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%.     

HPLC separation of catecholamines after derivatization with 9-fluorenylmethyl chloroformate

Summary Optimum conditions for the separation of 9-fluorenylmethyl chloroformate derivatized catecholamines by HPLC are described; three catecholamines (noradrenaline, adrenaline and dopamine) and an internal standard (epinine) were separated in less than 20 minutes under isocratic conditions. This method is 17 to 350 times more sensitive than electrochemical detection, depending on the test compounds. It has been applied to the analysis of catecholamines in urine. The sample was extracted by a metal-loaded silica prior to separation.     

Determination of the hydrolysis rate constants and activation energy of aesculin with capillary electrophoresis end-column amperometric detection

Indirect chemiluminescence (ICL) detection for capillary electrophoresis (CE) of monoamines and catechol using luminol-K3 [Fe(CN)6] system was described. A strong and stable background chemiluminescence (CL) signal can be generated by luminol-K3 [Fe(CN)6] reaction. Based on the principle of that some phenolic compounds may be oxidized in the presence of K3 [Fe(CN)6], quenching effect of catecholamines for luminol-K3[Fe(CN)6] CL reaction results in a quantifiable decrease in the background signal. The conditions for CE separation and the CL detection for four standard catecholamines were systematically investigated using a homemade CE-ICL system. Under the optimum conditions, the detection limits of dopamine (DA), epinephrine (EP), norepinephrine (NE) and catechol (CA) were determined to be 0.18 mciroM 0.39 microM 0.48 microM and 0.09 microM, respectively. It also has been successfully applied to analyze seven pharmaceutical samples and seven human urine samples.     

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.     

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.     

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.     

Determination of catecholamines and indoleamines in human urine based on intramolecular excimer-forming derivatization and fluorescence detection.

A liquid chromatographic (LC) determination of catecholamines and indoleamines is described. This is based on intramolecular excimer-forming fluorescence derivatization with 4-(1-pyrene)butanoyl chloride, followed by reversed-phase LC. The analytes, containing an amino moiety and phenolic hydroxyl moieties in a molecule, were converted to the corresponding polypyrene-labeled derivatives by one-step derivatization. They afforded intramolecular excimer fluorescence, which can clearly be discriminated from the normal fluorescence emitted from reagent blanks. The detection limits (S/N = 3) for catecholamines and indoleamines were femto-mole levels per 20-microL injection. Furthermore, this method was applied to a urine assay.     

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 chromatographic determination of L-3-(3,4-dihydroxyphenyl)-2-methylalanine (alpha-methyldopa) in human urine and plasma

A procedure is described for the determination of alpha-methyldopa (MD) [L-3-(3,4-dihydroxyphenyl)-2-methylalanine], its metabolite and catecholamines in the urine and plasma of patients undergoing MD therapy, by high-performance liquid chromatography with dual working electrode coulometric detection. An efficient sample preparation procedure is presented for the isolation of endogenous MD, its metabolite and catecholamines from plasma or urine. After deproteinization of a plasma sample with methanol containing 2% of 0.5 M perchloric acid and dilution of a urine sample (1:200), MD, dihydroxyphenylacetic acid (DOPAC), 3-O-methylmethyldopa (3-OMMD) and homovanillic acid (HVA) were separated with a Supelcosil LC-18 column. Catecholamines were extracted from the supernatant of deproteinized plasma or from urine by ion exchange on a Sephadex CM-25 column and subsequent adsorption on alumina. The use of the same mobile phase for the concurrent assay of MD, its metabolite and catecholamines increased considerably the efficiency of sample separation. Recoveries were close to 100% for MD, DOPAC, 3-OMMD and HVA and 70% for catecholamines. The effects of various experimental parameters related to mobile phase composition on chromatographic performance are reported. The purity of the eluted compounds was tested by recording both the first detector response (oxidation current) and the second detector response (reduction current). The ratio of the detector responses yielded a chemical reversibility ratio for the detected compound. A number of applications such as monitoring data from patients under MD therapy are presented.     

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.     

Study of catecholamines in patient urine samples by capillary electrophoresis.

Capillary zone electrophoresis with photodiode array detection at 220 nm was used for analysis of catechol compounds in human urine. The method was optimized with reference compounds 3,4-dihydroxybenzylamine, adrenaline, noradrenaline, normetanephrine, dopamine, dopac (homogensitic acid), methanephrine, vanillyl-mandelic acid, 5-hydroxyindoleacetic acid (5-HIAA), homovanillic acid and 3-methoxytyramic acid at pH 4.0 and 8.0 for their electrophoretic separation. The UV spectra of the catechols were detected at a concentration of 20 microM. Repeatability of the method calculated using the absolute migration times of the catechols was below 1.5% and using the peak areas below 5%. The patient samples were hydrolyzed by 0.5 M acid or base solutions. In the studies, a few patient samples were analyzed using 3,4-dihydroxybenzylamine as an internal standard. In the hydrolysis steps needed for their detection in urine, all the other catecholamines, except 5-HIAA, did not decompose to detectable species at 220 or 254 nm. The concentrations of the catecholamines observed in real samples were at nM levels.     

