5, of the compounds measured in urine are 300 pmol/ml for the two mandelic acids, 2-7 pmol/ml for the other acidic and alcoholic compounds, 12 pmol/ml for L-DOPA and 0.6-2 pmol/ml for the other amino compounds; the corresponding values for plasma samples are 80, 0.5-3, 10 and 0.6-3 pmol/ml, respectively.     

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.     

Analysis of catecholamines and related compounds in one whole metabolic pathway with high performance liquid chromatography based on derivatization

The simultaneous determination of Tyr, catecholamines and their metabolites in one whole metabolic pathway using high performance liquid chromatography coupled with fluorescence detection (HPLC–FLD) based on pre-column derivatization has been achieved successfully. 1,3,5,7-Tetramethyl-8-(N-hydroxysuccinimidyl butyric ester)-difluoroboradiaza-s-indacene (TMBB-Su), a highly reactive fluorescent reagent synthesized in our previous work, was used for the labeling of tyrosine (Tyr), l-3,4-dihydroxyphenylalanine (l-DOPA), dopamine (DA), norepinephrine (NE), epinephrine (E) and metanephrine (MN). Derivatization conditions including reagent concentration, buffer, reaction temperature and reaction time were also investigated to improve the derivatization efficiency and thus the sensitivity of the detection. The separation of the derivatives was obtained on C₁₈ column with the mobile phase of 20 mM pH 3.5 citric acid (H₃Cit)–sodium hydrogen phosphate (Na₂HPO₄) buffer and methanol. Good linearities with correlation coefficients square (R²) greater than 0.998 in the corresponding concentration ranges were observed and the detection limits (S/N = 3) were found in the range from 0.10 to 0.40 nM (l-DOPA: 1.45 nM). The proposed method has been applied to the detection of catecholamines and related compounds in mice liver and brain samples without tedious extraction or purification procedure, which exhibits excellent selectivity and sensitivity in the analysis of complex samples. This work provides an alternative approach in the metabolic research of catecholamines and is helpful for the study of catecholamine metabolism.     

A specific sensitive HPLC method for determination of plasma dopamine

Summary We describe here a sensitive, selective and rapid method to quantitate plasma catecholamines, especially dopamine, using high-performance liquid chromatography with electrochemical detection. This method requires a 10-minute run time and has a threshold for detection of 2 picograms, (10pg/ml).A number of commonly employed mobile phases for catecholamine analysis have been tested and have failed to detect dopamine in biological samples. Neither acetonitrile (3–7%) or methanol, (5–8%) in the mobile phase has produced consistently interpretable data either due to inability to detect or interference from co-eluting substances. Optimal detection was achieved with a mobile phase containing sodium acetate (6.8g), citric acid (5.9g), EDTA (48mg), di-n-butylamine (270l), Na-1-octane sulfate (850mg), methanol (100 ml) (amounts refer to 1 liter aqueous solution) (pH 4.3). The mobile phase was passed through a Waters 5 resolve C₁₈ column using a Waters 590 pump and m460 electrochemical detector and 740 data module, Flow rate was 0.9ml/min. Using this method, normal values in human and swine left ventricular myocardium and human and swine plasma have been established for norepinephrine, epinephrine, and dopamine.     

Separation and determination of epinephrine and dopamine in traditional Chinese medicines by micellar electrokinetic capillary chromatography with laser induced fluorescence detection

A micellar electrokinetic capillary chromatography method with laser-induced fluorescence detection was developed for the analysis of epinephrine and dopamine after derivatization with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole. The optimum derivatization conditions were: 30 mM sodium borate (pH adjusted to 8.0 with 1.0 M HCl), reaction time 30 min at 60 degrees C. Baseline separation was achieved within 14 min with a running buffer composed of 10 mM sodium borate + 25 mM sodium dodecyl sulfate (pH adjusted to 9.5 with 0.1 M NaOH) and an applied voltage of 15 kV. Good linearity relationships (correlation coefficients: 0.9991 for epinephrine and 0.9985 for dopamine) between peak areas and concentrations of the analytes were obtained. The detection limits and quantification limits for epinephrine and dopamine were 0.0038 mg/L and 0.013 mg/L, and 0.065 mg/L and 0.020 mg/L, respectively. The method was applied to the analysis of the two compounds in two Chinese medicines with recoveries in the range of 92.6-108.7%.     

A general method for the quantitative determination of catecholamines in body fluids using off-line sample pretreatment and HPLC-ED analysis

Summary Reversed phase HPLC with electrochemical detection (HPLC-ED) was used for quantitative determination of adrenaline, noradrenaline, and dopamine in several complex biological matrices, including plasma, uremic plasma, and urine. Three different methods of sample preparation for use in this clinical chemistry were tested. These were adsorption of catecholamines on alumina, organic solvent extraction after complex formation with diphenylborate, and adsorption of catecholamines on a cation exchange gel followed by organic solvent extraction of the elute. The selectivity and precision of the three methods were evaluated. The organic solvent extraction proved to be more precise and selective than adsorption on alumina (adrenaline: cv=3.80% vs. 7.58%; noradrenaline: cv=1.70% vs. 4.26%); it also proved suitable for use in the routine quantitative determination of catecholamines in plasma from patients with normal renal function (creatinine <1.2 mg/dl). However when working with uremic plasma or urine, a more selective sample preparation was required. In this case the adsorption of catecholamines on a cation exchange gel followed by organic solvent extraction of the elute was sufficiently selective and precise and thus allowed a reliable quantitative determination of adrenaline and noradrenaline from rather complex biological matrices (adrenaline: cv=6.2%; noradrenaline: cv=2.8%). Use of this specific method showed that basal plasma catecholamine levels in dialysis patients are comparable to those found in patients with normal renal function (adrenaline: 47.7±22.2 pg/ml; noradrenaline: 310.3±121.4 pg/ml).     

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.