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.     

Use of 2,6-dinitro-4-trifluoromethylbenzenesulfonic acid as a novel derivatizing reagent for the analysis of catecholamines, histamines and related amines by gas chromatography with electron-capture detection

We have found that 2,6-dinitro-4-trifluoromethylbenzenesulfonic acid reacts rapidly and specifically with primary amines at room temperature. We have used this reagent for derivatizing phenylethylamines, including catecholamines, and histamine and 1-methylhistamine. After the reaction, hydroxyl groups were derivatized to form the corresponding trimethylsilyl ethers, and the final derivatives were analyzed by gas chromatography with electron-capture detection. These derivatives are stable, possess excellent gas chromatographic properties and are detected with high sensitivity. We have applied this method to the analysis of histamine and 1-methylhistamine in human urine.     

Determination of cationic neurotransmitters and metabolites in brain homogenates by microchip electrophoresis and carbon nanotube-modified amperometry

An electrophoretic method for simultaneous determination of catecholamines and their O-methoxylated metabolites on the microchip as well as in the capillary is presented. A complex separation system employing sodium dodecyl sulfate (SDS) micelles, dendrimers forming a second pseudostationary phase and borate complexation is needed for the satisfactory separation of the selected compounds on the short migration length. A carbon nanotube-modified working electrode has been applied for the sensitive amperometric detection with submicromolar detection limits. The applicability of this new method for the analytics of real samples is demonstrated by analysis of mouse brain homogenate on the microchip and human urine by capillary electrophoresis.     

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.     

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.     

High-performance liquid chromatographic method for the determination of prolyl peptides in urine

A rapid and accurate method is described for the determination of prolyl peptides in urine, with specific reference to the dipeptide prolylhydroxyproline, and free hydroxyproline and proline. Free amino acids and peptides were isolated from urine on cation-exchange minicolumns, and free imino acids and prolyl-N-terminal peptides were selectively derivatized with 4-chloro-7-nitrobenzofurazan, after reaction of amino acids and N-terminal aminoacyl peptides with o-phthalaldehyde. The highly fluorescent adducts of imino acids and prolyl peptides were separated on a Spherisorb ODS 2 column by isocratic elution for 12 min using as mobile phase 17.5 mM aqueous trifluoracetic acid solution containing 12.5% acetonitrile (eluent A), followed by gradient elution from eluent A to 40% of 17.5 mM aqueous trifluoroacetic acid solution containing 80% acetonitrile in 20 min. Analytes of interest, in particular the dipeptide prolylhydroxyproline, can be easily quantified by fluorimetric detection (epsilon ex = 470 nm, epsilon em = 530 nm) without interference from primary amino-containing compounds.     

Determination of catecholamines by ion chromatography coupled to acidic potassium permanganate chemiluminescence detection

A simple,fast,sensitive,highly selective and eco-friendly analytical method for the determination of catecholamines in human urine by ion chromatography（IC） with chemiluminescence（CL） detection was described in this paper.Using 12 mmol/L H₂SO₄ without any organic additive as eluent,three catecholamines including epinephrine（EP）,norepinephrine（NE） and dopamine（DA） were well separated on a cation-exchange column.The CL detection was based on the reaction of analytes with acidic potassium permanganate in the presence of formaldehyde as an enhancer.The absence of methanol and acetonitrile in eluent made the proposed method more sensitive and eco-friendly.Under the optimal conditions,the linear range of the proposed method was in the range of 0.02-0.5μg/mL.The limit of detection（LOD） was in the range of 0.6 and 5.1μg/L.The relative standard deviations （RSD） for 0.1μg/mL mixed standard solution were in the range of 0.8-1.9%（n = 11）.The method has been applied to the determination of catecholamines in human urine successfully.Excellent spiked recoveries were achieved for catecholamines ranged from 91.2%to 112.7%.     

Determination of catecholamines by CE with direct chemiluminescence detection

A rapid and sensitive method to detect three catecholamines, isoprenaline, epinephrine, and dopamine, by CE coupled with direct luminol-potassium periodate chemiluminescence (CL) detection is described. The conditions for CE separation and CL reaction were systematically optimized. Under the optimum conditions, the baseline separation of three catecholamines was achieved within 6.5 min. The LODs obtained in standard solution were 5.3 x 10(-8 )mol/L for isoprenaline, 4.7 x 10(-8 )mol/L for epinephrine, and 1.5 x 10(-7 )mol/L for dopamine. The RSD of the migration time and peak area were less than 1.8 and 3.6% (n = 5), respectively. The present method was applied to the determination of the dopamine in urine samples of cigarette smokers and nonsmokers. The results obtained indicate that there is a close relationship between the content of dopamine in human urine and the amount of cigarettes smoked daily; the level of dopamine in smokers is higher than in nonsmokers.     

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.